Study of the ribosomal DNA intergenic spacer (IGS) region as a molecular tool for identification of vascular fungi associated with Olive Quick Decline Syndrome in Southern Italy: Phaeoacremonium italicum as a study model

This research concerns the first study of the ribosomal DNA intergenic spacer (IGS) region as a molecular target for detection and phylogenetic analyses of Phaeoacremonium spp. found associated with olive quick decline syndrome in southern Italy. The study follows a survey carried out during a period of three years (2015-2018), in different olive orchards of Salento (Apulia). Disease symptoms of wilt and dieback, browning and streaking under the bark and wood discoloration were observed. Three Phaeoacremonium species were isolated more frequently than other fungi. These Phaeoacremonium spp. were identified based on their morphological characteristics and by analyses of the β-tubulin and actin genes, namely Phaeoacremonium italicum, Phaeoacremonium scolyti and Phaeoacremonium minimum. Pm. italicum was the predominant Phaeoacremonium species isolated from olive wood discoloration and was used as a study model to explore originally, the ribosomal DNA intergenic spacer (IGS) region in these fungi. Pm. italicum isolates were subjected to PCR amplification of the intergenic spacer (IGS) region followed by cloning and sequencing. A structural analysis of the IGS sequences of Pm. italicum was performed but also for Pm. scolyti and Pm. minimum. The IGS region showed polymorphisms among Pm. italicum isolates and four categories of repeat elements of 12-15 nucleotides, organized in distinct patterns, were detected within a variable IGS region. A PCR-based assay, to provide a specific detection of Pm. italicum group was developed by designing a specific PCR primer on the basis of the nucleotide sequence variability of the rDNA intergenic spacer among Phaeoacremonium species. The primer pair specificity was checked and confirmed using 10 isolates of Pm. italicum isolates and other 14 non-target Phaeoacremonium spp. The PCR detection limit was up to 1 pg/μL. To understand whether the intergenic spacer (IGS) region could be used to improve phylogenetic resolution, within and among Phaeoacremonium species, this region was amplified from a collection of Phaeoacremonium spp. isolates, including the representative Pm. italicum, Pm. scolyti and Pm. minimum isolates collected from the field. Maximum parsimony analyses were performed on IGS, β-tubulin and actin gene dataset separately as β-tubulin and actin gene are considered as targeted markers for this genus. Multigenic analyses combining IGS-β-tubulin-actin sequences was also performed. The IGS region was informative and produced a phylogeny with essentialy the same topology of β-tubulin, actin and combined β-tubulin-actin trees in terms of species-level clades, however, they revealed conflicts related to the grouping of isolates within Pm. italicum, Pm scolyti and Pm. minimum clades. Specifically, IGS tree showed a clear division of Pm. italicum group into distinct subclades. Combined IGS-β-tubulin-actin phylogeny showed similar results as those obtained in the single IGS phylogeny, and the latter was actually consistent with the IGS structural organization of representative Pm. italicum isolates. The present study indicates that analysis of IGS sequences revealed inter- and intraspecific variation and proved a suitable molecular tool for detection and phylogenetic analyses, within and among Phaeoacremonium species.