Matrix approach to the simultaneous detection of multiple potato pathogens by real-time PCR

Abstract

Aim: Create a method for highly sensitive, selective, rapid and easy-to-use detection and identification of economically significant potato pathogens, including viruses, bacteria and oomycetes, be it single pathogen, or a range of various pathogens occurring simultaneously. Methods and Results: Test-systems for real-time PCR, operating in the unified amplification regime, have been developed for Phytophthora infestans, Pectobacterium atrosepticum, Dickeya dianthicola, Dickeya solani, Ralstonia solanacearum, Pectobacterium carotovorum, Clavibacter michiganensis subsp. sepedonicus, potato viruses Y (ordinary and necrotic forms as well as indiscriminative test system, detecting all forms), A, X, S, M, potato leaf roll virus, potato mop top virus and potato spindle tuber viroid. The test-systems (including polymerase and revertase) were immobilized and lyophilized in miniature microreactors (1Á2 ll) on silicon DNA/RNA microarrays (micromatrices) to be used with a mobile AriaDNA â amplifier. Conclusions: Preloaded 30-reaction micromatrices having shelf life of 3 and 6 months (for RNA-and DNA-based pathogens, respectively) at room temperature with no special conditions were successfully tested on both reference and field samples in comparison with traditional ELISA and microbiological methods, showing perfect performance and sensitivity (1 pg). Significance and Impact of the Study: The accurate, rapid and user-friendly diagnostic system in a micromatrix format may significantly contribute to pathogen screening and phytopathological studies.

Coinfección natural de virus de ARN en cultivos de papa (<i>Solanum tuberosum</i> subsp. Andigena) en Antioquia (Colombia)

“…En la actualidad, la prueba de ELISA se considera el test Gold Standard en los procesos de certificación de tubérculossemilla de papa y para el diagnóstico de virus de importancia cuarentenaria (Halterman et al, 2012;Frost et al, 2013;Stammler et al, 2018); sin embargo, en este estudio se encontró que la técnica de ELISA detectó un menor número de muestras infectadas con los virus PVY (55,5 %), PLRV (5,5 %) y PVX (0 %). Estos resultados no resultan sorpresivos, por cuanto existen reportes que señalan que la sensibilidad de la RT-qPCR para la detección de virus de plantas varía entre 10.000 y 100.000.000 veces más que la prueba de ELISA (Agindotan et al, 2007;Joo-jin et al, 2014;Nikitin et al, 2018;Stammler et al, 2018) y dicha observación se ha confirmado también en diferentes trabajos realizados en Antioquia sobre virus de papa. Así por ejemplo, García Ruíz et al, (2016) reportaron niveles de infección con el virus PVX en un 93,75 % de muestras de tubérculos-semilla de papa y papa criolla cuando emplearon RT-qPCR, mientras que al usar la técnica de ELISA, sólo detectaron el virus en el 50 % de éstas.…”

“…Sin embargo, este cultivo es afectado por un alto número de fitopatógenos e insectos plaga que reducen drásticamente sus rendimientos y calidad de los tubérculos, siendo recientemente calculado que las pérdidas de su producción alcanzan un promedio mundial de 17,2 % y que este valor puede ser aún mayor (21 %) en algunos países en vía de desarrollo (Savary et al, 2019). Adicionalmente, dado que comercialmente la papa se propaga en forma vegetativa a través de tubérculos, los patógenos sistémicos, especialmente aquellos de origen viral, son uno de los problemas fitosanitarios más limitantes para este cultivo (Nikitin et al, 2018). En el mundo, se han reportado más de 40 virus de papa, entre los que se destacan: Potato virus Y (PVY) (Potyvirus, Potyviridae), Potato virus S (PVS) (Carlavirus, Betaflexiviridae), Potato leafroll virus (PLRV) (Polerovirus, Luteoviridae), Potato yellow vein virus (PYVV) (Crinivirus, Closteroviridae) y Potato virus X (PVX) (Potexvirus, Alphaflexiviridae) (García-Ruíz et al, 2016;Zhang et al, 2017;Nikitin et al, 2018).…”

Application of tissue culture techniques in potato

“…The ELISA test (Enzyme-Linked Immunosorbent Assay) has been widely adopted for this purpose, since it is fast and allows detecting virus persistence in a large number of regenerated plants (NAIK; BUCKSETH, 2018). Molecular techniques based on the use of primers for specific regions of the virus genome, such as RT-PCR, are also largely used (LORENZEN et al, 2006;2008;MUÑOZ et al, 2018;NIKITIN et al, 2018).…”

“…It is a culture of outstanding importance worldwide for having socio-economic repercussions, and for being one of the major food sources for humankind. Potato is cultivated in about 150 countries (BASERA et al, 2018), and is the most important non-cereal food crop in the world (BAMBERG et al, 2016;NIKITIN et al, 2018). Last harvest season, Brazil produced 4.3 million tons of potato, in an area of 803,900 hectares (IBGE, 2017).…”

“…On a commercial scale, potato has vegetative propagation. However, this type of multiplication can accumulate several systemic fungi, bacteria and virus infections that provoke degeneration in the plants, culminating with yield and vigor losses (NIKITIN et al, 2018). Thus, the use of virus-free propagative material, with high phytosanitary, physiological and genetic quality is of great importance to ensure that the plant expresses its maximum yield potential.…”