Molecular Identification of Plenodomus species associated with Brassicaceae plant samples stored in the mycological herbarium LEP

Mycological herbaria represent unique and indispensable bioresource collections that play a key role in the study of fungal biodiversity. One of the largest collections of this kind is the herbarium of the All-Russian Institute of Plant Protection (VIZR, LEP), which houses the largest collection of microscopic phytopathogenic fungi in Russia. Over the course of long-term storage, some of the most valuable specimens lose their original properties and become unsuitable for studying by traditional morphological methods. Currently, molecular genetic methods are actively being applied to study herbarium collections, opening new opportunities for the analysis and preservation of biodiversity among phytopathogenic micromycetes. Phoma stem canker and Phoma leaf spot are one of the most widespread and harmfull diseases of cruciferous crops. The causal agents of this disease are closely related fungi, Plenodomus lingam and P. biglobosus. Within these species, two and seven phylogenetic lineages (subclades), respectively, have been identified. Reliable identification of these subclades is only possible using multilocus phylogenetic analysis of nucleotide sequences of the ITS locus and partial regions of actin (act) and β-tubulin (tub2). The aim of this study was to re-identify Plenodomus species in samples of cruciferous plants stored in the Mycological Herbarium LEP for 145–60 years using molecular phylogenetic methods. Nucleotide sequences of the ITS locus were determined in DNA extracted from 16 samples. According to the results of phylogenetic analysis based on ITS sequences, only four samples contained Plenodomus species as the causal agents of Phoma stem canker. In these four samples, in addition to the ITS locus, partial act and tub2 were successfully sequenced. In the phylogenetic tree constructed from nucleotide sequences of all three loci the samples clustered within a clade formed by representative strains of P. lingam subclade ‘brassicae’. Thus, it was reliably confirmed that the causal agent of Phoma stem canker in these four samples was P. lingam ‘brassicae’. Previously in Russia P. lingam ‘brassicae’ has been reliably identified in the Kaliningrad and Leningrad regions, P. biglobosus ‘brassicae’ in the Kaliningrad and Leningrad regions, the Republic of Adygea and Krasnodar Krai. As a result of this study, the presence of the fungus P. lingam ‘brassicae’ was confirmed in herbarium specimens collected in the Leningrad and Rostov regions and in Kazakhstan. To date, the earliest reliable finding of P. lingam in Russia is 114 years old.