RAPD Groups Research Articles

Inflammatory Responses in Periodontitis with or Without Rheumatoid Arthritis Alter Salivary Metallothionein and Zinc.

Periodontitis (PD) and rheumatoid arthritis (RA) are causally linked by their common inflammatory responses, yet it is largely unknown if these inflammatory responses might have an impact on salivary metallothionein (MT), zinc (Zn), and calcium (Ca) content. In this study, we analysed salivary concentrations of pro-inflammatory (IFN-γ, IL-6, and IL-17) and anti-inflammatory (IL-4 and IL-10) cytokines, as well as MT, Zn, and Ca in four groups of participants, namely control (without PD or RA, n = 21), PD (n = 21), RA (n = 21), or RAPD (n = 19). As expected, an increased amount of salivary pro-inflammatory cytokines were observed in the PD, RA, and RAPD groups. While Ca concentration was not significantly different between the groups, Zn concentration was lower in the PD, RA, and RAPD groups compared to the control group (p < 0.05). These groups also expressed higher MT/Zn ratios compared to the control group (p < 0.05). Unlike the control group, concentrations of inflammatory cytokines, MT, Zn, and Ca correlated with each other in the PD, RA, and RAPD groups (p < 0.05). Additionally, comorbidity of PD and RA appears to have a cumulative immuno-pathological impact that warrants further investigation. This study suggests that, in addition to inflammatory cytokines, salivary MT and Zn could reflect the severity of PD with or without RA, hence providing an important biomarker for diagnosis.

Investigating the relationship between the severity of periodontitis and rheumatoid arthritis: a cross-sectional study.

This paper evaluates the prevalence and severity of periodontitis (PD) in patients with rheumatoid arthritis (RA), focusing on the link between the severity of PD with RA disease activity/disability scores, the influence of RA treatment on PD, and levels of vitamin D. A total of 93 RA patients were enrolled in the cross-sectional study and analyzed accordingly as RA-PD (N = 63, 67.8%) and RA-only (N = 30, 32.2%) groups. A number of associations between rheumatological clinical data, i.e., Disease Activity Score (DAS28 CRP), health assessment questionnaires, and PD severity (measured by periodontal outcome parameters) with regard to serum levels of vitamin D were assessed. The outcome variables were compared by parametric and non-parametric tests. A total of 29% of RA patients were diagnosed with severe PD. The RA-PD group presented a higher mean DAS28 CRP score in moderate-severe PD compared to periodontally healthy-initial stage PD subjects (4.49 ± 1.22 vs. 3.86 ± 1.58, p = 0.033). RA patients treated with biologic disease-modifying antirheumatic drugs (bDMARDs) were less likely to be diagnosed with PD (p = 0.022) and revealed significantly lower PD outcome parameters, i.e., bleeding on probing (%) and bone loss (%) (p < 0.05). Vitamin D concentration was significantly lower in RA-PD group with diagnosed advanced severe PD (IV stage) compared to moderate PD (II stage) (39.61 ± 17.12 vs. 52.07 ± 18.23 nmol/l, p = 0.031). The study revealed a high prevalence of severe PD in RA patients, being significantly associated with higher RA disease activity and lower vitamin D level in RA-PD group, while bDMARD treatment was related to lower PD outcome parameters. Key Points • Severe PD is prevalent amongst RA patients and is associated with RA disease activity. The higher RA DAS28 CRP score is associated with moderate-severe PD compared to periodontally healthy-initial stage PD in RA patients. • Biologic DMARDs treatment used for RA is linked to lower PD rates and PD outcome parameters. • Significantly lower vitamin D level is found in advanced severe PD compared to moderate PD stage in RA-PD subjects.

Interspecies variation in biofilm-forming capacity of psychrotrophic bacterial isolates from Chinese raw milk

Biofilms on the surface of dairy processing equipment act as potential reservoirs of microbial contamination, which may affect product quality and cause substantial risks for consumer health. Biofilm-forming capacity of a high number of psychrotrophic bacterial species of dairy origin at low temperatures has been poorly reported. This work aimed to explore the biofilm-forming potential of bacterial strains (previously identified from Chinese raw milk) belonging to 72 RAPD based genetic groups (representing 11 predominant genera and 61 species) at 7 °C. Results showed that biofilm formation is a strain-dependent trait, and 25, 10, 19 RAPD groups were weak, moderate and high biofilm formers, respectively, based on results of the crystal violet staining. Isolates of Pseudomonas showed the high tendency to form biofilms. The cell numbers recovered on tryptic soy broth agar plates from surface biofilms ranged from 1.15 to 6.63 log CFU/cm2, and the maximum number was detected for Pseudomonas azotoformans (a) (6.63 log CFU/cm2) followed by Serratia quinivorans (6.39 log CFU/cm2) and Pseudomonas libanensis (6.34 log CFU/cm2). In the presence of skim milk, cell numbers decreased to a range from 1.04 to 6.1 log CFU/cm2, and high cell number was observed in case of P. azotoformans (a) (6.1 log CFU/cm2), P. libanensis (6.07 log CFU/cm2) and S. quinivorans (5.84 log CFU/cm2). A marked variation in biofilm-forming capacity was observed within the strains belonging to the same species. A moderate correlation (r = 0.42) between the crystal violet assay of polystyrene and methods based on cell enumeration on coupons was observed. Meanwhile, the cell number of Aeromonas hydrophila recovered from stainless steel increased in co-culture with Pseudomonas fluorescens (a). The results expand the knowledge of biofilm-forming capacity of psychrotrophic bacteria of dairy origin.

Propensity for biofilm formation by aerobic mesophilic and thermophilic spore forming bacteria isolated from Chinese milk powders

Biofilms on the surface of dairy manufacturing plants are potential reservoirs of microbial contamination. These microbial aggregates may harbour pathogenic and spoilage organisms which contaminate dairy products. The biofilm forming capacity of many spore forming isolates of dairy origin has not been given much attention. The present study explored the biofilm forming potential of 148 isolates, comprising mesophilic and thermophilic bacteria, with particular emphasis on Bacillus licheniformis on polystyrene and stainless steel (SS) surfaces. We concluded that only four species are of significance for biofilm development on the surface of SS in the presence of skimmed milk, namely, B. licheniformis, Geobacillus stearothermophilus, Geobacillus thermoleovorans group and Anoxybacillus flavithermus. The maximum number of cells recovered from the biofilms developed on SS coupons in the presence of skimmed milk for these four species was as follows: 4.8, 5.2, 4.5 and 5.3logCFU/cm2, respectively. Number of cells recovered from biofilms on 1cm2 SS coupons increased in the presence of tryptic soy broth (TSB) for all mesophiles including B. licheniformis, while decreased for G. stearothermophilus, G. thermoleovorans group and A. flavithermus. The crystal violet staining assay on polystyrene proved to be inadequate to predict cell counts on SS for the bacteria tested in our trial in the presence of either TSB or skimmed milk. The results support the idea that biofilm formation is an important part of bacterial survival strategy as only the most prevalent isolates from milk powders formed good biofilms on SS in the presence of skimmed milk.Biofilm formation also proved to be a strain-dependent characteristic and interestingly significant variation in biofilm formation was observed within the same RAPD groups of B. licheniformis which supports the previously reported genetic and phenotypic heterogeneity within the same RAPD based groups. The work reported in this manuscript will broaden our knowledge on biofilm formation of a large number of dairy isolates and emphasize strain and substrate dependence.

The heat resistance and spoilage potential of aerobic mesophilic and thermophilic spore forming bacteria isolated from Chinese milk powders

The propensity for aerobic bacilli and allied genera to produce highly heat-resistant spores and thermally stable spoilage enzymes are major bacteriological issues faced by the dairy industry. Most of the enzymes are able to survive any heat treatment applied during the manufacture of milk powders and have the potential to remain active in milk powders and other dairy products during storage, and may explain some of the sensory and functionality defects reported in dairy products. Despite many reports on the occurrence of spore-forming bacteria in dairy products, knowledge about food quality related properties of many aerobic sporeformers is still scarce. Therefore, the aim of this study was to determine thermal resistance and spoilage potential of a large pool of mesophilic and thermophilic sporeformers, representing 738 isolates and 31 different RAPD groups, recently isolated from Chinese milk powders. Spore formers producing highly heat resistant spores (surviving 125°C for 30min) included 2 thermophiles (Geobacillus thermoleovorans group and Geobacillus stearothermophilus) and one mesophilic species (Brevibacillus brevis). Paenibacillus macerans showed the highest proteolytic activity followed by members of the Bacillus cereus group, Br. brevis, Bacillus subtilis, G. thermoleovorans group and Virgibacillus proomii. The highest lipase producing strains belonged to Bacillus licheniformis. Phospholipase activity was only shown by members of the B. cereus group and Brevibacillus parabrevis. Ten strains showed positive β-galactosidase activity, while, 4 strains showed positive haemolytic activity. B. licheniformis strains, despite belonging to one RAPD group or sub-group showed markedly different phenotypic characters which support the previous findings of heterogeneity in RAPD-based B. licheniformis groups. The results of this study will broaden the knowledge about the spoilage potential and thermal resistance of many strains of dairy origin.

Identification of Acremonium isolates from grapevines and evaluation of their antagonism towards Plasmopara viticola

Some endophytic fungal genera in Vitis vinifera, including Acremonium, have been reported as antagonists of Plasmopara viticola. Endophytic Acremonium isolates from an asymptomatic grapevine cultivar Inzolia from Italy were identified by morphological features and multigene phylogenies of ITS, 18S and 28S genes, and their intra-specific genomic diversity was analyzed by RAPD analysis. Culture filtrates (CFs) obtained from Acremonium isolates were tested in vitro for their inhibitory activity against the P. viticola sporangia germination. Among 94 isolates, 68 belonged to the Acremonium persicinum and 26 to the Acremonium sclerotigenum. RAPD analysis grouped the A. persicinum isolates into 15 clusters and defined 31 different strains. The A. sclerotigenum isolates, instead, were clustered into 22 groups and represented 25 strains. All A. persicinum CFs inhibited sporangia germination of P. viticola, while not all those of A. sclerotigenum had inhibitory effect. A different degree of inhibition was observed between strains of the same species, while some strains of different species showed identical inhibitory effect. No correlation was found between RAPD groups and inhibitory activity in both Acremonium species.

Functional diversity of phosphate solubilizing plant growth promoting rhizobacteria isolated from apple trees in the Trans Himalayan region of Himachal Pradesh, India

A total of 206 phosphate solubilizing bacteria (PSBs) were isolated from rhizosphere soil (RS) (116) and endorhizosphere (90) of apple trees of four locations: Chamba, Shimla, Kinnaur and Kullu of Himachal Pradesh, India. The population count of PSBs was invariably higher in rhizosphere than in root endosphere (ER) of apple. The greatest Shannon diversity indices for PSBs from RS (1.77) and ER (1.69) were obtained from Kinnaur. Polymorphism among 85 PSBs was detected using PCR-RAPD analysis. Sixteen representative PSBs from RAPD groups were identified further by biochemical tests and 16S rRNA gene analysis. Bacillus was the dominant genus represented by six different species, B. subtilis and B. licheniformis followed by B. pumilus, B. methylotrophicus, B. firmus and B.altitudinis suggesting diversity among species of the genus Bacillus. Variation exists in RS and ER PSBs suggesting the hypothesis that root exudates of apple plant exert selective pressure on the proliferation of specific kind of rhizobacteria on the surface of the root as well as within the root. B. methylotrophicus SMa5A, B. pumilus ECS10A and B. altitudinis ECH8A from ER and B. firmus KR4A from RS have been reported for the first time as plant growth promoting rhizobacteria from apple trees.

Genetic Diversity of Fusarium oxysporum f. sp. dianthi in Southern Spain

The diversity of races and prevalence of pathogenic populations of Fusarium oxysporum f. sp. dianthi (Fod) were surveyed in an area in southern Spain. From 54 farms, 132 isolates were collected from wilted carnation plants. Isolates were characterized by RAPD-PCR, DNA sequence analysis of the TEF1-α gene, and race-specific molecular markers. Selected isolates from RAPD groups were phenotypically evaluated by pathogenicity tests. Data analysis showed that Fod race 2 was the most frequent and prevalent race in the study area, followed by race 1/8. Moreover, phylogenetic analyses showed similar results, which were different to those of the race-specific PCR assays. It was concluded that (i) seven isolates were not classified in groups where Fod testers were clustered; even they showed different results when race-specific markers were used, (ii) ten isolates with retarded race 1 or race 8 specific band were characterized as F. proliferatum by TEF1-α gene sequencing and clustered into an outgroup, and (iii) six isolates failed to generate an amplification signal using race-specific markers. Furthermore, three of them were grouped close to race 2 tester according to the phylogenetic analyses, showing the same differential pathogenicity as race 2. This may indicate a Fod race 2 subgroup in this region.

Corrigendum

Journal of Applied MicrobiologyVolume 113, Issue 6 p. 1564-1564 CORRIGENDUMFree Access Corrigendum This article corrects the following: Bacteriocinogenic and virulence potential of Enterococcus isolates obtained from raw milk and cheese P.M. Moraes, L.M. Perin, S.D. Todorov, A. Silva, B.D.G.M. Franco, L.A. Nero, Volume 113Issue 2Journal of Applied Microbiology pages: 318-328 First Published online: June 11, 2012 First published: 16 November 2012 https://doi.org/10.1111/jam.12011AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat The use of ‘α-haemolysis’ and ‘β-haemolysis’ was mistakenly transposed in the drafting of Moraes et al. (2012), and the authors would like to exchange ‘α’ and ‘β’ in the following locations: Page 318. Abstract. Last sentence of ‘Methods and Results’ section: change to ‘53·5% of the isolates presented α-haemolysis’. Page 321. Materials and Methods section. Characterization of virulence potential, phenotypic tests, lines 6–9: change to ‘… Clear halos around the colonies indicated total or β-haemolysis, and green halos around the colonies indicated partial or α-haemolysis’. Page 321. Results section. In the last sentence: change to ‘… (Table 7), and most of the profiles presented α-haemolysis’. Page 326. Table 7. Change the column heading ‘β-haemolysis’ to ‘α-haemolysis’, and the heading ‘α-haemolysis’ to ‘β-haemolysis’. (For Enterococcus, α-haemolysis is characterized by partial haemolysis, with the formation of greenish halos around the formed colonies in blood agar, and β-haemolysis is characterized by total haemolysis, with the formation of clear halos around the formed colonies in blood agar (Semedo et al. 2003). Following this, the majority of our Enterococcus isolates, considering the RAPD groups, presented α-haemolysis.). References Moraes, P.M., Perin, L.M., Todorov, S.D., Silva, A. Jr, Franco, B.D.G.M. and Nero, L.A. (2012) Bacteriocinogenic and virulence potential of Enterococcus isolates obtained from raw milk and cheese. J Appl Microbiol 113, 318– 328. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Semedo, T., Santos, M., Lopes, M., Figueiredo Marques, J., Barreto Crespo, M. and Tenreiro, R. (2003) Virulence factors in food, clinical and reference Enterococci: a common trait in the genus? Syst Appl Microbiol 26, 13– 22. CrossrefPubMedWeb of Science®Google Scholar Volume113, Issue6December 2012Pages 1564-1564 ReferencesRelatedInformation

Molecular genetic characterization of multidrug-resistant Acinetobacter baumannii strains and assessment of their sensitivity to phage AP22

Molecular genetic analysis of 130 multidrug-resistant nosocomial Acinetobacter baumannii strains was performed. The strains were obtained from patients admitted to different hospitals in large Russian cities (Chelyabinsk, Moscow, Nizhny Novgorod, and St. Petersburg) in 2005–2010. Species identification was performed by the amplified 16S rRNA gene restriction analysis and by determining the blaOXA-51-like genes intrinsic for A. baumannii using PCR. Genetic typing of the strains was performed by RAPD-PCR. All strains fell into two clusters, A and B, with the dominant RAPD groups A1 and B1, respectively, including 82% (107 out of 130) of all strains under study. Susceptibility of the strains to bacteriophage AP22 was determined. The phage was shown to infect specifically and to lyse 69% of 130 strains and 82% (88 out of 107) of A. baumannii strains from the dominant RAPD groups. The ability of bacteriophage AP22 to lyse a broad range of clinically relevant A. baumannii strains makes it an attractive candidate for designing phage cocktails intended to control the A. baumannii-associated nosocomial infections. Moreover, the phage can be used for identifying A. baumannii in the bacteriological tests of clinical samples.

Prevalent lactic acid bacteria in cider cellars and efficiency of Oenococcus oeni strains

Malolactic fermentation (MLF) is an important step in cider production in order to allowing for improvement of microbiological stability and organoleptic characteristics of cider. Induction of this fermentation by using starter cultures enables a better control over this bioprocess, but although it is a common practice in winemaking, starters specifically focussed for cider MLF are not yet commercially available. Proper starter cultures need to present the ability to degrade l-malic acid conferring pleasing sensory characteristics while avoiding toxicological risks. In this work, lactic acid bacteria (LAB) were first isolated from MLF industrial cider samples, obtained in a cellar in the main cider-producing region of Spain, Asturias. Isolates, identified by molecular tools, belonged to the Lactobacillus brevis and Oenococcus oeni species. After a phylogenetic analysis, representative strains of both identified species were evaluated in order to determine their fermentation capacity, showing O. oeni the best behaviour in this cider fermentation, as previously demonstrated for wine in the literature. Consequently, and with the aim to test the influence at strain level, selection of O. oeni isolates as starters for cider fermentation has been undergone. In order to check the influence of geography over biodiversity, O. oeni strains from six different industrial cellars representing the distinct producing areas in the region (located in a ratio of 30 km) were analyzed by using a specific RAPD method. In this way, isolates were typed in five distinct groups, mainly corresponding to each producing area. All strains isolated from the same cellar showed the same RAPD profile revealing the significance of geographical origin in the indigenous cider LAB. Molecular tools were applied to reject those isolates exhibiting presence of genes related to organoleptic spoilage (exopolysaccharides and acrolein production) or food safety (biogenic amine production), as key selection criteria. Representative strains of each of the five O. oeni RAPD groups were tested as pure cultures to evaluate their technological utility for cider production. Experimental data of malic acid degradation and cell concentration obtained were fitted to previously selected kinetic models aimed to optimization and prediction of bioprocess performance. Four strains revealed as suitable potential starter cultures for conducting MLF in cider production.

Genetic relatedness of Brazilian Colletotrichum truncatum isolates assessed by vegetative compatibility groups and RAPD analysis.

The genetic variation among nine soybean-originating isolates of Colletotrichum truncatum from different Brazilian states was studied. Nitrate non-utilizing (nit) mutants were obtained with potassium chlorate and used to characterize vegetative compatibility reactions, heterokaryosis and RAPD profile. Based on pairings of nit mutants from the different isolates, five vegetative complementation groups (VCG) were identified, and barriers to the formation of heterokaryons were observed among isolates derived from the same geographic area. No complementation was observed among any of the nit mutants recovered from the isolate A, which was designed heterokaryon-self-incompatible. Based on RAPD analysis, a polymorphism was detected among the wild isolate C and their nit1 and NitM mutants. RAPD amplification, with five different primers, also showed polymorphic profiles among Brazilian C. truncatum isolates. Dendrogram analysis resulted in a similarity degree ranging between 0.331 and 0.882 among isolates and identified three RAPD groups. Despite the lack of a correlation between the RAPD analysis and the vegetative compatibility grouping, results demonstrated the potential of VCG analysis to differentiate C. truncatum isolates genotypically similar when compared by RAPD.

Genetic relatedness of Brazilian Colletotrichum truncatum isolates assessed by vegetative compatibility groups and RAPD analysis

The genetic variation among nine soybean-originating isolates of Colletotrichum truncatum from different Brazilian states was studied. Nitrate non-utilizing (nit) mutants were obtained with potassium chlorate and used to characterize vegetative compatibility reactions, heterokaryosis and RAPD profile. Based on pairings of nit mutants from the different isolates, five vegetative complementation groups (VCG) were identified, and barriers to the formation of heterokaryons were observed among isolates derived from the same geographic area. No complementation was observed among any of the nit mutants recovered from the isolate A, which was designed heterokaryon-self-incompatible. Based on RAPD analysis, a polymorphism was detected among the wild isolate C and their nit1 and NitM mutants. RAPD amplification, with five different primers, also showed polymorphic profiles among Brazilian C. truncatum isolates. Dendrogram analysis resulted in a similarity degree ranging between 0.331 and 0.882 among isolates and identified three RAPD groups. Despite the lack of a correlation between the RAPD analysis and the vegetative compatibility grouping, results demonstrated the potential of VCG analysis to differentiate C. truncatum isolates genotypically similar when compared by RAPD.

Molecular characterization of nitrogen-fixing bacteria isolated from brazilian agricultural plants at São Paulo state

Fourteen strains of nitrogen-fixing bacteria were isolated from different agricultural plant species, including cassava, maize and sugarcane, using nitrogen-deprived selective isolation conditions. Ability to fix nitrogen was verified by the acetylene reduction assay. All potentially nitrogen-fixing strains tested showed positive hybridization signals with a nifH probe derived from Azospirillum brasilense. The strains were characterized by RAPD, ARDRA and 16S rDNA sequence analysis. RAPD analyses revealed 8 unique genotypes, the remaining 6 strains clustered into 3 RAPD groups, suggesting a clonal origin. ARDRA and 16S rDNA sequence analyses allowed the assignment of 13 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Azospirillum, Herbaspirillum, Pseudomonas and Enterobacteriaceae. Two strains were classified as Stenotrophomonas ssp. Molecular identification results from 16S rDNA analyses were also corroborated by morphological and biochemical data.

Caracterización molecular y genética de aislamientos patógenos de Pyricularia grisea del trigo ( Triticum aestivum Lam.) y triticale ( x Triticosecale Wittmack) en la Provincia de Paraná, Brasil

Isolates of Pyricularia grisea from wheat (Triticum aestivum Lam.) and triticale (x Triticosecale Wittmack) spikes with blast symptoms were analyzed by classical (VCG) and molecular (RAPD) techniques. P. grisea mutants, unable to use sodium nitrate (nit) as nitrogen source, were obtained with potassium chlorate. For vegetative compatibility (VCG) tests, genetically complementary nit mutant pairs were inoculated in a medium with sodium nitrate as a single nitrogen source. P. grisea isolates were divided into two vegetative compatibility groups and two RAPD groups. Since vegetative compatible strains may mutually exchange genetic and cytoplasmatic material, the contribution of the parasexual cycle in the genetic variability of Brazilian P. grisea isolates is discussed.

Virulence markers and genetic relationships of Shiga toxin-producing Escherichia coli strains from serogroup O111 isolated from cattle

Shiga toxin-producing Escherichia coli (STEC) strains isolated from healthy cattle (O111:NM, seven strains; O111:H8, three strains) in Brazil were studied and compared to previously characterized human strains in regard to their phenotypic and genotypic characteristics to evaluate their pathogenic potential. Most bovine STEC O111 strains were isolated from dairy calves, and strains with genotypes stx1 alone and stx1/ stx2 (variant stx2) occurred in different regions. Irrespective of the stx genotype, all strains were positive for eae θ, α variants of tir, espA and espB, and for ler, qseA, iha, astA and efa1 genes. Only one strain was negative for EHEC- hlyA and all strains were negative for iha, saa and espP genes and for EAF and bfpA, genetic markers of EPEC. Except for the presence of stx2, bovine strains showed the same profile of putative virulence genes found among the human strains. Similar biochemical behavior was identified among the strains analysed. Two bovine STEC strains produced the localized adherence (LA) phenotype in 6-h tests with Caco-2 (human enterocyte) cells. Intimate attachment (judged by the FAS test) was found in 9 out of 10 bovine strains as it was observed for the human STEC strains. RAPD-PCR analysis showed two distinct RAPD groups among the STEC O111 strains examined. Despite the relative low frequency of STEC O111 strains recovered from cattle no differences in their pathogenic potential were observed compared to some strains isolated from human diarrhea, suggesting that healthy cattle may be a potential source of infection for humans in Brazil.

Pathogenicity, molecular characterization, and cercosporin content of Brazilian isolates of Cercospora kikuchii

Cercospora kikuchii, involved with the defoliation of soybean (Glycine max) plants, is normally associated with Septoria glycines in late season. Seventy-two isolates from different regions in Brazil, obtained mainly from purple stained seeds, showed phenotypic variation. Cercosporin content and rate of colony growth was higly variable among isolates. A strong correlation between cercosporin content and virulence was identified. Genetic variation among and within populations was evaluated based on 86 RAPD loci. The RAPD analysis clustered all isolates into seven groups. No relationship was observed between RAPD groups and geographic origin or cercosporin content. The sequence of the internal spacer regions (ITS1-5.8S-ITS2) from 13 isolates chosen according to the previous RAPD and clustering analysis showed high similarity (97%-100%) to the GenBank sequences of C. kikuchii (AY266160, AY266161, AY152577 and AF291708). It is clear from this work that Brazilian isolates of C. kikuchii from different geographic regions, are variable in relation to virulence, RAPD profiles and cercosporin content. Cercosporin content could be a good parameter for choosing an adequate isolate for screening resistant or tolerant cultivars. Considering that this pathogen is easily seed-borne, findings are expected to show the same haplotypes in different regions. Migration could be favoured by infected seeds as demonstrated by RAPD analysis.

Selection and Identification of Autochthonous Potential Probiotic Bacteria from Turbot Larvae (Scophthalmus maximus) Rearing Units

The purpose of this study was to select, identify and characterise bacteria as a disease control measure in the rearing of marine fish larvae (turbot, Scophthalmus maximus). Thirty-four out of 400 marine bacterial strains exhibited in vitro anti-bacterial activity against three fish larval pathogens. Two strains originated from culture collections and thirty two strains were isolated directly from turbot larvae rearing units using a pre-selection procedure to facilitate detection of antagonists. Approximately 8,500 colonies from colony-count plates were replica-plated on agar seeded with Vibrio anguillarum, and 196 of them caused zones of clearing in the V. anguillarum agar layer. Of these, 32 strains exhibited reproducible antibacterial properties in vitro when tested against the fish pathogens V. anguillarum 90-11-287, V. splendidus DMC-1 and a Pseudoalteromonas HQ. Seventeen antagonists were identified as Vibrio spp. and four of twelve tested were lethal to yolk-sac larvae. The 15 remaining strains were identified as Roseobacter spp. based on phenotypic criteria and 16S rDNA gene sequence analysis of two strains representing the two major RAPD groups. Most of the remaining 164 strains selected in the initial replica plating were identified as Vibrionaceae or Pseudoalteromonas. Roseobacter spp. were not lethal to egg yolk sac turbot larvae and in two of three trials, the mortality of larvae decreased (p > 0.001) in treatments where 10(7) cfu/ml Roseobacter sp. strain 27-4 was added, indicating a probiotic potential.

Characterisation of Verticillium dahliae Kleb. isolates from Olea europea using RAPD markers

The random amplification of polymorphic DNA fragments (RAPD) was used as a tool both for estimating the genetic variability of Verticillium dahliae Kleb. and for characterising this fungus. Forty V. dahliae isolates were examined, 33 from olive in the main olive-growing areas, 2 from tomato, one from aubergine, one from the soil of an infected olive grove in Morocco and 3 from olive in Algeria. A total of 95 polymorphic RAPD bands were obtained with ten primers selected among the 50 tested. The results revealed a clear polymorphism between the isolates, which were assigned to 4 RAPD groups. No correlation between RAPD markers, the geographic origin or the host plants of the isolates was observed.

Toxigenic diversity of two different RAPD groups ofStachybotrys chartarumisolates analyzed by potential for trichothecene production and for boar sperm cell motility inhibition

Thirty-one isolates of Stachybotrys chartarum from indoor and outdoor environments were analyzed for the presence of the trichodiene synthase (Tri5) gene, trichothecenes, boar sperm cell motility inhibition, and randomly amplified polymorphic DNA banding patterns (RAPDs). Twenty-two S. chartarum isolates tested positive for the Tri5 gene and nine were negative when tested using novel Tri5 gene-specific PCR primer pair. The Tri5 gene positive isolates contained satratoxins (five isolates) or the simple trichothecene, trichodermol (11 isolates). The Tri5 gene negative isolates did not produce satratoxins or trichodermol. Nineteen S. chartarum isolates, distributed among the Tri5 gene negative and positive groups, inhibited boar spermatozoan motility at concentrations of < or = 60 microg of crude cell extract/mL. The inhibition of motility was independent of satratoxins or atranones. Unweighted pair group method of arithmetic averages (UPGMA) cluster analysis of RAPD fragments clustered the 31 S. chartarum isolates in two distinct groups designated as RAPD groups 1 and 2. The grouping of S. chartarum isolates obtained by UPGMA cluster analysis of RAPD fragments was identical to the grouping obtained by Tri5 gene-specific PCR. This indicates that the S. chartarum isolates belonging to different groups were genetically distinct in a much wider area than just the Tri5 gene.