Genetic Diversity and a PCR-Based Method for Xanthomonas axonopodis Detection in Passion Fruit

Xanthomonas axonopodis pv. passiflorae causes bacterial spot in passion fruit. It attacks the purple and yellow passion fruit as well as the sweet passion fruit. The diversity of 87 isolates of pv. passiflorae collected from across 22 fruit orchards in Brazil was evaluated using molecular profiles and statistical procedures, including an unweighted pair-group method with arithmetical averages-based dendrogram, analysis of molecular variance (AMOVA), and an assigning test that provides information on genetic structure at the population level. Isolates from another eight pathovars were included in the molecular analyses and all were shown to have a distinct repetitive sequence-based polymerase chain reaction profile. Amplified fragment length polymorphism technique revealed considerable diversity among isolates of pv. passiflorae, and AMOVA showed that most of the variance (49.4%) was due to differences between localities. Cluster analysis revealed that most genotypic clusters were homogeneous and that variance was associated primarily with geographic origin. The disease adversely affects fruit production and may kill infected plants. A method for rapid diagnosis of the pathogen, even before the disease symptoms become evident, has value for producers. Here, a set of primers (Xapas) was designed by exploiting a single-nucleotide polymorphism between the sequences of the intergenic 16S-23S rRNA spacer region of the pathovars. Xapas was shown to effectively detect all pv. passiflorae isolates and is recommended for disease diagnosis in passion fruit orchards.

Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp citri

Genetic transformation with genes that code for antimicrobial peptides has been an important strategy used to control bacterial diseases in fruit crops, including apples, pears, and citrus. Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri Schaad et al. (Xcc) is a very destructive disease, which affects the citrus industry in most citrus-producing areas of the world. Here, we report the production of genetically transformed Natal, Pera, and Valencia sweet orange cultivars (Citrus sinensis L. Osbeck) with the insect-derived attacin A (attA) gene and the evaluation of the transgenic plants for resistance to Xcc. Agrobacterium tumefaciens Smith and Towns-mediated genetic transformation experiments involving these cultivars led to the regeneration of 23 different lines. Genetically transformed plants were identified by polymerase chain reaction, and transgene integration was confirmed by Southern blot analyses. Transcription of attA gene was detected by Northern blot analysis in all plants, except for one Natal sweet orange transformation event. Transgenic lines were multiplied by grafting onto Rangpur lime rootstock plants (Citrus limonia Osbeck) and spray-inoculated with an Xcc suspension (10(6) cfu mL(-1)). Experiments were repeated three times in a completely randomized design with seven to ten replicates. Disease severity was determined in all transgenic lines and in the control (non-transgenic) plants 30 days after inoculation. Four transgenic lines of Valencia sweet orange showed a significant reduction in disease severity caused by Xcc. These reductions ranged from 58.3% to 77.8%, corresponding to only 0.16-0.30% of leaf diseased area as opposed to 0.72% on control plants. One transgenic line of Natal sweet orange was significantly more resistant to Xcc, with a reduction of 45.2% comparing to the control plants, with only 0.14% of leaf diseased area. Genetically transformed Pera sweet orange plants expressing attA gene did not show a significant enhanced resistance to Xcc, probably due to its genetic background, which is naturally more resistant to this pathogen. The potential effect of attacin A antimicrobial peptide to control ACC may be related to the genetic background of each sweet orange cultivar regarding their natural resistance to the pathogen.

Culture-Independent Assessment of Rhizobiales-Related Alphaproteobacteria and the Diversity of Methylobacterium in the Rhizosphere and Rhizoplane of Transgenic Eucalyptus

The rhizosphere is an ecosystem exploited by a variety of organisms involved in plant health and environmental sustainability. Abiotic factors influence microorganism-plant interactions, but the microbial community is also affected by expression of heterologous genes from host plants. In the present work, we assessed the community shifts of Alphaproteobacteria phylogenetically related to the Rhizobiales order (Rhizobiales-like community) in rhizoplane and rhizosphere soils of wild-type and transgenic eucalyptus. A greenhouse experiment was performed and the bacterial communities associated with two wild-type (WT17 and WT18) and four transgenic (TR-9, TR-15, TR-22, and TR-23) eucalyptus plant lines were evaluated. The culture-independent approach consisted of the quantification, by real-time polymerase chain reaction (PCR), of a targeted subset of Alphaproteobacteria and the assessment of its diversity using PCR-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Real-time quantification revealed a lesser density of the targeted community in TR-9 and TR-15 plants and diversity analysis by principal components analysis, based on PCR-DGGE, revealed differences between bacterial communities, not only between transgenic and nontransgenic plants, but also among wild-type plants. The comparison between clone libraries obtained from the transgenic plant TR-15 and wild-type WT17 revealed distinct bacterial communities associated with these plants. In addition, a culturable approach was used to quantify the Methylobacterium spp. in the samples where the identification of isolates, based on 16S rRNA gene sequences, showed similarities to the species Methylobacterium nodulans, Methylobacterium isbiliense, Methylobacterium variable, Methylobacterium fujisawaense, and Methylobacterium radiotolerans. Colonies classified into this genus were not isolated from the rhizosphere but brought in culture from rhizoplane samples, except for one line of the transgenic plants (TR-15). In general, the data suggested that, in most cases, shifts in bacterial communities due to cultivation of transgenic plants are similar to those observed when different wild-type cultivars are compared, although shifts directly correlated to transgenic plant cultivation may be found.

Phytoplasma closely related to the pigeon pea witches`-broom phytoplasma (16Sr IX) is associated with citrus huanglongbing symptoms in the state of Sao Paulo, Brazil

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of Sao Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fDI/rPI was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the Pigeon pea witches`-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these result.,;. With two primers D7f2/D7r2 designed based oil the 16S rDNA Sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and `Candidatus Liberibacter asiaticus`. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results Show that a phytoplasma of group IX is associated with citrus HLB symptoms ill northern, central, and Southern SPs. This phytoplasma has very probably been transmitted to citrus from an external Source of inoculum, but the Putative insect vector is not yet known.

Cloning and characterization of transcripts differentially expressed in the pulp of ripening papaya

Papaya (Carica papaya) is a relevant tropical crop and physico-chemical changes take place very quickly, as a consequence of activation of biochemical pathways by de nova synthesis of several proteins. Thus, in order to have information on the changes in gene expression in ripening papaya, transcripts from the pulp of unripe and ripe fruit were profiled by differential-display RT-PCR (DDRT-PCR). Seventy transcript derived fragments (TDFs) isolated from gels were re-amplified by PCR and differential expression of 40 papaya genes was confirmed by reverse northern blotting. Twenty-nine positively cloned TDFs were sequenced, and 17 were putatively identified by homology search. Ten of these genes were downregulated during ripening and UDP-glucose glucosyltransferase, alpha-2 importin, RNase L inhibitor-like protein, and a syntaxin protein were identified. Among the up-regulated genes there was a carboxylesterase, an integral membrane Yip1 family protein, a glycosyl hydrolase family-like protein and an endopolygalacturonase. Considering their relatedness to papaya quality, the fragments of genes potentially implicated in carbohydrate metabolism and pulp softening may be considered of interest for further studies. According to the results, differential display was a feasible approach to investigate differences in gene expression during fruit ripening, and can provide interesting information about those fruits whose genomic data is scarce, as is the case of papayas. (c) 2009 Elsevier B.V. All rights reserved.

Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples

Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C-stretch and CA repeat. To observe which CA ""alleles"" were present in each tissue, PCR products were cloned and re-sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

P>Thirty-five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Parana, Brazil. Twenty-two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff`s method and processed for acid-fast bacilli staining, culture in Stonebrink and Lowenstein-Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR-positive results (54.5%) was similar to that of culture-positive results (51.5%, P > 0.05). The percentage of PCR-positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR-negative were reanalysed using 2.5 mu l DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.

Iron deficiency and frequency of HFE C282Y gene mutation in Brazilian blood donors

Limited data are available about iron deficiency (ID) in Brazilian blood donors. This study evaluated the frequencies of ID and iron-deficiency anaemia (IDA) separately and according to frequency of blood donations. The protective effect of the heterozygous genotype for HFE C282Y mutation against ID and IDA in female blood donors was also determined. Five hundred and eight blood donors were recruited at the Blood Bank of Santa Casa in Sao Paulo, Brazil. Haemoglobin and serum ferritin concentrations were measured. The genotype for HFE C282Y mutation was determined by polymerase chain reaction followed by restriction fragment length polymorphism analysis. The ID was found in 21 center dot 1% of the women and 2 center dot 6% of the men whereas the IDA was found in 6 center dot 8 and 0 center dot 3%, respectively. The ID was found in 11 center dot 9% of the women in group 1 (first-time blood donors) and the frequency increased to 38 center dot 9% in women of the group 3 (blood donors donating once or more times in the last 12 months). No ID was found in men from group 1; however the ID frequency increased to 0 center dot 9% in group 2 (who had donated blood before but not in the last 12 months) and 5 center dot 0% in group 3. In summary, the heterozygous genotype was not associated with reduction of ID or IDA frequencies in both genders, but in male blood donors it was associated with a trend to elevated ferritin levels (P = 0 center dot 060). ID is most frequent in Brazilian women but was also found in men of group 3.

Identification of Staphylococcus aureus Carrying the mecA Gene in Ready-to-Eat Food Products Sold in Brazil

Since Staphylococcus aureus can cause several types of diseases, the development of antibiotic resistance poses an even greater threat to public health. S. aureus is known to possess the adaptive capability to promptly respond to antibiotics, making it resistant and increasingly difficult to treat; methicillin-resistant strains of S. aureus are a major concern with regard to this species. Previous studies reported the identification of methicillin-resistant S. aureus in food, demonstrating that this can represent a source of S. aureus which may carry the mecA gene. Fifty-seven S. aureus isolates, previously obtained from different types of food, were screened by polymerase chain reaction with specific primers for the mecA gene, which mediates methicillin resistance. Five (9%) isolates showed the presence of mecA gene, demonstrating that food may contain microorganisms possessing resistance genes. This study emphasizes the need to include food as a possible source of S. aureus carrying mecA gene and the need to monitor these products. Moreover, this is the first report of the presence of mecA genes in S. aureus isolated from ready-to-eat food in Brazil and Latin America.

Molecular identification of naturally occurring bacteriocinogenic and bacteriocinogenic-like lactic acid bacteria in raw milk and soft cheese

Lactic acid bacteria ( LAB) are currently used by food industries because of their ability to produce metabolites with antimicrobial activity against gram-positive pathogens and spoilage microorganisms. The objectives of this study were to identify naturally occurring bacteriocinogenic or bacteriocinogenic-like LAB in raw milk and soft cheese and to detect the presence of nisin-coding genes in cultures identified as Lactococcus lactis. Lactic acid bacteria cultures were isolated from 389 raw milk and soft cheese samples and were later characterized for the production of antimicrobial substances against Listeria monocytogenes. Of these, 58 (14.9%) LAB cultures were identified as antagonistic; the nature of this antagonistic activity was then characterized via enzymatic tests to confirm the proteinaceous nature of the antimicrobial substances. In addition, 20 of these antagonistic cultures were selected and submitted to genetic sequencing; they were identified as Lactobacillus plantarum (n = 2) and Lactococcus lactis ssp. lactis (n = 18). Nisin genes were identified by polymerase chain reaction in 7 of these cultures. The identified bacteriocinogenic and bacteriocinogenic-like cultures were highly variable concerning the production and activity of antimicrobial substances, even when they were genetically similar. The obtained results indicated the need for molecular and phenotypic methodologies to properly characterize bacteriocinogenic LAB, as well as the potential use of these cultures as tools to provide food safety.

ABCB1 and ABCC1 expression in peripheral mononuclear cells is influenced by gene polymorphisms and atorvastatin treatment

This study investigated the effects of atorvastatin on ABCB1 and ABCC1 mRNA expression on peripheral blood mononuclear cells (PBMC) and their relationship with gene polymorphisms and lowering-cholesterol response. one hundred and thirty-six individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). Blood samples were collected for serum lipids and apolipoproteins measurements and DNA and RNA extraction. ABCB1 (C3435T and G2677T/A) and ABCC1 (G2012T) gene polymorphisms were identified by polymerase chain reaction-restriction (PCR)-RFLP and mRNA expression was measured in peripheral blood mononuclear cells by singleplex real-time PCR. ABCB1 polymorphisms were associated with risk for coronary artery disease (CAD) (p < 0.05). After atorvastatin treatment, both ABCB1 and ABCC1 genes showed 50% reduction of the mRNA expression (p < 0.05). Reduction of ABCB1 expression was associated with ABCB1 G2677T/A polymorphism (p = 0.039). Basal ABCB1 mRNA in the lower quartile (<0.024) was associated with lower reduction rate of serum low-density lipoprotein (LDL) cholesterol (33.4 +/- 12.4%) and apolipoprotein B (apoB) (17.0 +/- 31.3%) when compared with the higher quartile (>0.085: LDL-c = 40.3 +/- 14.3%; apoB = 32.5 +/- 10.7%; p < 0.05). ABCB1 substrates or inhibitors did not affect the baseline expression, while ABCB1 inhibitors reversed the effects of atorvastatin on both ABCB1 and ABCC1 transporters. In conclusion, ABCB1 and ABCC1 mRNA levels in PBMC are modulated by atorvastatin and ABCB1 G2677T/A polymorphism. and ABCB1 baseline expression is related to differences in serum LDL cholesterol and apoB in response to atorvastatin. (C) 2008 Elsevier Inc. All rights reserved.

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirao Preto, Sao Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTET (TM) QPCR SYBR (R) Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fist and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method. (C) 2009 Elsevier Ltd. All rights reserved.

Predominance of CTX-M-type extended-spectrum beta-lactamase genes among enterobacterial isolates from outpatients in Brazil

Two hundred fifty-seven nalidixic acid-resistant enterobacterial isolates were collected in a Brazilian community from January 2000 to May 2005 to determine the prevalence of plasmid-encoded extended-spectrum beta-lactamases. The bla(CTX-M) genetic environment was determined by polymerase chain reaction and sequencing. Eleven isolates (4.2%) harbored a bla(CTX-M-2) gene, 3 isolates bla(CTX-M-9), 2 isolates bla(CTX-M-8), and 6 isolates bla(SHV-5). Two novel bla(CTX-M-2) variants, namely, bla(CTX-M-74) and bla(CTX-M-75), were identified. (C) 2009 Elsevier Inc. All rights reserved.

A rapid single-step multiplex method for discriminating between Trichogramma (Hymenoptera: Trichogrammatidae) species in Australia

Inaccurate species identification confounds insect ecological studies. Examining aspects of Trichogramma ecology pertinent to the novel insect resistance management strategy for future transgenic cotton, Gossypium hirsutum L., production in the Ord River Irrigation Area (ORIA) of Western Australia required accurate differentiation between morphologically similar Trichogramma species. Established molecular diagnostic methods for Trichogramma identification use species-specific sequence difference in the internal transcribed spacer (ITS)-2 chromosomal region; yet, difficulties arise discerning polymerase chain reaction (PCR) fragments of similar base pair length by gel electrophoresis. This necessitates the restriction enzyme digestion of PCR-amplified ITS-2 fragments to readily differentiate Trichogramma australicum Girault and Trichogramma pretiosum Riley. To overcome the time and expense associated with a two-step diagnostic procedure, we developed a “one-step” multiplex PCR technique using species-specific primers designed to the ITS-2 region. This approach allowed for a high-throughput analysis of samples as part of ongoing ecological studies examining Trichogramma biological control potential in the ORIA where these two species occur in sympatry.

The Genus Wolbachia

Numerous invertebrate species form long lasting symbioses with bacteria (Buchner, 1949; Buchner, 1965). One of the most common of these bacterial symbionts is Wolbachia pipientis, which has been estimated to infect anywhere from 15–75% of all insect species (Werren et al., 1995a; West et al., 1998; Jeyaprakash and Hoy, 2000; Werren and Windsor, 2000) as well as many species of arachnids, terrestrial crustaceans and filarial nematodes (O’Neill et al., 1997a; Bandi et al., 1998). In most arthropod associations, Wolbachia act as reproductive parasites manipulating the reproduction of their hosts to enhance their own vertical transmission. There appears to be little direct fitness cost to the infected host besides the costs arising from the reproductive manipulations. However instances have been reported where Wolbachia can be either deleterious (Min and Benzer, 1997; Bouchon et al., 1998) or beneficial (Girin and Boultreau, 1995; Stolk and Stouthamer, 1995; Wade and Chang, 1995; Vavre et al., 1999b; Dedeine et al., 2001) to their hosts. Wolbachia were first described as intracellular Rickettsia-like organisms (RLOs), infecting the gonad cells of the mosquito, Culex pipiens (Hertig and Wolbach, 1924), and were later named 'Wolbachia pipientis' (Hertig, 1936). It was not until the work of Yen and Barr (Yen and Barr, 1971; Yen and Barr, 1973) that Wolbachia were implicated in causing crossing incompatibilities between different mosquito populations (Laven, 1951; Ghelelovitch, 1952). When polymerase chain reaction (PCR) diagnostics for Wolbachia became available, it became clear that this agent was both extremely widespread and also responsible for a range of different reproductive phenotypes in the different hosts it infected (O’Neill et al., 1992; Rousset et al., 1992; Stouthamer et al., 1993). The most common of these are cytoplasmic incompatibility, inducing parthenogenesis, overriding host sex-determination, and male-killing (O’Neill et al., 1997a). As of the time of this writing, more than 450 different Wolbachia strains with unique gene sequences, different phenotypes, and infecting different hosts have been deposited in GenBank and the Wolbachia host database (http://www.wolbachia.sols. uq.edu.au).