Induced proteome of Trichoderma harzianum by Botrytis cinerea

As a notable biocontrol agent, Trichoderma harzianum can antagonize a diverse array of phytopathogenic fungi, including Botrytis cinerea, Rhizoctonia solani and Fusarium oxysporum. Elucidating the biocontrol mechanism of T. harzianum in response to the pathogens enables it to be exploited in the control of plant diseases. Two-dimensional gel electrophoresis (2-DE) was performed to obtain secreted protein patterns of T. harzianum ETS 323, grown in media that contained glucose, a mixture of glucose and deactivated B. cinerea mycelia, deactivated B. cinerea mycelia or deactivated T. harzianum mycelia. Selected protein spots were identified using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Ninety one out of 100 excised protein spots were analyzed and some proteins were sequence identified. Of these, one l-amino acid oxidase (LAAO) and two endochitinases were uniquely induced in the media that contained deactivated B. cinerea mycelia as the sole carbon source. Activities of the cell wall-degrading enzymes (CWDEs), including β-1,3-glucanases, β-1,6-glucanases, chitinases, proteases and xylanases, were significantly higher in media with deactivated B. cinerea mycelia than in other media. This finding suggests that the cell wall of B. cinerea is indeed the primary target of T. harzianum ETS 323 in the biocontrol mechanism. The possible roles of LAAO and xylanase were also discussed.     

Susceptibility of two fishes (Oreochromis niloticus and Cyprinodon variegatus) to Pfiesteria shumwayae and its associated toxin: Influence of salinity

Researchers examining the mechanisms of ichthyotoxicity of Pfiesteria shumwayae have come to different conclusions about the role of toxin in this process. Some attribute fish mortality solely to direct attack by these pedunculate dinoflagellates on exposed fish tissue while others have provided evidence for a role of a soluble toxin. Detection of toxin, especially in low concentrations, is a function of the sensitivity of the selected bioassay methods and the various groups addressing this question have utilized different methods. One notable difference in fish bioassay methods utilized to detect Pfiesteria-associated toxin (PfTx) is the species of fish tested. Studies that have not detected PfTx in bioassays generally have used Cyprinodon variegatus (sheepshead minnow) as the test fish while those that have detected toxin generally used Oreochromis spp. (Tilapia). In this study response of these two fish species was compared to determine their relative sensitivity to physical attack by P. shumwayae and to PfTx. The results indicate that Oreochromis niloticus is more susceptible to P. shumwayae and its associated toxin than C. variegatus and implicate differences in the ability these species to osmoregulate as a contributing factor for this phenomenon. Salinity stress enhanced susceptibility of O. niloticus to PfTx and thus improved the sensitivity of the bioassay. The observation that salinity stress enhances toxicity to O. niloticus provides additional information regarding the mechanism of PfTx toxicity although the conditions utilized are not representative of the natural habitat of these freshwater fish.     

Effect of salinity and body weight on ecophysiological performance of the Pacific white shrimp (Litopenaeus vannamei)

Ecophysiological responses of Litopenaeus vannamei were evaluated as functions of environmental salinity and animal size. Growth rate, routine metabolic rate, limiting oxygen concentration, and marginal metabolic scope were determined for L. vannamei acclimated to, and tested at, salinities of 2, 10, and 28 ppt, all at 28 °C. Routine metabolic rate (RMR), estimated as oxygen-consumption rate per unit body weight for fasted, routinely-active shrimp, was independent of salinity but decreased with increasing shrimp weight. Limiting oxygen concentration for routine metabolism (LOCr) decreased with increased shrimp weight for the 10 and 28 ppt treatments, but not for the 2 ppt treatment. Marginal metabolic scope (MMS = RMR/LOCr) also decreased with increasing shrimp weight and was independent of salinity. Growth rate was significantly less at 2 ppt than at either 10 or 28 ppt, which gave similar growth rates.     

The use of warmed water treatment to induce protective immunity against the bacterial cold-water disease pathogen Flavobacterium psychrophilum in ayu (Plecoglossus altivelis)

We investigated the induction of protective immunity against bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum by warmed water treatment in ayu (Plecoglossus altivelis). Fish were immersed in a live bacterial suspension (10⁷ CFU mL⁻¹) for 30 min and placed in 700 L concrete tanks. The 28 °C warmed water treatment lasted 3 days and began 1, 6, and 24 h after immersion in the live bacterial suspension. A naïve control fish group was immersed in a sterilized modified Cytophaga (MCY) broth instead of the bacterial suspension. Fourteen days after the immersion, agglutination antibody titers against F. psychrophilum were measured by using micro-titer methods. Fish were then exposed to a bacterial bath to infect them with live F. psychrophilum, and cumulative mortality was monitored. Fish treated with warmed water at 1, 6, and 24 h after immersion in the live bacterial suspension had cumulative mortalities of 36%, 30%, and 18%, respectively, all of which were significantly lower than the cumulative mortality of the naïve control fish (90%). Treated fish also showed high antibody titers against F. psychrophilum in agglutination tests. These results demonstrate that warmed water treatment could not only cure BCWD but also immunize the fish against the causative agent F. psychrophilum.     

The effect of environmental salinity on the proteome of the sea bass (Dicentrarchus labrax L.)

The European sea bass, Dicentrarchus labrax L., tolerates a range of salinities from freshwater to hyper-saline. To study differences in protein expression, fish were reared in both freshwater and seawater. After 3-month acclimation, gill and intestine epithelia were collected and the soluble protein extracted. In all, 362 spots were differentially expressed in the gills and intestines of fishes reared in seawater compared to those from freshwater. Fifty differential protein spots were excised from a colloidal Coomassie-stained gel. Nine separate protein spots were identified unambiguously by mass spectrometry and database searching. Among the six proteins over-expressed in gill cells in seawater, five were cytoskeleton proteins and one was the aromatase cytochrome P450. In gill cells under freshwater conditions, the two over-expressed proteins identified were the prolactin receptor and the major histocompatibility complex class II β -antigen. In intestinal cells under freshwater conditions, the Iroquois homeobox protein Ziro5 was upregulated over ninefold. The expression of these proteins, their possible direct or indirect roles in the adaptation of D. labrax to salinity, and their correspondences with a previous study are discussed.     

Pattern of synaptic connections in the pineal organ of the ayu,Plecoglossus altivelis (Teleostei)

Summary Synaptic connections were studied by means of electron microscopy in the sensory pineal organ of the ayu, Plecoglossus altivelis, a highly photosensitive teleost species. Three types of specific contacts were observed in the pineal end-vesicle: 1) symmetrically organized gap junctions between the basal processes of adjacent photoreceptor cells; 2) sensory synapses endowed with synaptic ribbons, formed by basal processes of photoreceptor cells and dendrites of pineal neurons; 3) conventional synapses between pineal neurons, containing both clear and dense-core vesicles at the presynaptic site. Based on these findings, the following interpretations are given: (i) The gap junctions may be involved in an enhancement of electric communication and signal encoding between pineal photoreceptor cells. (ii) The sensory synapses transmit photic signals from the photoreceptor cells to pineal nerve cells. (iii) The conventional synapses are assumed to be involved in a lateral interaction and/or summation of information in the sensory pineal organ. A concept of synaptic relationships among the sensory and neuronal elements in the pineal organ of the ayu is presented.Fellow of the Alexander von Humboldt Foundation, Federal Republic of Germany     

盐度对长牡蛎和近江牡蛎及其杂交稚贝生长和存活的影响

2011年8月以长牡蛎自繁组GG(Crassostrea gigas♀×C.gigas♂),近江牡蛎自繁组AA(C.ariakensis♀×C.ariakensis♂)、正交组GA(Crassostrea gigas♀×C.ariakensis♂)、反交组AG(C.ariakensis♀×C.gigas♂)为实验材料,开展了稚贝对盐度的适应性研究。结果发现长牡蛎的最适生存盐度为15—35,最适生长盐度为25—35;近江牡蛎的最适生存盐度为10—25,最适生长盐度为20—25;GA的最适生存盐度为15—30,最适生长盐度为15—30,AG的最适生存盐度为20—30,最适生长盐度为20—25。GG对低盐度敏感,AA对高盐度敏感,AG具有高盐度存活的杂种优势,在盐度30时,中亲杂种优势HG×A为13.32,单亲杂种优势HGA和HAG分别为1.89和27.88,在盐度40时,HAG上升到400,GA和AG都不具有生长优势。杂种稚贝对盐度适应介于双亲之间,且表现出一定程度的父系遗传特点。     

The effect of environmental factors on the growth rate of Karenia brevis (Davis) G. Hansen and Moestrup

The red tide dinoflagellate Karenia brevis (Davis) G. Hansen and Moestrup is noted for causing mass mortalities of marine organisms in the Gulf of Mexico. Most research has focused on culture isolates from the eastern Gulf of Mexico. In this investigation, we examine the effects of light, temperature and salinity on the growth rate of K. brevis from the western Gulf of Mexico. Growth rates of K. brevis were determined under various combinations of irradiance (19, 31, 52, 67, and 123 μmol m⁻² s⁻¹), salinity (25, 30, 35, 40 and 45), and temperature (15, 20, 25, and 30 °C). Maximum growth rates varied from 0.17 to 0.36 div day⁻¹ with exponential growth rates increasing with increasing irradiance. Little or no growth was supported at 19 μmol photons m⁻² s⁻¹ for any experiment. Maximum growth rates at 15 °C were much lower than at other temperatures. Maximum growth rates of the Texas clone (SP3) fell within the range of Florida clones reported in the literature (0.17–0.36 div day⁻¹ versus 0.2–1.0 div day⁻¹). The Texas clone SP3 had a very similar light saturation point compared to that of a Florida isolate (Wilson's clone) (67 μmol m⁻² s⁻¹ versus 65 μmol m⁻² s⁻¹), and light compensation (20–30 μmol m⁻² s⁻¹1). The upper and lower salinity tolerance of the Texas clone was similar than that of some Florida clones (45 versus 46 and 25 versus 22.5, respectively). In our study, the Texas clone had the same temperature tolerance reported for Florida clones (15–30 °C). While individual clones can vary considerably in maximum growth rates, our results indicate only minor differences exist between the Texas and Florida strains of K. brevis in their temperature and salinity tolerance for growth. While the literature notes lower salinity occurrences of K. brevis in nearby Louisiana, our isolate from the southern Texas coast has the higher salinity requirements typical of K. brevis in the eastern Gulf of Mexico.     

Morphological and molecular genetic characterization of Cryptoperidiniopsis brodyi (Dinophyceae) from Australia-wide isolates

Cryptoperidiniopsis brodyi is a common heterotrophic dinoflagellate known to often co-occur with Pfiesteria species in eastern U.S. estuaries. In this study, C. brodyi from Australia and Pfiesteria piscicida from ballast water from Indonesia were characterized by morphological and genetic analyses. Two P. piscicida strains originating from ballast water samples showed little genetic differences compared to P. piscicida from other countries and their morphology was identical. This finding indicates a potential inflow of P. piscicida into Australian estuaries via ballast water. Nine cultures of C. brodyi were established from Tasmania, South Australia and Western Australia. All C. brodyi cultures exhibited identical thecal plate patterns and could not be discriminated from other non-Australian strains. In contrast, two distinct genotypes could be identified by rDNA sequence analyses which were distinct from the U.S. genotype of C. brodyi. A previous survey using PCR-based methods reported a wide distribution of Pfiesteria shumwayae in Australia. However, the present study demonstrated that SSU rDNA-based P. shumwayae-specific primers produce false-positive PCR reactions with Australian C. brodyi. These results suggest that genetic variants of C. brodyi are widely distributed in Australia and Australian genotypes of C. brodyi had previously been misidentified as P. shumwayae. This finding also indicates that previous Australian distribution studies of P. shumwayae using SSU rDNA-based primers are potentially erroneous and need to be revisited.     

Phytochemical differentiation of Galanthus nivalis and Galanthus elwesii (Amaryllidaceae): A case study

The alkaloid patterns of two occasionally sympatric Galanthus nivalis and Galanthus elwesii populations were studied by GC/MS. Thirty-seven alkaloids were detected, 25 for G. nivalis and 17 for G. elwesii. Only five alkaloids were found to occur in both species. The populations of Galanthus differed in their alkaloid biosynthetic pathways. Thus, the alkaloid pattern of G. nivalis was dominated by compounds coming from a para–para′ oxidative coupling of O-methylnorbelladine. The predominant alkaloids in the roots of this species were found to belong to the lycorine and tazettine structural types; bulbs were dominated by tazettine, leaves by lycorine and flowers by haemanthamine type alkaloids. In contrast, the alkaloid pattern of G. elwesii was dominated mainly by compounds coming from an ortho–para′ oxidative coupling. The predominant alkaloids in G. elwesii roots, bulbs and leaves were those of homolycorine type, whereas the flowers accumulated mainly tyramine type compounds. The chemotaxonomical value of the alkaloids found in the studied species is discussed.     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.     

Germination responses of Diplotaxis harra to temperature and salinity

Diplotaxis harra (Forssk.) Boiss, an annual herb in the family of Brassicaceae, is widely distributed in many sandy and gypseous areas in southern Tunisia. Laboratory experiments were carried out to assess the effects of temperature and salinity on seed germination and recovery responses after seed transfer to distilled water. The germination responses of the seeds in complete darkness were determined over a wide range of temperatures (5, 10, 15, 20, 25 and 30 °C) and salinities (0, 50, 100, 150 and 200 mM NaCl). Germination was inhibited by either an increase or decrease in temperature from the optimal temperature (15 °C). Highest germination percentages were obtained under non-saline conditions and an increase in NaCl concentrations progressively inhibited seed germination. Rate of germination decreased with an increase in salinity at all temperatures but comparatively higher rates were obtained at 15 °C. Salt stress decreased both the percentage and the rate of germination. An interaction between salinity and temperature yielded no germination at 200 mM NaCl. Seeds were transferred from salt solution to distilled water after 20 days, and those from low salinities recovered at all temperatures. At NaCl concentration of 200 mM, the recovery of germination was completely inhibited.     

The effects of short term salinity exposure on the sublethal stress response of Vallisneria americana Michx. (Hydrocharitaceae)

Short term exposure of Vallisneria americana to elevated salinity was found to induce a stress response that could be quantified by a series of metabolic assays measuring oxidative stress, photosynthetic efficiency, and dark adapted respiration. Plant specimens exposed to elevated salinity for 24 h displayed signs of oxidative damage represented by the accumulation of reactive oxygen species (ROS) and lipid hydroperoxides in blade tissue (noted at salinities of 10 and 15, respectively). Respiratory demand nearly doubled (140 nanomoles O₂ consumed min⁻¹ g⁻¹) when plants were placed in a salinity of 15 for 24 h versus control specimens maintained at 0. After 1 week of exposure a significant increase in respiration and lipid hydroperoxide content was detected in plants incubated at or above a salinity of 13. In addition, effective quantum yield () dropped significantly compared to plants maintained at a salinity below 13. These results highlight the use of cellular stress assays to monitor salt-induced sublethal responses in V. americana.     

Existence of an extra-retinal and extra-pineal photoreceptive organ that regulates photoperiodism in gonadal development of an Osmerid teleost, ayu (Plecoglossus altivelis)

Ayu (Plecoglossus altivelis) is an Osmerid teleost whose gonadal development exhibits clear photoperiodism: it is stimulated and prevented under short and long photoperiod, respectively. However, the photoreceptor organ involved in this process remains to be identified. In the present study, we examined whether gonads of ophthalmectomized (Ex) and pinealectomized (Px) ayu respond to short photoperiod to test whether photoreceptors other than lateral eyes and pineal complex are involved in the photoperiodic response of gonadal development. Gonadosomatic index (GSI) and plasma levels of sex steroids (testosterone and 11-ketotestosterone for males and testosterone and estradiol 17-beta for females) were significantly increased in the Ex+Px ayu kept under short photoperiod in both males and females as compared with the initial control. On the other hand, there were no significant increases in GSI and sex steroids in the Ex+Px ayu kept under long photoperiod. Histological observation of gonads in the Ex+Px ayu revealed that oocytes undergoing final maturation in females and proliferation of germ cells in males were observed under short photoperiod but not under long photoperiod. These results indicate that extra-retinal and extra-pineal photoreceptive organ regulates photoperiodic gonadal development in this species.     

Effects of water salinity on melatonin levels in plasma and peripheral tissues and on melatonin binding sites in European sea bass (Dicentrarchus labrax)

Sea bass is an euryhaline fish that lives in a wide range of salinities and migrates seasonally from lagoons to the open sea. However, to date, the influence of water salinity on sea bass melatonin levels has not been reported. Here, we evaluated the differences in plasma and tissue melatonin contents and melatonin binding sites in sea bass under four different salinity levels: seawater (36‰), isotonic water (15‰), brackish water (4‰) and freshwater (0‰). The melatonin content was evaluated in plasma, whole brain, gills, intestine and kidney, while melatonin binding sites were analyzed in different brain regions and in the neural retina. Plasma melatonin levels at mid-dark varied, the lowest value occurring in seawater (102 pg/mL), and the highest in freshwater (151 pg/mL). In gills and intestine, however, the highest melatonin values were found in the seawater group (209 and 627 pg/g tissue, respectively). Melatonin binding sites in the brain also varied with salinity, with the highest density observed at the lower salinities in the optic tectum, cerebellum and hypothalamus (30.3, 13.0, and 8.0 fmol/mg protein, respectively). Melatonin binding sites in the retina showed a similar pattern, with the highest values being observed in freshwater. Taken together, these results reveal that salinity influences melatonin production and modifies the density of binding sites, which suggests that this hormone could play a role in timing seasonal events in sea bass, including those linked to fish migration between waters of different salinities for reproduction and spawning.     

The effects of mullein plants (Verbascum thapsus) on the population dynamics of Dicyphus hesperus (Heteroptera: Miridae) in tomato greenhouses

The response of Dicyphus hesperus Knight (Heteroptera: Miridae) to whitefly populations in tomato greenhouses was measured in the presence and absence of mullein (Verbascum thapsus L.) as an alternative host plant. The dynamics of the D. hesperus population on tomato (Lycopersicon esculentum Mill.) and on mullein plants were followed through an entire growing season. In houses with mullein plants, more predators occurred on mullein when whitefly density was low on tomato. A mark-release-recapture experiment where rabbit IgG was used as an external marker showed that D. hesperus adults moved from mullein plants to tomato plants. D. hesperus was always more abundant in houses with mullein than in the houses with tomato plants alone. Movements between tomato and mullein plants are discussed as a strategy to optimize predator foraging. The use of mullein as an alternative host plant may contribute to the establishment of D. hesperus and help to preserve the predator population when prey on tomato crops is scarce.     

Molecular and morphological characterization of Leveillula (Ascomycota: Erysiphales) on monocotyledonous plants

Leveillula on monocotyledonous plants have been recorded as L. taurica by several authors, whereas the fungus on Allium has been described as an independent species, namely L. allii, by some authors. We sequenced ca 600 bp of the rDNA ITS region for two Leveillula specimens from Allium and Polianthes (both from monocotyledons) and compared them with several already published sequences from Leveillula isolates from dicotyledons. Pair-wise percentages of sequence divergences were calculated for all Leveillula isolates. The ITS sequence of the Polianthes isolate was identical to L. taurica on Helianthus and Vicia. The sequence of the Allium isolate was 99.5 % identical to L. taurica on Euphorbia, Haplophylum, Peganum, etc. These results suggest close relationships between monocot and dicot pathogenic Leveillula species. The identity between two monocot isolates was 98.4 %. Phylogenetic analysis revealed that the two monocot isolates do not group into a clade together. This result suggests that Leveillula acquired parasitism to monocots at least twice independently.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

家蚕雌性附腺及其Ng突变体的蛋白质组差异研究(英)

家蚕雌蛾性附腺在化蛾前2到3天开始大量分泌胶状粘性蛋白,其贮存部迅速地膨大,而其Ng突变体的雌蛾性附腺不能正常分泌胶状粘性物质.分别对家蚕（Bombyx mori）的正常及Ng突变体雌蛾性附腺分泌部组织的蛋白质进行提取,并采用双向凝胶电泳和计算机辅助分析方法,对提取的蛋白质混合物进行分离和比较分析,并对主要差异表达的蛋白质用质谱鉴定.实验结果表明,用银染法,平均每张电泳图谱可以分离约700个蛋白质点,其中大部分的蛋白质点分布在pH 4～8 范围内,其分子质量主要集中在30～70 ku区域.比较分析发现一些差异表达蛋白,其中No2, 3蛋白质点经质谱鉴定为肌动蛋白A3,该蛋白质只在化蛹后期正常雌性附腺组织中特异表达,而Ng突变体中肌动蛋白A3的缺失,暗示了肌动蛋白A3可能与家蚕雌性附腺的胶状粘性物质的胞外分泌有关.