RESUMO
Coffee berry disease is caused by Colletotrichum kahawae, a quarantine fungus still absent from most coffee-producing countries. Given the potential adverse effects on coffee berry production, it is a severe worldwide threat to farmers and industry. Current biosecurity management focuses on exclusion by applying quarantine measures, including the certification of coffee plants and their products. However, methods for detecting C. kahawae by National Plant Protection Organization (NPPO) laboratories still need approval. This research aims to functionally demonstrate, standardize, and validate a method for detecting and discriminating C. kahawae from other Colletotrichum species that may be present in coffee plant samples. The method proposes to use an end-point PCR marker for the mating type gene (MAT1-2-1) and a confirmatory test with a real-time quantitative PCR (qPCR) marker developed on the glutamine synthetase gene. The C. kahawae amplicons for the Cen-CkM10 qPCR marker exhibited specific melting temperature values and high-resolution melt profiles that could be readily differentiated from other tested species, including their relatives. Given the fungus's quarantine status, specificity was tested using artificial mixtures of DNA of C. kahawae with other Colletotrichum species and coffee plant DNA. The described method will enable NPPOs in coffee-producing and exporting countries, especially Colombia, to prevent this pathogen's entry, establishment, and spread.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Assuntos
Coffea , Colletotrichum , Doenças das Plantas , Colletotrichum/genética , Colletotrichum/isolamento & purificação , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Coffea/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase/métodosRESUMO
Passiflora virus Y was detected naturally infecting soybean (Glycine max) for the first time in Brazil. Here, we report the nearly complete genome sequence and molecular and biological properties of the PaVY-Br isolate. The nearly complete genome sequence is 9679 nt long and shares 84.4% nt sequence identity with a previously reported PaVY isolate from Passiflora sp. PaVY-Br induced chlorotic spots and systemic mosaic on soybean and chlorotic local lesions on yellow passion fruit (Passiflora edulis) and sesame (Sesamum indicum). The virus was successfully transmitted by Myzus persicae, indicating that this aphid vector can contribute to the spread of PaYV from passion fruit to soybean plants. Additional epidemiological research is in progress to investigate the distribution of PaVY in soybean production areas in Brazil.
Assuntos
Passiflora , Potyvirus , Potyvirus/genética , Glycine max , Doenças das Plantas , FilogeniaRESUMO
RESUMEN La extracción de ARN de calidad constituye el primer paso para el análisis de la expresión génica. Sin embargo, su obtención no es sencilla debido a la susceptibilidad de esta molécula a la presencia de contaminantes como ARNasas, proteínas y polisacáridos. Adicionalmente, debido a la diversa composición de la pared celular de los hongos se requiere optimizar los procesos de extracción de ARN para organismos específicos. Este estudio evalúo el uso de diferentes metodologías de homogeneización de tejido (nitrógeno líquido y liofilización) y extracción de ARN (Trizol, CTAB y RNeasy mini kit) a partir del hongo nativo ascomiceto Xylaria sp. Se determinó la pureza, concentración e integridad del ARN obtenido por medio de espectrofotometría y electroforesis. Adicionalmente, se diseñaron cebadores de referencia para el gen β-Tubulina a partir del alineamiento de secuencias de este gen obtenidas de diferentes ascomicetes. Estos cebadores fueron utilizados para evaluar si el ARN extraído es amplificable mediante RT-PCR. Se determinó que la homogeneización de tejido por medio de liofilización generó mayores rendimientos de extracción independientemente del protocolo de extracción utilizado; sin embargo, éstos alteraron la integridad del ARN. Se obtuvo un ARN con mayor pureza con el protocolo CTAB y un mayor rendimiento con el RNeasy mini kit. Los resultados indican que el ARN extraído, independientemente de la metodología de homogeneización y extracción utilizada, es amplificable mediante RT-PCR. No obstante, se recomienda homogeneizar el tejido con nitrógeno líquido y extraer con RNeasy mini kit por la brevedad del protocolo de extracción y calidad obtenida.
ABSTRACT Obtaining high quality RNA is the first step for gene expression analysis. However, the low stability of this molecule and high presence of contaminants such as RNases, proteins and polysaccharides may trouble extractions. Fungi cell wall composition is highly diverse; therefore optimizing RNA extraction procedures is necessary when studying specific organisms. In this study, different methods of tissue homogenization (liquid nitrogen and lyophilization) and RNA extraction (Trizol, CTAB and RNeasy mini kit) were assessed with a native ascomycete, Xylaria sp. RNA purity, concentration and integrity were determined by spectrophotometry and electrophoresis. In addition, a set of housekeeping gene primers was designed targeting the β-tubulin gene. The primers were used to determine if the RNA extracted allowed RT-PCR amplification. It was demonstrated that homogenization of tissue by lyophilization allowed higher yields of RNA regardless of the extraction protocol used, however, the RNA integrity was affected. The higher RNA purity was obtained using CTAB and the higher yields using the RNeasy mini kit. The extracted RNA is amplifiable by RT-PCR regardless of the homogenization and extraction methodology used. However, it is recommended to homogenize the tissue with liquid nitrogen and to extract RNA with the RNeasy mini kit due the shortness and efficiency of these protocols.