Rapid Detection of Alternaria Species Involved in Pear Black Spot Using Loop-Mediated Isothermal Amplification.

Alternaria species are the most important fungal pathogens that attack various crops as well as fruit trees such as pear and cause black spot disease. Here, a loop-mediated isothermal amplification (LAMP) assay is developed for the detection of Alternaria species. A. alternata cytochrome b (cyt-b) gene was used to design two pairs of primers and amplified a 229-bp segment of Aacyt-b gene. The results showed that LAMP assay is faster and simpler than polymerase chain reaction (PCR). LAMP assay is highly sensitive method for the detection of about 1 pg of genomic DNA of A. alternata by using optimized concentration of MgCl2 (4 mM) in final LAMP reaction. In contrast, the limit of detection was 1 ng of target DNA via conventional PCR. Among the genomic DNA of 46 fungal species, only the tubes containing DNA of Alternaria spp. except A. porri, A. solani, and A. infectoria changed color from orange to yellowish green with SYBR Green I including the main pathogens of pear black spot. The yellowish green color was indicative of DNA amplification. Moreover, LAMP assay was used for testing infected tissues among 22 healthy and diseased pear tissues; the orange color changed to yellowish green for infected tissues only. Altogether, we conclude that cyt-b gene can be used for the detection of Alternaria spp. via LAMP assay, which is involved in pear black spot disease.

Rapid Detection of Ustilaginoidea virens from Rice using Loop-Mediated Isothermal Amplification Assay.

Ustilaginoidea virens is an important fungus that causes rice false smut disease. This disease significantly reduces both grain yield and quality. Various methods have been developed for the detection of U. virens but most of these methods need sophisticated equipment such as a thermal cycler. Here, we present a loop-mediated isothermal amplification (LAMP) assay for the specific detection of U. virens. This assay used a specific region of the UvG-ß1 gene (212-bp region) to design six LAMP primers. The LAMP assay was optimized by the combination of rapidity, simplicity, and high sensitivity for the detection of about 1 pg of target genomic DNA in the reaction whereas, with polymerase chain reaction (PCR), there was no amplification of DNA with concentrations less than 1 ng. Among the genomic DNA of 22 fungus species and two strains of U. virens, only the tube containing the DNA of U. virens changed to yellowish green with SYBR Green I. The color change was indicative of DNA amplification. No DNA was amplified from either the other 22 fungus species or the negative control. Moreover, 20 spikelets and 22 rice seed samples were used for the detection of rice false smut via LAMP. The results were comparable with conventional PCR. We conclude that gene UvG-ß1 coupled with LAMP assay, can be used for the detection and identification of U. virens gene via LAMP.

TaMDAR6 acts as a negative regulator of plant cell death and participates indirectly in stomatal regulation during the wheat stripe rust-fungus interaction.

We identified a new monodehydroascorbate reductase (MDAR) gene from wheat, designated TaMDAR6, which is differentially affected by wheat-Puccinia striiformis f. sp. tritici (Pst) interactions. TaMDAR6 is a negative regulator of plant cell death (PCD) triggered by the Bax gene and Pst. Transcript levels of TaMDAR6 are significantly upregulated during a compatible wheat-Pst interaction, indicating that TaMDAR6 may contribute to plant susceptibility. In addition, H2 O2 production and PCD are significantly induced and initial pathogen development is significantly reduced in the TaMDAR6 knocked-down plants upon Pst infection. Thus, the suppression of TaMDAR6 enhances wheat resistance to Pst. Besides, the suppression of TaMDAR6 during an incompatible interaction induces a change in the morphology of stomata, which leads to poor stoma recognition and as a consequence to reduced infection efficiency. The percentage of infection sites that develop substomatal vesicles decreases in the TaMDAR6 knocked-down plants during the incompatible interaction presumably due to the increase in ROS accumulation, which is likely to activate other resistance mechanisms that have a negative effect on substomatal vesicle formation. TaMDAR6 can therefore be considered a negative regulator of PCD and of wheat defense to Pst.

Predictors of a sustained virological response in patients with genotype 4 chronic hepatitis C.

OBJECTIVES: To determine the clinical, biological, virological and histological predictive factors associated with a sustained virological response (SVR) to combined interferon therapy among Egyptian patients infected by genotype 4 hepatitis C virus (HCV). PATIENTS AND METHODS: Individual data from 250 patients with genotype 4 chronic hepatitis C, treated with different regimens of combined interferon, were analysed. The primary end point was SVR defined as undetectable HCV RNA by polymerase chain reaction (PCR) 24 weeks after the end of treatment. Multivariate logistic regression analysis was performed to select the independent prognostic parameters associated with SVR. RESULTS: A sustained virological response was achieved among 137/250 (54.8%) patients. Baseline factors independently and negatively associated with SVR were serum alpha-fetoprotein (AFP) level (above 0.3 upper limit of normal) [odds ratio (OR)=0.5, 95% confidence interval (CI): 0.2-0.8], severe fibrosis (Metavir score >F2) (OR=0.4, 95% CI: 0.2-0.8), presence of steatosis (OR=0.5, 95% CI: 0.3-0.97) and standard interferon treatment (OR=0.4, 95% CI: 0.2-0.8). CONCLUSIONS: Among genotype 4 chronic hepatitis C patients, severe fibrosis, severe steatosis, treatment with standard interferon and a high serum AFP level were all negatively associated with SVR. Pretreatment serum AFP level should be considered in the routine assessment of factors predictive of a treatment response.

HCV-related morbidity in a rural community of Egypt.

The origin of the hepatitis C virus (HCV) epidemic in Egypt has been attributed to intravenous schistosomiasis treatment in rural areas in the 1960s to 70s. The objective of this study was to estimate the HCV-related morbidity in a rural area where mass schistosomiasis treatment campaigns took place 20-40 years before. The study sample included 2,425 village residents aged 18-65 years recruited through home-based visits. Overall, HCV antibody prevalence was 448/2,425 = 18.5% (95% CI = 16.9-20.1%), reaching 45% in males over 40 years, and 30% in females over 50 years. Of those with HCV antibodies, 284/448 (63.4%, 95% CI = 58.7-67.9%) had chronic HCV infection, among which 107/266 (40.2%, 95% CI = 34.3-46.4%) had elevated alanine aminotransferase (ALT). As part of pre-treatment screening, 26 consenting patients had a liver biopsy: 13 (50.0%) had a treatment indication. Thus, of all patients with HCV antibodies, 13 (2.9%) were eligible for treatment and willing to be treated. The relatively low level of morbidity observed in this study is discussed in view of co-factors of HCV infection progression, such as young age at infection, absence of alcohol intake, the prevalence of Schistosoma mansoni infection, and the prevalence of chronic hepatitis B.