Esca is a destructive disease that affects vineyards leading to important losses in wine production. Information about the response of Vitis vinifera plants to this disease is scarce, particularly concerning changes in plant metabolism. In order to study the metabolic changes in Vitis plants affected by esca, leaves from both infected and non-affected cordons of V. vinifera cv. Alvarinho (collected in the Vinho Verde region, Portugal) were analysed. The metabolite composition of leaves from infected cordons with visible symptoms [diseased leaves (dl)] and from asymptomatic cordons [healthy leaves (hl)] was evaluated by 1D and 2D (1)H-nuclear magnetic resonance (NMR) spectroscopy. Principal component analysis (PCA) of the NMR spectra showed a clear separation between dl and hl leaves, indicating differential compound production due to the esca disease. NMR/PCA analysis allowed the identification of specific compounds characterizing each group, and the corresponding metabolic pathways are discussed. Altogether, the study revealed a significant increase of phenolic compounds in dl, compared with hl, accompanied by a decrease in carbohydrates, suggesting that dl are rerouting carbon and energy from primary to secondary metabolism. Other metabolic alterations detected comprised increased levels of methanol, alanine, and gamma-aminobutyric acid in dl, which might be the result of the activation of other defence mechanisms.

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The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy. The novelty of this work lies in the design and optimization of two MBs for detection of SARS-CoV-2, namely, concentration, fluorescence plateaus of hybridization, reaction temperature and real-time results. We also identify putative G-quadruplex (G4) regions in the genome of SARS-CoV-2. A total of 458 nasopharyngeal and throat swab samples (426 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity, and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract.

The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to, detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy, using MBs to detect the presence of target amplicons by fluorescence analysis. Two MBs were designed, optimized in terms of concentration, fluorescence plateaus of hybridization, reaction temperature and best real-time results. A total of 450 nasopharyngel and throat swab samples (418 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract. Our two-stage testing strategy is suitable for further development into a point-of-care assay and potentially scalable to population level.

Ocimum minimum L. (fine-leaved basil) is cultivated along the Douro Litoral Region in Portugal. In June 2009, a disease was observed in fine-leaved basil fields in three geographically separated locations: Maia, Rio Tinto, and São Mamede Infesta. Affected plants showed wilt symptoms, chlorotic leaves, and stem necrosis. Discolored vascular tissue was observed when the stems were cut longitudinally. For isolation, crown and stem sections (10 cm long) were surface disinfected for 1 min in 1.5% NaOCl and washed twice with sterile distilled water. The sections were cut longitudinally and small pieces of discolored vascular tissue were plated onto potato dextrose agar (PDA) amended with streptomycin sulfate (0.5 g liter–1). Plates were incubated at 25°C in the dark. Fusarium colonies were consistently isolated from symptomatic plants sampled from the three different locations and transferred to PDA and Spezieller Nährstoffarmer agar (SNA) culture media for morphological species identification (2). After 10 days of incubation at 25°C, all isolates were identified as F. oxysporum. A PCR-based assay was conducted with nine single-spored isolates (F2, F3, F4, F7, F8, F9, F10, F11, and F13) using the F. oxysporum f. sp. basilici specific primer pair, Bik 1 and Bik 2 (1). A single DNA fragment of 382 bp was amplified in all isolates, which confirmed the identification of F. oxysporum f. sp. basilici. Pathogenicity of all nine isolates was determined on 2-month-old fine-leaved basil seedlings growing in sterile peat moss. Plants were inoculated by watering the roots with 20 ml of a conidial suspension (106 conidia ml–1) harvested from 3-week-old cultures grown on PDA. Thirty-six replicates (each one in individual pots) for each isolate were used, with an equal number of control plants. Plants were maintained in a greenhouse at 15 to 20°C. Within 2 weeks of inoculation, all inoculated plants wilted and exhibited severe leaf and stem necrosis. The fungus was reisolated from vascular tissues of the crown area and the stems of symptomatic seedlings, fulfilling Koch's postulates. Control plants remained healthy. To our knowledge, this is the first report of F. oxysporum f. sp. basilici infecting fine-leaved basil in Portugal. References: (1) A. Chiocchetti et al. Plant Dis. 85:607, 2001. (2) J. F. Leslie et al. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.