Grapevine yellows in Northern Italy: molecular identification of Flavescence dorée phytoplasma strains and of Bois Noir phytoplasmas

AIMS: Verify the presence and the molecular identity of phytoplasmas in Northern and Central Italy vineyards where yellows diseases are widespread. METHODS AND RESULTS: Phytoplasma presence and identity were determined by PCR/RFLP analyses on 16S ribosomal gene testing 1424 symptomatic samples. The 65% of samples resulted phytoplasma infected; in particular 256 samples were found positive to phytoplasmas belonging to group 16SrV (mainly Flavescence dorée associated), and the remaining 37% was infected by phytoplasmas belonging to ribosomal subgroup 16SrXII-A (Stolbur or Bois Noir associated). 16SrV ribosomal group representative strains were further typed for variability in SecY and rpS3 genes. The results showed the presence of phytoplasmas belonging to 16SrV-C, 16SrV-D and to a lesser extent, 16SrV-A subgroup. CONCLUSIONS: Possible relationships between genetic polymorphisms of phytoplasma strains belonging to subgroup 16SrV-C and their geographic distribution and/or epidemic situations were detected. SIGNIFICANCE AND IMPACT OF THE STUDY: Bois Noir and Flavescence dorée phytoplasmas are present in significant percentages in the areas under investigation. Molecular tools allowed to identify phytoplasma-infected plants and the genes employed as polymorphism markers resulted useful in distinguishing and monitoring the spreading of the diseases associated with diverse phytoplasmas belonging to 16SrV subgroup in vineyards.     

First detection of a human astrovirus type 8 in a child with diarrhea in Belém, Brazil: comparison with other strains worldwide and identification of possible three lineages

This study describes the genetic relationships of the first human astrovirus type-8 (HAstV-8) detected in Belém-Brazil, during a public hospital-based study. This strain was compared with other HAstV-8 strains identified elsewhere which have sequences available at GeneBank. The regions ORF1a (primers Mon348/Mon340) and ORF2 (primers Mon269/Mon270) were analyzed by nucleotide sequencing and a high similarity rate was observed among the Belém strain and other HAstV-8 strains. In ORF1a, homology values of 93-100% were detected, and in ORF2 96-99%. Considering the sequence variation (7%) observed in ORF2 region, it was suggested that HAstV-8 strains could be divided in three different lineages.     

Neurofibromatosis type 2 attributable to gonosomal mosaicism in a clinically normal mother, and identification of seven novel mutations in the NF2 gene

Neurofibromatosis type 2 (NF2) is an autosomal dominant cancer syndrome that predisposes to the development of bilateral vestibular schwannomas sometimes associated with schwannomas at other locations, meningiomas, ependymomas and juvenile posterior subcapsular lenticular opacities. This disease is caused by inactivating mutations in the NF2 tumour-suppressor gene, located in 22q12. Recently, somatic mosaicism has been demonstrated in some "de novo" NF2 patients. We here report the genetic study of 33 NF2 patients from 33 unrelated Italian families. Twelve mutations were characterised, including seven newly identified mutations and five recurrent ones. Furthermore, we describe one patient with an inactivating mutation that lies in exon 13 but that is present in only a portion of the lymphocytes and, more importantly, a clinically normal individual carrying a somatic/germinal mosaicism for a nonsense mutation in exon 10 of the NF2 gene. Our results confirm the relatively high percentage of mosaicism for mutations in the NF2 gene and establish the importance of evaluating genomic DNA from several tissues, in addition to lymphocytes, so as to identify mosaicism in "de novo" NF2 patients and their relatives. In addition, the demonstration of somatic and/or gonadal mosaicism is an important tool for accurate genetic counselling in families with sporadic cases of NF2.     

Antibiotic resistance and plasmid pattern of enterotoxigenic ST-a strains of Escherichia coli isolated in Puebla, México

Antibiotic susceptibilities of 22 strains of Escherichia coli isolated from children from 0 to 3 years old at the University Hospital of Puebla were determined. Almost all strains were resistant to ampicillin, tetracycline, streptomycin, and kanamycin. Gel electrophoresis of DNA from 10 clinical strains of E. coli revealed a heterogeneous plasmid population. Plasmid DNA, ranging in molecular mass from 1.8 to 120 megadaltons, was demonstrated in 10 strains. Moreover, the frequency of antibiotic transfer ranged from 1.6/10(8) to 2/10, and the simultaneous transfer of the gene encoding heat-stable enterotoxin was also determined. Six out of 10 strains tested were able to cotransfer ST-a as demonstrated by the suckling mouse test. It is possible that antibiotic selective pressure may increase the isolation of enterotoxigenic E. coli strains.     

Comparison of Recent Siliceous and Carbonate Mat Development on the Shore of Hyper-Alkaline Lake Van and Mt. Nemrut Soğuk Lake,NE Anatolia,Turkey

This article focuses on the molecular geochemistry of previously unreported carbonate- and siliceous-mat assemblages at two adjacent lake-shore environments. The first study area, on the Lake Van shore, is an intersection of hyper-alkaline lake water and seep water discharge, both of which represent an acid-base geochemical barrier. Comparing alkaline lake water with seep water samples collected from a biomineralization site reveals that seep water has an acidic character and is rich in Ca and Si cations. The second study area, Mt. Nemrut, is the largest volcanic caldera located west of Lake Van in eastern Anatolia, and is the only volcano that has erupted in historical time. While the main lithified-mat components of Lake Van's shore environments are Ca-carbonate and diatoms, Na-carbonate and diatom assemblages represent recent biomineralization in the Mt. Nemrut So?uk Lake shores. In particular, the normal habitat for the development of these unique microbial mat assemblages can be changed in a short time by the decreasing seep water discharge into the Lake Van shore environment. A detailed study on these lake shore microbialites, before they are lost, would provide a great opportunity to understand the biogeochemical interactions that produce unique sediment fabrics.     

Comparison between conventional methods, ChromAgar Candida® and PCR method for the identification of Candida species in clinical isolates

The increase in the incidence of yeast species causing fungemia in susceptible immunocompromised patients in the last two decades and the low sensitivity of conventional blood culture has led to the need to develop alternative approaches for the early detection and identification of causative species. The aim of this study was to compare the usefulness of molecular testing by the polymerase chain reaction (PCR) and conventional methods to identify clinical isolates of different species, using the ID32C ATB system (bioMérieux, France), chromogenic culture Chromagar Candida? (CHROMagar, France) and morphogenesis in corn meal agar. We studied 79 isolates, in which the most prevalent species using the system ID32C and PCR was C. albicans, followed by C. tropicalis, C. glabrata and C .krusei. PCR patterns obtained for the identification of clinical isolates were stable and consistent in the various independent studies and showed good reproducibility, concluding that PCR with species-specific primers that amplify genes ITS1 and ITS2 for rRNA or topoisomerase II primers is a very specific and sensitive method for the identification of C. glabrata, C. krusei, C. albicans, and with less specificity for C. tropicalis.     

Comparison of the small heat shock proteins αB-crystallin, MKBP, HSP25, HSP20, and cvHSP in heart and skeletal muscle

Seven members of the small heat shock protein (sHSP) family are exceptional with respect to their constitutive high abundance in muscle tissue. It has been suggested that sHSPs displaying chaperone-like properties may stabilize myofibrillar proteins during stress conditions and prevent them from loss of function. In the present study five sHSPs (B-crystallin, MKBP, HSP25, HSP20, and cvHSP) were investigated with respect to similarities and differences of their expression in heart and skeletal muscle under normal and ischemic conditions. In ischemic heart and skeletal muscle these five sHSPs translocated from cytosol to the Z-/I-area of myofibrils. Myofibrillar binding of all sHSPs was very tight and resisted for the most part extraction with 1 M NaSCN or 1 M urea. MKBP and HSP20 became extracted by 1 M NaSCN to a significant extent indicating that these two sHSPs may bind partially to actin-associated proteins which were completely extracted by this treatment. Ultrastructural localization of B-crystallin showed diffuse distribution of immunogold label throughout the entire I-band in skeletal muscle fibers whereas in cardiomyocytes B-crystallin was preferentially located at the N-line position of the I-band. These observations indicate different myofibrillar binding sites of B-crystallin in cardiomyocytes versus skeletal muscle fibers. Further differences of the properties of sHSPs could be observed regarding fiber type distribution of sHSPs. Thus sHSPs form a complex stress–response system in striated muscle tissue with some common as well as some distinct functions in different muscle types.     

A new morphological character for the identification of Stomoxys calcitrans and S. niger (Diptera: Muscidae). Comparison of La Réunion island populations

On La Reunion Island (France), two morphological closely related species of stable flies (Diptera: Muscidae), living in the same environment, Stomoxys calcitrans and S. niger, are involved in the transmission of blood parasites to the livestock. To facilitate a rapid identification of both species in the field conditions, we highlighted a diagnostic morphological character not yet described: the length of the maxillary palpus. The study of three populations of S. calcitrans and two populations of S. niger, collected at various elevations, showed that the maxillary palpi of S. niger were significantly longer than in S. calcitrans, independent of sex. This character, easily visible in the field with a simple magnifying glass, has been confirmed on individuals of both species from West Africa.     

Mutational analysis of 85 mucopolysaccharidosis type I families: frequency of known mutations, identification of 17 novel mutations and in vitro expression of missense mutations

The lysosomal storage disorder, mucopolysaccharidosis type I (MPS I), is caused by a deficiency of the enzyme alpha-L-iduronidase, which is involved in the breakdown of dermatan and heparan sulphates. There are three clinical phenotypes, ranging from the Hurler form characterised by skeletal abnormalities, hepatosplenomegaly and severe mental retardation, to the milder Scheie phenotype where there is aortic valve disease, corneal clouding, limited skeletal problems, but no mental retardation. In this study, 85 MPS I families (73 Hurler, 5 Hurler/Scheie, 7 Scheie) were screened for 9 known mutations (Q70X, A75T, 474-2a>g, L218P, A327P, W402X, P533R, R89Q, 678-7g>a). W402X was the most frequent mutation in our population (45.3%) and Q70X was the second most frequent (15.9%). In 30 families, either one or both of the mutations were not identified, which accounted for 25.9% of the total alleles. Therefore, all 14 exons of the alpha-L-iduronidase gene were screened in these patients and 23 different sequence changes were found, 17 of which were previously unknown. The novel sequence changes include 4 deletions (153delC, 628del5, 740delC, 747delG), 5 nonsense mutations (Q60X, Y167X, Q400X, R619X, R628X), 6 missense mutations (C205Y, G208V, H240R, A319V, P496R, S633L), a splice site mutation (IVS12+5g>a), and a rare polymorphism (A591T). The polymorphism and novel missense mutations were transiently expressed in COS-7 cells and all of them except the polymorphism showed complete loss of enzyme activity. In total, 165 of the 170 mutant alleles were identified in this study and despite the high frequency of W402X and Q70X, the identification of many novel mutations unique to individual families further highlights the genetic heterogeneity of MPS I.     

Genetic identification of a small and highly isolated population of fin whales (Balaenoptera physalus) in the Sea of Cortez, México

For many years, researchers have speculatedthat fin whales are year-round residents in theSea of Cortez (= Gulf of California). Previouswork by Bérubé and co-workers has shownthat the degree of genetic diversity among finwhales in the Sea of Cortez at nuclear andmitochondrial loci is highly reduced. However,the relatively unobstructed connection with theNorth Pacific Ocean argues that Sea of Cortezfin whales are part of a much larger easternNorth Pacific population given the extensivemigratory ranges observed in fin whales andbaleen whales in general. The low degree ofgenetic variation might thus simply be due tohistoric fluctuations in the effectivepopulation size of an eastern North Pacificpopulation. In order to test if the reducedgenetic variation detected among fin whales inthe Sea of Cortez is due to small populationsize or a past bottleneck in an otherwise largeeastern North Pacific population, we analyzedthe geographic distribution of geneticvariation at a single mitochondrial (controlregion) and 16 nuclear loci in samplescollected from fin whales in the eastern NorthPacific (n = 12) as well as the Sea of Cortez(n = 77). Our results showed that fin whalesobserved in the Sea of Cortez constitute ahighly isolated and thus evolutionary uniquepopulation, which warrants special conservationmeasures given the current low estimate ofabundance of approximately 400 individuals.     

Comparison of ageing methods and growth rates for largemouth bass,Micropterus salmoides Lacépède,from northern latitudes

Synopsis Ages determined by counts of apparent annuli on scales, sagittae, vertebrae, pectoral fin ray and dorsal fin spine cross sections of largemouth bass from northern populations, which are older and slower growing fish than in the southern parts of its native range, were compared to establish the accuracy of each method. Linear regression techniques indicated strong agreement (r> 0.9) among ages assigned from the examination of scales, sagittae, and vertebrae. The pattern of growth zones on pectoral fin ray and dorsal fm spine cross sections proved too variable for accurate age determination. Limited data suggest that ages greater than 7+ assigned from scales were more likely to underestimate true ages than the other body parts used, although none of these methods gave satisfactory results. Examination of scales from recovered tagged fish, and the similarity between back-calculated lengths of fish through age 7+ to annulus I and observed lengths of juvenile largemouth bass near the end of their first growing season, support the validity of ages determined from scales. Despite a very limited amount of habitat suitable for largemouth bass and severe climatic conditions, growth of this species in Tadenac Lake was similar to growth in other waters north of the Great Lakes. Differences in physical characteristics among these waters does not appear to influence growth rates of largemouth bass, but probably affects production and biomass.     

Steroid-induced regulation of 3α,20β- and 3β,17β-hydroxysteroid dehydrogenase activity in wild type and mutants of Streptomyces hydrogenans

The effect of estradiol, hydrocortisone and progesterone on 3,20-and 3,17-hydroxysteroid dehydrogenase (HSD) in mutants of Streptomyces hydrogenans was compared to the steroid response of the wild type. Mutants were defective in arginine biosynthesis and/or aerial mycelial formation and lacked both enzymes or only 17-HSD. Some 17-HSD mutants had lost the ability to be induced by estradiol, by progesterone or by both. Some 20-HSD mutants had lost the ability to be induced by hydrocortisone, by progesterone or by both. Non-inducibility of 17-and 20-HSD by progesterone was not co-ordinate. An additional study of the growth phase-dependent enzyme activity of the wild type after induction with estradiol, hydrocortisone and progesterone was performed.Non-standard abbreviations 17-HSD 3,17-Hydroxysteroid dehydrogenase (EC 1.1.1.51) - 20-HSD 3,20-hydroxysteroid dehydrogenase (EC 1.1.1.53) - AO acridine orange - EBr ethidium bromide - EMS ethyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Light quality and explant type modulate growth,antioxidant properties and bioactive compounds production of calluses of Passiflora setacea cv BRS Pérola do Cerrado

Plant Cell, Tissue and Organ Culture (PCTOC) - Induction of somatic embryogenesis from in vitro-cultivated anthers represents a recent but poorly understood regeneration pathway for passion fruit...     

Comparison of the social systems of primates and feral horses: data from a newly established horse research site on Serra D’Arga,northern Portugal

Horses are phylogenetically distant from primates, but considerable behavioral links exist between the two. The sociality of horses, characterized by group stability, is similar to that of primates, but different from that of many other ungulates. Although horses and primates are good models for exploring the evolution of societies in human and non-human animals, fewer studies have been conducted on the social system of horses than primates. Here, we investigated the social system of feral horses, particularly the determinant factors of single-male/multi-male group dichotomy, in light of hypotheses derived from studies of primate societies. Socioecological data from 26 groups comprising 208 feral horses on Serra D’Arga, northern Portugal suggest that these primate-based hypotheses cannot adequately explain the social system of horses. In view of the sympatric existence of multi- and single-male groups, and the frequent intergroup transfers and promiscuous mating of females with males of different groups, male–female relationships of horses appear to differ from those of polygynous primates.     

‘Harem’ and ‘non-harem’ type mating systems in two species of subsocial spider mites (Acari,Tetranychidae)

The mating system of a subsocial spider mite, Schizotetranychus miscanthi Saitō, which is closely related to Schizotetranychus longus Saitō (the long seta form of Schizotetranychus celarius (Banks) is a synonym of the latter) was studied in comparison with that of S. longus. Comparisons between nesting patterns of the two related species, S. miscanthi and S. longus revealed a difference in distribution of males among nests. Although more than one male sometimes occurred in the large nests of S. miscanthi, most nests were occupied only by a single male. On the other hand, many nests of S. longus included several males. Behavioral experiments revealed that the male and females of S. miscanthi which cohabited in a nest defended their offspring from phytoseiid predators. Observations and a census of the nesting pattern in a wild population indicate that this is the second example of biparental defense and of a subsocial life-pattern in spider mites. Differences in mating systems were experimentally demonstrated in the two species. Only a single male of S. miscanthi survived in a nest, as a result of highly aggressive male-male combat, while two males of S. longus cohabited in a nest. The mating system of the former species is thus considered as harem polygyny, while that of the latter as scramble type polygyny. Furthermore, observations by video recording and scanning electron microscopy showed that the winning male in the male-male combat in S. miscanthi often preyed on the loser, suggesting cannibalism among them.     

A general and flexible methodology to define thresholds for heat health watch and warning systems, applied to the province of Québec (Canada)

Several watch and warning systems have been established in the world in recent years to prevent the effects of heat waves. However, many of these approaches can be applied only in regions with perfect conditions (e.g., enough data, stationary series or homogeneous regions). Furthermore, a number of these approaches do not account for possible trend in mortality and/or temperature series, whereas others are generally not adapted to regions with low population densities or low daily mortality levels. In addition, prediction based on multiple days preceding the event can be less accurate if it attributes the same importance to each of these days, since the forecasting accuracy actually decreases with the period. The aim of the present study was to identify appropriate indicators as well as flexible and general thresholds that can be applied to a variety of regions and conditions. From a practical point of view, the province of Québec constitutes a typical case where a number of the above-mentioned constraints are present. On the other hand, until recently, the province’s watch and warning system was based on a study conducted in 2005, covering only the city of Montreal and applied to the whole province. The proposed approach is applied to each one of the other health regions of the province often experiencing low daily counts of mortality and presenting trends. The first constraint led to grouping meteorologically homogeneous regions across the province in which the number of deaths is sufficient to carry out the appropriate data analyses. In each region, mortality trends are taken into account. In addition, the proposed indicators are defined by a 3-day weighted mean of maximal and minimal temperatures. The sensitivity of the results to the inclusion of traumatic deaths is also checked. The application shows that the proposed method improved the results in terms of sensitivity, specificity and number of yearly false alarms, compared to those of the existing and other classical approaches. An additional criterion based on the Humidex is applied in a second step and a local validation is applied to historical observations at reference forecasting stations. An integrated heat health watch and warning system with thresholds that are adapted to the regional climate has thus been established for each sub-region of the province of Quebec and became operational in June 2010.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains associated with the production of cachaça: identification and characterization by traditional and molecular methods (PCR,PFGE and mtDNA-RFLP)

A study of the genetic diversity of populations of Saccharomyces cerevisiae was conducted in ten different cachaça producers (alambiques) in the southern state of Minas Gerais, Brazil. A total of 106 isolates were identified by PCR using the primer SCREC114, specific to S. cerevisiae, by pulsed-field gel electrophoresis (PFGE) and by restriction fragment polymorphism of mitochondrial DNA analysis (RFLP-mtDNA). PCR showed a product of amplification to 61 isolates, enabling a rapid identification of S. cerevisiae in different alambiques. Nine different profiles were found by PFGE; all the yeasts identified as S. cerevisiae by PCR had profiles similar to that of the marker S. cerevisiae, highlighting the specificity of primer SCREC114. RFLP-mtDNA, using four different enzymes, enabled the grouping of strains of S. cerevisiae, with 80%–100% similarity. Some alambiques that had a higher frequency of S. cerevisiae characterized by PCR and PFGE, had a lower level of genetic diversity determined by RFLP-mtDNA, indicating the ability of these strains to lead the fermentative process.