Environmental and Experimental Biology (2018) 16: 307–314 Original Paper DOI: 10.22364/eeb.16.22 Genetic differentiation ofPhoma sp. isolates using retrotransposon-based iPBS assays Vilnis Šķipars1*, Maryna Siaredzich2, Viktorija Belevich1, Natālija Bruņeviča1, Lauma Brūna1, Dainis E. Ruņģis1 1Latvian State Forest Research Institute „Silava”, Rīgas 111, Salaspils LV–2169, Latvia 2Forest Protection and Wood Science Department, Belarusian State Technological University, Sverdlova 13A, Minsk 220006, Belarus *Corresponding author, E-mail: [email protected] Abstract Phoma blight is a disease affecting Norway spruce, Scots pine and other conifer seedlings in many forest tree nurseries throughout the world. Members of the Phoma genus, the causatives of this disease, are difficult to distinguish morphologically and genetically. In this study the use of a retrotransposon-based polymerase chain reaction approach using iPBS amplification for intra-species genetic discrimination between Phoma samples is described. Eight retrotransposon-based iPBS primers were used to genotype DNA from pure cultures of several Phoma species. The utilised markers were able to discriminate betweenPhoma species, but not all of them were able to differentiate all Phoma sp. isolates investigated. Belarusian samples were found to be distinct from the Latvian Phoma isolates. The Belorussian isolates were very similar to each other. A combination of three iPBS markers (2001, 2076 and 2242) enabled partial differentiation of the investigated Belarusian Phoma isolates. Key words: genetic discrimination, inter primer binding site (iPBS) markers, Phoma sp. Abbreviations: iPBS, inter primer binding site. Introduction “Silava” phytopathology and mycology department from Betula pendula samples collected in local tree nurseries Phoma blight is an infectious disease associated with (Brūna, unpublished results), and members of the Phoma several species of Phoma that affect several species of firs genus have also been isolated from grey alder and Norway and pines and cause significant damage in tree nurseries spruce samples taken from Latvian forest ecosystems, in the USA (Srago et al. 1989). Phoma eupyrena is also (Arhipova et al. 2011a; Arhipova et al. 2011b). Interestingly, associated with upper stem canker in Douglas fir (Hamm P. herbarum has been described as potentially beneficial et al. 1989). Phoma blight causes considerable economic for plant growth (Muhammad et al. 2009), including in damage in Belarusian tree nurseries, affecting 5 to 15% of Scots pine (Sanz-Ros et al. 2015), and has also been used Pinus sylvestris and Picea abies seedlings (Siaredzich 2017). as a biological control agent against Taraxacum officinale Phoma spp. was found to be the most common pathogen of (Neumann Brebaum 1998). P. glomerata has been described P. sylvestris, Larix sibirica, P. abies, Pinus sibirica and Abies as having mycoparasitic properties (Sullivan, White 2000), sibirica in Russian forest nurseries of the Novosibirsk region as an endophyte (Deng et al. 2011), as a pathogen causing (Larionova et al. 2017). In contrast, in Latvian coniferous boxwood tip blight (Horst 2001), cankers of peach trees forest tree nurseries Phoma sp. is not considered a threat or (Thomidis et al. 2011) and, according to the American is successfully managed. This is inferred from the absence Phytopathological Society, phoma canker in elm (https:// of Phoma sp. related disease outbreaks in conifer tree www.apsnet.org/publications/commonnames/Pages/Elm. nurseries in Latvia (Brūna, unpublished results). A study aspx). Phoma macrostoma var. incolorata has been reported about root-associated fungi in healthy-looking P. sylvestris to inhibit the growth of the ash pathogen Hymenoscyphus and P. abies seedlings in Swedish forest nurseries showed fraxineus (Haňáčková et al. 2017). These reports show that members of Phoma genus as commonly present on roots Phoma species can play vastly different roles in different of healthy-looking samples not excluding a possibility conditions and host species. of latent infection that could activate after outplanting Phoma species are difficult to identify due to the within- (Stenström et al. 2014). However, representatives of Phoma species variation of morphological features when cultivated herbarum, Phoma glomerata and Phoma adonidicola in vitro (Aveskamp et al. 2008). The available information as well as an unidentified Phoma sp. were isolated by about the genetics of Phoma is increasing. The genome of a members of the Latvian State Forest Research Institute Phoma member called Phoma sp 1 has been sequenced by Environmental and Experimental Biology ISSN 2255-9582 307 V. Šķipars, M. Siaredzich, V. Belevich, N. Bruņeviča, L. Brūna, D.E. Ruņģis the Forest Institute of the National Academy of Sciences of Materials and methods Belarus (Baranov et al. 2015). The genotype has not been definitely assigned to a species, and whole genome shotgun Material sequences of the P. herbarum strain JCM 15942 have been DNA from twelve pure cultures of Phoma sp., each made available by Manabe et al. from RIKEN Center for obtained from a different forest tree nursery in Belarus Life Science Technologies, Japan (NCBI SRA database and five Latvian Phoma isolates, obtained from trees of accession numbers DRX033246 & DRX029297). Another several species growing in Latvian forests was extracted for sequencing project involving Phoma tracheiphila, a citrus genetic analyses of these isolates. Sequences of 12 Latvian pathogen, is under way in U.S. Department of Energy Phoma DNA samples (Z9B – Z300) previously obtained Joint Genome Institute (NCBI SRA database accession by N. Bruņeviča (unpublished data) were used in the numbers SRX1728765, SRX1728766 and SRX1728771). analysis (Table 1). DNA isolation was carried out using the Presently the identification of Phoma species as well Genomic DNA purification kit (ThermoFisher Scientific) as discrimination between isolates and species is still according to the manufacturer’s protocol. According to difficult and time consuming. This is because the DNA morphological characteristics, the Belarusian samples were regions used for species differentiation show low sequence inferred to be P. glomerata or P. macrostoma, but the species polymorphism, and therefore several DNA regions have to could not be determined conclusively. be analysed. One of the most detailed reports of the genetic discrimination of taxa of the Phoma genus used sequencing Sequencing analysis of three different loci: the ITS1-5.8S-ITS2 region (ITS) of DNA sequences of intergenic transcribed spacer region of the nuclear ribosomal DNA operon, part of the actin gene, ribosomal RNA genes were obtained from PCR amplicons and part of the β-tubulin gene (Aveskamp et al. 2009). Additional use of the RNA polymerase II second largest Table 3. Phoma isolates analysed in the present study subunit (rpb2) was employed by Chen et al. (2015b) to increase resolution. Translation elongation factor 1 subunit Isolate Taxon Origin (tef1) has also been used for phylogenetic studies of Phoma N04 Phoma sp. Belarus (Irinyi et al. 2007). A short yet comprehensive review N04.1 Phoma sp. Belarus regarding identity determination of Phoma by multiple N06 Phoma sp. Belarus approaches, including additional DNA markers, is provided N07 Phoma sp. Belarus by Rai et al. (2014). Use of large numbers of samples both N10 Phoma sp. Belarus for pathogen screening in nurseries and for population N12 Phoma sp. Belarus genetics studies is time-consuming and expensive. N13 Phoma sp. Belarus The iPBS method (Kalendar et al. 2010) might serve N14 Phoma sp. Belarus as a tool for differentiation between Phoma sp. isolates. N16 Phoma sp. Belarus This method relies on the non-uniform distribution of N17 Phoma sp. Belarus retrotransposon elements in the genomes of different N19 Phoma sp. Belarus isolates and species and allows for greater discriminatory N20 Phoma sp. Belarus power. This procedure is cost-effective, less time- LV07 Phoma glomerata Latvia consuming and allows differentiation between isolates LV07v Phoma sp. Latvia of the same or different species. In addition to providing LV08k Phoma herbarum Latvia information on genetic diversity, retrotransposons can be LV09v Phoma herbarum Latvia used for identification of a certain pathogen if sufficient LV249 Phoma herbarum Latvia genetic information is available (Fernandez et al. 1998), Z9B Phoma herbarum Latvia differentiation between isolates (Pasquali et al. 2007) and Z18 Phoma adonidicola Latvia have also been shown to influence pathogenicity of plant Z47 Phoma herbarum Latvia pathogens (Mouyna et al. 1996) and plant resistance against them (McDowell, Meyers 2013). The aim of the study was Z78 Phoma sp. Latvia to utilise iPBS markers to investigate the genetic diversity Z94 Phoma sp. Latvia of Phoma sp. isolates collected in several Belarusian forest Z130 Phoma sp. Latvia nurseries, and to compare the Belarusian samples with Z158 Phoma herbarum Latvia Phoma samples isolated from Latvian forests. Sequencing Z163 Phoma herbarum Latvia of the intergenic transcribed spacer region of ribosomal Z178 Phoma sp. Latvia RNA genes was also performed for the Belarusian samples Z215 Phoma glomerata Latvia to obtain additional data for phylogenetic comparison to Z268 Phoma herbarum Latvia publicly available Phoma sp. sequences. Z300 Phoma glomerata Latvia 308 Genetic differentiation of Phoma sp. isolates using iPBS assays obtained with primers ITS1-F and ITS4-B (Gardes, Bruns 1993) from the