Temperature- and salinity-dependent development of a Nordic strain of Ichthyophthirius multifiliis from rainbow trout

During the twentieth century evidence was presented which suggested the presence of various strains and races of the parasite Ichthyophthirius multifiliis Fouquet. However, ecological profiles of various parasite isolates from different climatic zones are sparse. Such stringent characterizations of parasite development at defined abiotic conditions could provide valuable criteria for the different races: profile comparison from various localities is one way to differentiate these strains. Baseline investigations were therefore performed on the associations between abiotic factors (temperature/salinity) and the development of theronts in tomocysts of I. multifiliis isolated from rainbow trout in a Danish trout farm. It was shown that tomocyst formation and theront development took place between 5 and 30degreesC. Development rates and sizes of theronts were clearly affected by temperature: theronts escaped tomocysts already after 16-27 h at 25degreesC and 30degreesC, whereas this process took 8-9 days at 5degreesC. Likewise, theront size decreased steadily from a maximum of 57.4 x 28.6 mum at 5degreesC to 28.6 x 20.0 mum at 30degreesC. This size variation was only partly associated with the number of theronts that appeared at different temperatures. The lowest number of theronts escaping from one tomocyst was indeed found at 5-7degreesC (mean 329-413). At 11.6, 17.0 and 21degreesC. the highest number of theronts appeared (mean 546-642). However, at 25 and 30degreesC, the number decreased (458 and 424, respectively). Additional studies on the salinity dependent development of the parasite (at 11.6degreesC) showed that salinities above 5 p.p.t. totally inhibited development. Even at 5 p.p.t. the developmental time significantly increased and the number of theronts produced from one tomocyst decreased.

小麦抗白粉病新基因PmCD1和抗条锈病基因YrCD的鉴定及分子标记筛选

本研究对自育小麦白粉病抗源“07鉴126”和条锈病抗源CD1437、CD0534-5进行抗性遗传分析和微卫星引物的筛选，建立了与PmCD1和YrCD抗病基因连锁的SSR分子标记，主要研究结果如下： 1.小麦白粉抗源“07鉴126”抗白粉病基因的鉴定和分子标记的建立 品系“07鉴126”对我国目前白粉菌强优势生理小种E09、E11和其它多种小种表现免疫或高度抵抗。Pm-sus是07鉴126的自然突变感病株。利用“07鉴126”和Pm-sus的F2抗病性分离群体进行抗条锈病性遗传分析和分子标记定位，结果表明，“07鉴126”的白粉抗性为显性单基因控制的全生育期抗性，暂命名为PmCD1；并筛选到了与PmCD1共分离的显性SSR分子标记Xbarc183。系谱分析和分子标记分析表明PmCD1来源于荆州黑麦。抗谱分析表明PmCD1不同于已知的黑麦抗白粉基因，是一个新的抗白粉病基因。Xbarc183这一分子标记的建立为PmCD1的分子标记辅助选择和抗病基因累加提供了方便。 2.小麦条锈抗源CD1437抗条锈病基因的鉴定和分子标记的建立 利用对优势条锈菌小种条中32免疫的小麦品系CD1437及其自然突变感病株Yr-sus杂交构建F2、F3抗病性分离群体。抗条锈病性遗传分析结果显示，1437的抗条锈性为显性单基因控制的全生育期抗性，该基因暂命名为YrCD。SSR分析发现，位于1B染色体上的7个SSR标记Xcfd65、Xgwm11、Xgwm18、Xbarc187、Xwmc406、 Xwmc419、Xwmc216依次分布在YrCD的一侧，与YrCD的遗传距离在1.7 cM至9.2 cM。YrCD和YrCH42的等位性分析显示二者可能为等位基因。YrCD和Yr24、Yr26的抗谱相似。系谱分析和分子标记分析表明贵农20是YrCD的供体。本研究推测YrCD、Yr24、Yr26和YrCH42可能是等位基因，并推测Yr-sus是缺失突变体。 3. 小麦条锈抗源CD0534-5抗条锈病基因的鉴定 利用对条中32免疫的小麦抗条锈病品系CD0534-5及其感病重组自交系CD0534-4建立F2抗病性分离群体。抗条锈病性遗传分析表明，CD0534-5的条锈抗性由两对独立的显性主效基因控制。用BSK法分析，发现其中一对基因与SSR分子标记Xgwm11、Xgwm18、Xwmc128、Xwmc419连锁，该基因是来源于贵农20的YrCD。另一抗性基因来源贵农19，是极有利用价值的未知抗性基因。 This study focused on the investigation and identification of a novel powdery mildew resistant gene PmCD1 in wheat lines 07jian126 and stripe rust resistant gene YrCD in wheat lines CD1482 and CD0534-5, and screened SSR markers tightly linked to them. The main results were as follows: 1.Identification and SSR markers screening of a novel powdery mildew PmCD1 in wheat line 07jian126. Using a Pm resistant wheat line 07jian126 and its Pm susceptible mutant, a F2 population was constructed. Pedigree and genetic analyses indicated that the Pm resistance in 07jian126 was tranderred from rye (Secale cereale L.) cv. Jinzhou and was controlled by a single dominant gene. Differential test using 21 Bgt isolates revealed that the Pm resistant gene in 07jian126 is novel and was temporarily designated as PmCD1. A dominant SSR marker Xbarc183/130 bp was found co-segregated with PmCD1 in the F2 population. The diagnostic band of Xbarc183/130 bp co-segregating with PmCD1 could be used as an ideal marker in marker-assisted-selection during wheat breeding program. 2. Identification and SSR markers mapping of yellow rust resistant gene YrCD in wheat line CD1437. Wheat line CD1437 was highly resistant to predominant Chinese stripe rust race CYR32 at both seedling and adult stages. A F2 population was developed from the cross of CD1437 and its Yr susceptible mutant Yr-sus. Genetic analysis indicated line CD1437 contains a single dominant gene, temporarily designated YrCD. Seven SSR markers on the chromosome 1BS including Xcfd65, Xgwm11, Xgwm18, Xbarc187, wmc406, Xwmc419and Xwmc216 were close linked to YrCD with a genetic dsitance 1.7 cM to 9.2 cM. YrCD came from wheat cultivar Guinong 20. Allelic test of CD1437 and Chinese cultivar Chuanmai 42 indicated that YrCD and YrCH42 were allelic. Reaction patterns of YrCD and Yr24, Yr26 to 21 PST isolates were the same. These results suggested that YrCD and Yr24, Yr26, YrCH42 might be allelic. 3.Detection and identification of yellow rust resistance genes in wheat line CD0534-5 Wheat line CD0534-5 was highly resistant to predominant Chinese stripe rust race CYR32, while its recombinant inbred line CD0534-4 was susceptible. Genetic analysis with a F2 population developed from the cross of CD0534-5 and CD0534-4 indicated line CD0534-5 contains two independent dominant genes. Four SSR markers on the chromosome 1BS including Xgwm11, Xgwm18, Xwmc128, Xwmc419 were found to linked with one gene in CD0534-5. According the locations of makers and pedigree, this gene in CD0534-5 was YrCD, from cultivar Guinong 20. Another resistant gene was from cultivar Guinong 19, different with those genes on 1B such as Yr10, Yr15, Yr5 etc, was a valuable resistant gene in wheat breeding.

The Development of a Marine Natural Product-based Antifouling Paint

Problems with tin and copper antifouling compounds have highlighted the need to develop new environmentally friendly antifouling coatings. Bacteria isolated from living surfaces in the marine environment are a promising source of natural antifouling compounds. Four isolates were used to produce extracts that were formulated into ten waterbased paints. All but one of the paints showed activity against a test panel of fouling bacteria. Five of the paints were further tested for their ability to inhibit the settlement of barnacle larvae, Balanus amphitrite, and algal spores of Ulva lactuca, and for their ability to inhibit the growth of U. lactuca. Two paints caused a significant decrease in the number of settled barnacles. One paint containing extract of Pseudomonas sp. strain NUDMB50-11, showed excellent activity in all assays. The antifouling chemicals responsible for the activity of the extract were isolated, using bioassay guided fractionation, and their chemical structures determined.

Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

Aims: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. Methods nand Results: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. Conclusions: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. Significance and Impact of the Study: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.

Ulva diversity in the Yellow Sea during the large-scale green algal blooms in 2008-2009

P>In the Yellow Sea of China, large-scale green tides have broken out for three consecutive years from 2007 to 2009. As part of the efforts to localize the algal source, two cruises were conducted in the early stage and the outbreak stage of the bloom in 2009. We analyzed the morphological and genetic diversity of drifting Ulva specimens and culture-derived isolates from seawater sampled in different localities. For phylogenetic analyses, the nuclear encoded ribosomal DNA internal transcribed spacer region (ITS nrDNA) and the plastid encoded large subunit of ribulose-1, 5-bisphosphate carboxylase/oxgenase gene (rbcL) were used. Our molecular and morphological data indicate that the dominant free-floating Ulva species in 2008 and 2009 possibly belonged to a single strain of the U. linza-procera-prolifera (LPP) clade. The ITS sequences from bloom-forming algal samples with dense branches were identical to those from U. linza-like specimens without branches derived from the Yellow Sea. Microscopic individuals of the dominant Ulva strain were detected in eight stations, revealing that spore dispersal in the water helped to enlarge biomass in the water during the outbreak stage of green tide in the Yellow Sea.

Identification of vhhP2, a novel genetic marker of Vibrio harveyi, and its application in the quick detection of V. harveyi from animal specimens and environmental samples

Aims: To investigate the species-specific prevalence of vhhP2 among Vibrio harveyi isolates and the applicability of vhhP2 in the specific detection of V. harveyi from crude samples of animal and environmental origins. Methods and Results: A gene (vhhP2) encoding an outer membrane protein of unknown function was identified from a pathogenic V. harveyi isolate. vhhP2 is present in 24 V. harveyi strains isolated from different geographical locations but is absent in 24 strains representing 17 different non-V. harveyi species, including V. parahaemolyticus and V. alginolyticus. A simple polymerase chain reaction method for the identification of V. harveyi was developed based on the conserved sequence of vhhP2. This method was demonstrated to be applicable to the quick detection of V. harveyi from crude animal specimens and environmental samples. The specificity of this method was tested by applying it to the examination of two strains of V. campbellii, which is most closely related to V. harveyi. One of the V. campbellii strains was falsely identified as V. harveyi. Conclusions: vhhP2 is ubiquitously present in the V. harveyi species and is absent in most of the non-V. harveyi species; this feature enables vhhP2 to serve as a genetic marker for the rapid identification of V. harveyi. However, this method can not distinguish some V. campbellii strains from V. harveyi. Significance and Impact of the Study: the significance of our study is the identification of a novel gene of V. harveyi and the development of a simple method for the relatively accurate detection of V. harveyi from animal specimens and environmental samples.

Genetic mechanisms of multi-antimicrobial resistance in a pathogenic Edwardsiella tarda strain

TX01, a pathogenic Edwardsiella tarda strain isolated from diseased fish at an epidemic-inflicted fish farm in China, exhibits resistance to multiple classes of antimicrobial agents. The genes (kn(R). catA3, and tet(A), respectively) encoding resistance to kanamycin, chloramphenicol, and tetracycline were cloned and found to be 99-100% identical to the corresponding genes carried by known plasmids and transposons of human, animal, and environmental isolates. Further study demonstrated that TX01 harbors a plasmid, pETX, which proved to be (i) the carrier of the tet and cut operons; (ii) a mobile genetic element that is capable of transferring between bacteria of different genera. These results, which, to our knowledge, documented for the first time the co-existence of chloramphenicol and tetracycline resistance determinants on a conjugative plasmid in a pathogenic E tarda strain, indicated that gene acquisition via horizontal transferring of pETX-like mobile genetic entities may have played an important part in the dissemination of antimicrobial resistance and that there have existed for some time widespread genetic exchanges between bacteria of human, animal/fish, and environmental origins. (C) 2008 Elsevier B.V. All rights reserved.

Regulation of the Edwardsiella tarda Hemolysin Gene and luxS by EthR

Edwardsiella tarda is a pathogen with a broad host range that includes human and animals. The E. tarda hemolysin (Eth) system, which comprises EthA and EthB, is a noted virulence element that is widely distributed in pathogenic isolates of E. tarda. Previous study has shown that the expression of ethB is regulated by iron, which suggests the possibility that the ferric uptake regulator (Fur) is involved in the regulation of ethB. The work presented in this report supports the previous findings and demonstrates that ethB expression was decreased under conditions when the E. tarda Fur (Fur(Et)) was overproduced, and enhanced when Fur(Et) was inactivated. We also identified a second ethB regulator, EthR, which is a transcription regulator of the GntR family. EthR represses ethB expression by direct interaction with the ethB promoter region. In addition to ethB, EthR also modulates, but positively, luxS expression and AI-2 production by binding to the luxS promoter region. The expression of ethR itself is subject to negative autoregulation; interference with this regulation by overexpressing ethR during the process of infection caused (i) drastic changes in ethB and luxS expressions, (ii) vitiation in the tissue dissemination and survival ability of the bacterium, and (iii) significant attenuation of the overall bacterial virulence. These results not only provide new insights into the regulation mechanisms of the Eth hemolysin and LuxS/AI-2 quorum sensing systems but also highlight the importance of these systems in bacterial virulence.

Diverse tetracycline resistant bacteria and resistance genes from coastal waters of Jiaozhou Bay

Environmental microbiology investigation was carried out in Jiaozhou Bay to determine the source and distribution of tetracycline-resistant bacteria and their resistance mechanisms. At least 25 species or the equivalent molecular phylogenetic taxa in 16 genera of resistant bacteria could be identified based on 16S ribosomal deoxyribonucleic acid sequence analysis. Enterobacteriaceae, Pseudomonadaceae, and Vibrionaceae constituted the majority of the typical resistant isolates. Indigenous estuarine and marine Halomonadaceae, Pseudoalteromonadaceae, Rhodobacteraceae, and Shewanellaceae bacteria also harbored tetracycline resistance. All the six resistance determinants screened, tet(A)-(E) and tet(G), could be detected, and the predominant genes were tet(A), tet(B), and tet(G). Both anthropogenic activity-related and indigenous estuarine or coastal bacteria might contribute to the tet gene reservoir, and resistant bacteria and their molecular determinants may serve as bioindicators of coastal environmental quality. Our work probably is the first identification of tet(E) in Proteus, tet(G) in Acinetobacter, tet(C) and tet(D) in Halomonas, tet(D) and tet(G) in Shewanella, and tet(B), tet(C), tet(E), and tet(G) in Roseobacter.

Phylogeography of the Northern Atlantic species Chondrus crispus (Gigartinales, Rhodophyta) inferred from nuclear rDNA internal transcribed spacer sequences

Eighteen isolates of the red algae Chondrus crispus were collected from Northern Atlantic sites, together with C. ocellatus, C. yendoi and C. pinnulatus from the North Pacific. The nuclear rDNA internal transcribed spacer (ITS) was sequenced and compared, spanning both the ITS regions and the 5.8S rRNA gene. Percentage of nucleotide variation for C. crispus ranged from 0.3% to 4.0%. Phylogenetic analyses were performed using maximum parsimony (MP), neighbor-joining (NJ) and minimum evolution methods. They showed that two main clades existed within the C. crispus samples examined and that suggested C. crispus had a single Atlantic origin. The clustering however did not follow the geographic origin. We hypothesized that the current distribution of C. crispus populations might be a result of three main factors: temperature boundaries, paleoclimate and paleoceanography. ITS data exhibited abundant molecular information not only for phylogeographical investigation but also for systematics studies.

Molecular characterizations of oxytetracycline resistant bacteria and their resistance genes from mariculture waters of China

Oxytetracycline-resistant bacteria were isolated from a mariculture farm in China, and accounted for 32.23% and 5.63% of the total culturable microbes of the sea cucumber and the sea urchin rearing waters respectively. Marine vibrios, especially strains related to Vibrio splendidus or V. tasmaniensis, were the most abundant resistant isolates. For oxytetracycline resistance, tet(A), tet(B) and tet(D) genes were detected in both sea cucumber and sea urchin rearing ponds. The dominant resistance type for V. tasmaniensis-like strains was the combination of both tet(A) and tet(B) genes, while the major resistance type for V. splendidus-like strains was a single tet(D) gene. Most of the sea cucumber tet-positive isolates harbored a chloramphenicol-resistance gene, either cat IV or cat II, while only a few sea urchin tet-positive isolates harbored a cat gene, actually cat IV. The coexistence of tet and cat genes in the strains isolated from the mariculture farm studied was helpful in explaining some of the multi-resistance mechanisms. (c) 2006 Elsevier Ltd. All rights reserved.

Concurrence of cat and tet genes in multiple antibiotic-resistant bacteria isolated from a sea cucumber and sea urchin mariculture farm in China

A basic understanding of abundance and diversity of antibiotic-resistant microbes and their genetic determinants is necessary for finding a way to prevent and control the spread of antibiotic resistance. For this purpose, chloramphenicol and multiple antibiotic-resistant bacteria were screened from a mariculture farm in northern China. Both sea cucumber and sea urchin rearing ponds were populated with abundant antibiotic-resistant bacteria, especially marine vibrios. Sixty-five percent chloramphenicol-resistant isolates from sea cucumber harbored a cat gene, either cat IV or cat II, whereas 35% sea urchin isolates harbored a cat gene, actually cat II. The predominant resistance determinant cat IV gene mainly occurred in isolates related to Vibrio tasmaniensis or Pseudoalteromonas atlantica, and the cat II gene mainly occurred in Vibrio splendidus-like isolates. All the cat-positive isolates also harbored one or two of the tet genes, tet(D), tet(B), or tet(A). As no chloramphenicol-related antibiotic was ever used, coselection of the cat genes by other antibiotics, especially oxytetracycline, might be the cause of the high incidence of cat genes in the mariculture farm studied.

Extracellular hydrolytic enzyme screening of culturable heterotrophic bacteria from deep-sea sediments of the Southern Okinawa Trough

The Southern Okinawa Trough is an area of focused sedimentation due to particulate matter export from the shelf of the East China Sea and the island of Taiwan. In order to understand the geomicrobiological characteristics of this unique sedimentary environment, bacterial cultivations were carried out for an 8.61 m CASQ core sediment sample. A total of 98 heterotrophic bacterial isolates were characterized based on 16S rRNA gene phylogenetic analysis. These isolates can be grouped into four bacterial divisions, including 13 genera and more than 20 species. Bacteria of the gamma-Proteobacteria lineage, especially those from the Halomonas ( 27 isolates) and Psychrobacter ( 20 isolates) groups, dominate in the culturable bacteria assemblage. They also have the broadest distribution along the depth of the sediment. More than 72.4% of the isolates showed extracellular hydrolytic enzyme activities, such as amylases, proteases, lipases and Dnases, and nearly 59.2% were cold-adapted exoenzyme-producers. Several Halomonas strains show almost all the tested hydrolases activities. The wide distribution of exoenzyme activities in the isolates may indicate their important ecological role of element biogeochemical cycling in the studied deep-sea sedimentary environment.

Molecular characterizations of chloramphenicol- and oxytetracycline-resistant bacteria and resistance genes in mariculture waters of China

In order to gain an understanding of the diversity and distribution of antimicrobial-resistant bacteria and their resistance genes in maricultural environments, multidrug-resistant bacteria were screened for the rearing waters from a mariculture farm of China. Both abalone Haliotis discus hannai and turbot Scophthalmus maximus rearing waters were populated with abundant chloramphenicol-resistant bacteria. These bacteria were also multidrug resistant, with Vibrio splendidus and Vibrio tasmaniensis being the most predominant species. The chloramphenicol-resistance gene cat II, cat IV or floR could be detected in most of the multidrug-resistant isolates, and the oxytetracycline-resistance gene tet(B), tet(D), tet(E) or tet(M) could also be detected for most of the isolates. Coexistence of chloramphenicol- and oxytetracycline-resistance genes partially explains the molecular mechanism of multidrug resistance in the studied maricultural environments. Comparative studies with different antimicrobial agents as the starting isolation reagents may help detect a wider diversity of the antimicrobial-resistant bacteria and their resistance genes. (C) 2009 Elsevier Ltd. All rights reserved.

Dominant chloramphenicol-resistant bacteria and resistance genes in coastal marine waters of Jiaozhou Bay, China

Studies of abundance, diversity and distribution of antibiotic-resistant bacteria and their resistance determinants are necessary for effective prevention and control of antibiotic resistance and its dissemination, critically important for public health and environment management. In order to gain an understanding of the persistence of resistance in the absence of a specific antibiotic selective pressure, microbiological surveys were carried out to investigate chloramphenicol-resistant bacteria and the chloramphenicol acetyltransferase resistance genes in Jiaozhou Bay after chloramphenicol was banned since 1999 in China. About 0.15-6.70% cultivable bacteria were chloramphenicol resistant, and the highest abundances occurred mainly in the areas near river mouths or sewage processing plants. For the dominant resistant isolates, 14 genera and 25 species were identified, mostly being indigenous estuarine or marine bacteria. Antibiotic-resistant potential human or marine animal pathogens, such as Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Shewanella algae, were also identified. For the molecular resistance determinants, the cat I and cat III genes could be detected in some of the resistant strains, and they might have the same origins as those from clinical strains as determined via gene sequence analysis. Further investigation about the biological, environmental and anthropogenic mechanisms and their interactions that may contribute to the persistence of antibiotic-resistance in coastal marine waters in the absence of specific antibiotic selective pressure is necessary for tackling this complicated environmental issue.