Analysis of mycobiome in damaged potato (Solanum tuberosum L.) leaves by using metagenomic approaches

The problem of potato diseases caused by fungi and fungi-like organisms is relevant for all regions of the world cultivating this crop, since it is mycoses that cause the most significant damage to plants (A. Bernreiter, 2017). The traditional approaches for identification of potato pathogens are aimed at identifying a specific pathogen and do not take into account neither other, often unknown pathogens, nor the other most important component - the beneficial microbiota of the phyllosphere community whose alterations can also become one of the causes of diseases. The novelty of this work lies in the fact that the high-throughput sequencing methods we used here is free of this disadvantages and makes it possible to identify virtually all plant microorganisms, including the phyllosphere and endosphere. The purpose of this study was to use metagenomic approaches to analyze the total fungal and fungi-like community in potato leaves that have morphological markers of damage by pathogens of the genus Alternaria and Phytophthora which are the causative agents of early blight and late blight. For fungal and fungi-like communities analysis, DNA samples was extracted from leaves of potato ( Solanum tuberosum L.) cultivar Nikulinsky, affected by “alternaria” and “phytophthora” diseases types, which were later used to create amplicon libraries of ITS1 and ITS2 fragments and high-throughput sequencing on the Illumina MiSeq platform (Illumina, Inc., USA). During the bioinformatic data processing with the Illumina software and the PIPITS software package (H.S. Gweon et al, 2015), 187 OTE, 113 phylotypes for the ITS1 library and 249 OTE, 127 phylotypes for ITS2 were identified. Subsequent annotation of OTE and taxonomic analysis of the resulting communities were carried out with the QIIME program (J.G. Caporaso et al., 2010), the diversity coefficients within the community were calculated using the PAST software package (Ø. Hammer et al., 2001). Comparison of the fungal communities obtained for both types of lesion using different universal primers for the ITS1 regions (M. Usyk et al., 2017) and ITS2 (T.J. White et al., 1990) showed that only the first pair is suitable for the detection of phytophthora, and in general gives a more even community structure. The tools of automatic annotation turned out to be insufficient for objective identification of alternaria in samples, as a result we had to use the methods of manual search with the BLASTn program (S.F. Altschul et al., 1990). Since the primer pair ITS2 does not allow identification of Phytophthora in the samples, the further comparative analysis of the fungal communities of the two types of lesion was carried out using data only from the ITS1 library. The data of taxonomic analysis showed that in the affected areas for both types of mycoses a rich fungal community is formed, and, in the case of “late blight”, the fraction of the pathogen is about 30 % in the community, and in the variant with “early blight”, only 2.07 % with a significant part (about 15 %) accounted for by Phytophthora , which does not exclude the case of secondary lesion. Thus, it was shown that in the fungal and fungi-like communities formed in the areas affected by disease, the proportion of pathogens is no more than 30 %, which indicates a pronounced dynamics of the taxonomic composition of fungi in the affected area. It is obvious that high-throughput sequencing methods have a very high potential in fundamental and applied research on plant diseases of a microbiological nature.