The thermotherapy of sugarcane setts is currently the primary management method for Leifsonia xyli subsp. xyli (Lxx), in Brazil. When they are immersed, the enzymes and proteins of the bacterial cell are denatured without harming the setts buds. Due to possible escapes from detection and consequent bacterium survival to thermotherapy, what may result in asymptomatic seedlings, this study aimed to detect the Leifsonia xyli subsp. xyli bacterium in sugarcane setts using molecular techniques and different time and temperature combinations, with or without the addition of antibiotics. The conventional PCR method detected the Lxx bacterial DNA only in the positive control, consisting of a highly susceptible plant with a high bacterial concentration. Using the nested-PCR, the Lxx DNA was detected in all the treatments used. Thus, none of the treatments adopted in the thermotherapy was able to eliminate the Lxx from the setts, and the use of kasugamicin also did not eliminate the bacterium, but reduced the bacterial population in the tested treatments. These results confirm that the nested-PCR is a useful tool to detect the presence of this phytobacterium in setts that will be used as seedlings.

The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike gene. Monitoring the S gene mutations is crucial for successful controlling measures and detecting variants that can evade vaccine immunity. Even after the costs reduction resulting from the pandemic, the new generation sequencing methodologies remain unavailable to a large number of scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S gene, this work describes a new feasible protocol for complete nucleotide sequencing of the S gene using the Sanger technique. Such a methodology could be easily adopted by any laboratory with experience in sequencing, adding to effective surveillance of SARS-CoV-2 spreading and evolution.

Resumo: O objetivo deste trabalho foi avaliar a reação de 48 acessos de tomateiro (Solanum lycopersicum), inclusive de espécies selvagens, a diferentes isolados das três raças de Fusarium oxysporum f. sp. lycopersici (FOL). Utilizaram-se marcadores moleculares ligados aos genes de resistência I-1, I-2 e I-3. A combinação de bioensaios e marcadores moleculares específicos mostrou elevada correlação para a maioria dos acessos. Acessos de S. peruvianum e S. corneliomuelleri apresentaram resistência contra todas as raças de FOL; a introgressão de fatores de resistência destes genótipos em germoplasma-elite de tomateiro é de elevado interesse para o melhoramento genético desta cultura.

Culex Flavivirus (CxFV) is a classical insect-specific virus, which has aroused interest after the first indication that it can produce in nature superinfection exclusion of viruses of medical interest such as West Nile. Despite the detection of CxFV in different regions, CxFV ecology and the influence of co-circulation of arboviruses remains poorly understood. Therefore, our primary goals are to observe the occurrence of CxFV infection in mosquitoes trapped in an urban area of Rio de Janeiro, Brazil, characterize the virus circulating, and provide isolates. A prospective study was carried out for eight months on the campus of the Federal University of Rio de Janeiro, trapping adult mosquitoes. The CxFV minimum infection rates were determined in this period, and the virus isolation process is fully described. Samples from this study were grouped into genotype 2, along with CxFV sequences from Latin America and Africa.

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A loop-mediated isothermal amplification (LAMP) assay was developed and compared to polymerase chain reaction (PCR), nested PCR, and selective isolation assays for detection of Xanthomonas albilineans, the causal agent of sugarcane leaf scald. The pathogen was isolated on selective medium from 44 out of 45 (98%) samples taken from symptomatic stalks, and from 44 out of 70 (63%) samples from asymptomatic stalks that were collected from plots with symptomatic stalks. Forty-two (93%), 41 (91%), and 42 (93%) symptomatic samples tested positive by LAMP, PCR and nested PCR, respectively. The pathogen was detected in 19 (27%), 8 (11%), and 25 (36%) of the 70 asymptomatic samples by LAMP, PCR and nested PCR, respectively. Symptomatic stalks were mainly, but not always, associated with high populations of the pathogen (10 7-10 9 CFU/ml), and asymptomatic stalks with low populations (<10 3 CFU/ml) or no bacteria. Although our LAMP and nested PCR methods detected 10 CFU/ml of X. albilineans in suspensions prepared with pure culture, they sometimes failed to detect the pathogen in samples with low pathogen populations. Isolation on selective medium along with another method should therefore be used for detection of the pathogen in asymptomatic stalks, especially in quarantine programs.

Vascular wilting in tomato plants is an important disease caused by soil-inhabiting pathogens, especially Verticillium dahliae, which results in significant production losses. Control measures against this disease are difficult to implement due to intrinsic pathogen characteristics, such as high adaptability to the subterranean environment, in association with the host, and development of resistance structures that remain viable in the soil for long periods. The introgression of genes that express resistance is the main control measure and requires a continuous characterization program of resistant accessions. This study aimed at identifying tomato (Solanum lycopersicum) accessions resistant to V. dahlia, by using the phenotypic and genotypic methods. The reaction of 33 tomato accessions to different V. dahliae isolates was reinforced by molecular analysis, through markers linked to Ve resistance genes. The combination of bioassays and specific molecular markers showed a high correlation (94.3 %), with the selection of 25 accessions resistant to V. dahliae.

RESUMO -A pinta preta ou mancha preta dos citros, causada pelo fungo Guignardia citricarpa, é considerada uma doença quarentenária, que impõe restrições ao transporte de frutas frescas para países da União Europeia. A ocorrência de infecções latentes e o tempo para o diagnóstico por métodos convencionais levam à necessidade de validar protocolos moleculares rápidos, eficientes e reprodutíveis para detecção do patógeno em tecidos assintomáticos. Assim, este trabalho visou detectar G. citricarpa em tecidos de frutos sintomáticos e em folhas assintomáticas de laranja Pêra por PCR convencional e por PCR em tempo real.A especificidade e o limite de detecção foram avaliados em amostras de tecidos de lesões em frutos e em folhas assintomáticas. Em folhas assintomáticas a presença do fungo foi detectada em baixas concentrações, nessas condições, a PCR em tempo real demonstrou ser viável, reprodutível e altamente sensível para a detecção do patógeno. Termos para indexação: Citricultura, diagnose molecular, Phyllosticta citricarpa. DETECÇÃO MOLECULAR DE Guignardia citricarpa EM TECIDOS ASSINTOMÁTICOS DE LARANJA-PERAABSTRACT -Citrus black spot, a fungal disease caused by the quarantine fungus Guignardia citricarpa, restricts the exportation of fresh fruit to countries in the European Union. The occurrence of latent infections and the time required for diagnosis using conventional methods have brought about the need to validate fast, efficient and reproducible molecular techniques to detect the pathogen in asymptomatic tissue. As such, this study aims to detect G. citricarpa in the symptomatic fruit and asymptomatic leaf tissue of sweet oranges by conventional and real-time polymerase chain reaction (PCR). Specificity and limit of detection (LOD) were assessed in tissue samples of fruit lesions and asymptomatic leaves. Low concentrations of the fungus were found in asymptomatic leaves. Under these conditions, real-time PCR proved to be viable, reproducible and highly sensitive to detection of the pathogen.