Development of new qPCR-based identification systems for non-quarantine potato (Solanum tuberosum L.) pathogens distributed in the territory of Russia

Abstract

Russia is among the largest potato producers in the world. According to statistics for 2018, the sown area of potatoes amounted to 310.7 thousand ha, which is 3.5 % more than in 2017, and the gross harvest in the industrial sector is 7157 thousand tons. At the same time, potatoes are susceptible to infection by various plant pathogens of different taxonomic groups. In modern potato growing, the progressive spread of viral and bacterial diseases, which, in addition to reducing the yield, causes a catastrophic deterioration of tubers' quality, leads to a serious problem for commercial production. Not all dangerous pathogens belong to quarantine organisms in Russia. However, the need for their accurate and highly specific identification is not less than for quarantine organisms. Currently, common diagnostic methods in potato growing are indicator plants, serological and cytological tests. They are relatively reliable, but not always sensitive enough, time-consuming and their use requires highly qualified personnel. A modern alternative to these methods are diagnostic systems based on polymerase chain reaction (PCR), in particular, its real-time modification (quantitative PCR, qPCR). In the present study, for the first time in Russia, qPCR-based tests were developed for six non-quarantine pathogens - necrotic strains of potato virus Y (PVYN-NTN and PVYN:O), tobacco rattle virus (TRV), and pathogenic bacteria Dickeya solani, D. dianthicola, and Pectobacterium atrosepticum. Primers and fluorescent-labeled probes were designed based on nucleotide sequences deposited in the NCBI GenBank international database for the amplicon size not more than 500 bp. The specificity of the proposed systems was shown in tests with the genetic material of pathogens that infect potatoes, which are taxonomically close or occupy similar ecological niches, such as the ordinary strain PVY, potato mop-top virus, Pectobacterium carotovorum, and D. zeae. The quality of the proposed test systems was also evaluated using plant material, presumably infected with the analyzed pathogens. Nucleic acids were isolated using Proba-NK (for RNA) and Proba-GS (for DNA) reagents (AgroDiagnostica LLC, Russia). For the reverse transcription reaction, the RevertAid Premium First Strand cDNA Synthesis Kit (Thermo Scientific, USA) was used. PCR was carried out in the Tertsik thermocycler (DNA-technology, Russia), and quantitative PCR was performed in the DT-96 detection thermocycler (DNA-technology, Russia). To assess possible inhibition, an internal control sample (IC, a plasmid with a specific 560 bp insert) was added to the reaction mixture. Positive control samples (PCs) were cloned using the Quick-TA kit (Evrogen, Russia). Plasmid DNA concentration was determined (a NanoVue spectrophotometer, GE HealthCare, USA). Specific amplification products were sequenced (an ABI PRISM 3730 automated sequencer, Applied Biosystems, USA). Analytical sensitivity was evaluated by quantitative PCR, in which sequential 10-fold dilutions of plasmid DNA (PC in four independent replicates) in the range of 107 to 100 copies per reaction were used as templates. High sensitivity of the developed test systems, ranging from 10 to 500 copies of specific DNA per reaction, as well as high reproducibility (Cv 1.5-2.0%) were shown. The maximum fluorescence increase for the developed hydrolyzed probes ranged from 1200 to 2000 units of background. The universality of the proposed amplification profiles can serve as the basis for adapting test systems to the multiplex PCR format. The obtained results indicate that these systems detect the analyzed pathogens with high specificity and sensitivity and can be used as part of phytosanitary control and routine detection of 6 non-quarantine pathogens in plants, planting material and food products.