The fungus Phyllosticta citricarpa is a quarantine pathogen in the EU and is of high economic importance in many parts of the world where favourable climate conditions drive the development of citrus ...black spot (CBS) disease. Disease symptoms include necrotic lesions on leaves and fruits. Low disease pressure can reduce crop marketability, while higher disease pressure can cause premature fruit drop, significantly increasing crop losses. The wind‐dispersed spores of P. citricarpa are especially problematic for rapid pathogen dispersal, but also provide an opportunity for early detection of the disease spreading into a new area. In this study we have developed and validated a quantitative PCR (qPCR) assay based on the TEF1‐α sequence. Specificity testing demonstrated that it is currently the only qPCR assay that does not cross‐react with closely related Phyllosticta species. The assay is sensitive and can detect a single copy of the TEF1 gene in a reaction, it is highly repeatable and reproducible and can be used for testing of the sticky tapes from spore traps as well as citrus fruit samples. High‐throughput sequencing (HTS) of the DNA barcodes ITS1 and TEF1 was also explored for the detection and discrimination of P. citricarpa. The limit of detection of the HTS was 1000 spores on a daily spore trap tape. This study makes an important improvement to the diagnostics of the CBS and the methods developed can also be applied to improve the surveillance and early detection of the pathogen when linked to spore samplers in the field.
Targeted and non‐targeted approach to detect causal agent of citrus black spot disease in air samples: specific qPCR assay and high‐throughput sequencing of two barcodes for detection and identification of Phyllosticta citricarpa.
The dihydropteroate sulfate (DHPS) gene is associated with resistance to sulfa/sulfone drugs in Pneumocystis jirovecii. We investigated the DHPS mutation rate in three groups of Iranian HIV-positive ...and HIV-negative patients by polymerase chain reaction-restricted fragment length polymorphism analysis. Furthermore, an association between P. jirovecii DHPS mutations and strain typing was investigated based on direct sequencing of internal transcribed spacer region 1 (ITS1) and ITS2. The overall P. jirovecii DHPS mutation rate was (5/34; 14.7%), the lowest rate identified was in HIV-positive patients (1/16; 6.25%) and the highest rate was in malignancies patients (3/11; 27.3%). A moderate rate of mutation was detected in chronic obstructive pulmonary disease (COPD) patients (1/7; 14.3%). Most of the isolates were wild type (29/34; 85.3%). Double mutations in DHPS were detected in patients with malignancies, whereas single mutations at codons 55 and 57 were identified in the HIV-positive and COPD patients, respectively. In this study, two new and rare haplotypes were identified with DHPS mutations. Additionally, a positive relationship between P. jirovecii strain genotypes and DHPS mutations was identified. In contrast, no DHPS mutations were detected in the predominant (Eg) haplotype. This should be regarded as a warning of an increasing incidence of drug-resistant P. jirovecii strains.
Nemea and Mantinia are famous wine regions in Greece known for two indigenous grape varieties, Agiorgitiko and Moschofilero, which produce high quality PDO wines. In the present study, indigenous ...Saccharomyces cerevisiae yeast strains were isolated and identified from spontaneous alcoholic fermentation of Agiorgitiko and Moschofilero musts in order to evaluate their oenological potential. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) recovered the presence of five distinct profiles from a total of 430 yeast isolates. The five obtained strains were evaluated at microvinifications trials and tested for basic oenological and biochemical parameters including sulphur dioxide and ethanol tolerance as well as H2S production in sterile grape must. The selected autochthonous yeast strains named, Soi2 (Agiorgitiko wine) and L2M (Moschofilero wine), were evaluated also in industrial (4000L) fermentations to assess their sensorial and oenological characteristics. The volatile compounds of the produced wines were determined by GC-FID. Our results demonstrated the feasibility of using Soi2 and L2M strains in industrial fermentations for Agiorgitiko and Moschofilero grape musts, respectively.
Abstract Validation of the MycoAlign assay, a newly developed Mycobacterium spp. identification system based on internal transcribed spacer-1 sequencing, was performed using 50 acid-fast bacilli ...(AFB)–positive clinical laboratory specimens. Forty-three (86%) diagnostic-level results were obtained, including 38 Mycobacterium spp. and 5 other AFB-positive genera. Three isolates (6%) had suboptimal identity scores with high probability (81–87% identity score). Four (8%) mixed-pattern results were obtained. Forty-five (90%) observations were concordant with the species identification by standard methods, including all controls.
Nucleotide variation of the internal transcribed spacer 2 region of nuclear ribosomal DNA was used to estimate the phylogeny of the moss family Hylocomiaceae. Both parsimony and neighbour-joining ...analyses support the monophyly of this family, as well as all seven of the genera within the family. Rhytidium appears as the basal taxon and a North American clade comprising Rhytidiopsis and Thelia is indicated. The analyses suggest that Hylocomium s. l., a polyphyletic genus, should be divided into three separate genera. The rate of nucleotide substitutions of the ITS-2 region in the Hylocomiaceae is consistent with the hypothesis of a molecular clock based on a relative rate tests. A nearly constant rate of 6.42 × 10—10 substitutions per site and per year is postulated for the family, a rate close to that of the Winteraceae and slower than that of Lopezia.
Fusarium wilt (FW), caused by Fusarium oxysporum Schlechtend f. sp. melonis (Leach & Currence) Snyd. & Hans (Fom) is a substantial threat to muskmelon (Cucumis meloL.) cultivation around the world. ...Identification of new sources of resistance and transfer of the resistance genes to elite cultivars is the effective breeding strategy to reduce the FW-associated crop losses in muskmelon. In the present study, inheritance studies and molecular mapping for FW resistance were conducted in a muskmelon inbred line KP
4
HM-15 with FW resistance introgressed from snapmelon. Inheritance studies in the F
2
population derived from the cross Punjab Sunehri//KP
4
HM-15 indicated that FW resistance in KP
4
HM-15 is governed by a single dominant gene. Bulked segregant analysis was conducted to map the Fom-5
(t)
gene in KP
4
HM-15 using 527 SSR primers. Four primer pairs, CMCTN35, DM0096, CSWCTT02, and ECM181, were found to show differential polymorphism in the resistant and susceptible bulks and were analysed in the whole of the population. Two SSR markers, DM0096 and CSWCTT02, mapped close to the Fom-5
(t)
gene at a genetic distance of 1.4 and 2.5 cM, respectively. These markers can potentially be used to transfer Fom-5
(t)
in elite muskmelon genotypes through marker-assisted backcross breeding until more tightly linked markers are available.
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