Introduction

Ashes are woodland tree species in Hungary and widely cultivated as ornamental plants. These species are often infected with powdery mildew (PM), and until recently (Heluta et al. 2017), only Phyllactinia PM species were described from ash trees, such as P. fraxini, P. fraxinicola, P. japonica, and P. fraxini-longicuspis (Maeda et al. 2021). However, a new causal agent, Erysiphe salmonii, has been discovered in several European countries since 2015 (Heluta et al. 2017). This species was originally described from Japan, and was known also from China, infecting different Fraxinus and Syringa species (Braun and Cook 2012). In Europe, E. salmonii was first found on F. excelsior and F. pennsylvanica in Ukraine by Heluta et al. (2017). Later on, it was also described in Switzerland (Beenken and Brodtbeck 2020) infecting F. ornus; in Austria on F. excelsior and F. ornus (Voglmayr et al. 2021); in Romania (Chinan and Dascălu 2022); in Italy (Hofbauer and Braun 2023), and in Slovenia (Brglez et al. 2023).

In 2022 and 2023, powdery mildew symptoms were observed on the adaxial side of leaves of F. ornus and F. excelsior plants in several locations in Hungary. The present study aimed to identify and characterize the causal agent of these symptoms on Fraxinus plants in Hungary.

Materials and methods

The first infected F. ornus leaves were collected in Budapest in October 2022, and the others in the vicinity of Budapest in September 2023 from forested areas. The infected F. excelsior leaves were sampled in Budapest in September 2023 from urban areas (Table 1). All but one of the infected plants were young, no more than two years old, and heavily infected. The fifth sample, PM339 was collected from a mature tree, which was moderately infected. Samples were placed in plastic bags and transferred to the laboratory for further investigation. Chasmothecia were examined covered in lactic acid; while the anamorphs were studied after boiling in lactic acid (Shin and La 1993). The morphological characteristics of the fungal structures were examined with phase contrast microscopy using a ZEISS AxioScope2 microscope (Germany) equipped with an AxioCam ICc5 camera (Zeiss). At least, 20 measurements were made for each fungal structure. The pathogen was identified based on morphology (Braun and Cook 2012) and based on sequences of the nrDNA internal transcribed spacer (ITS) (see below). The PM infected leaves were deposited at the Herbarium of the Hungarian Natural History Museum, Budapest, Hungary (Table 1).

Table 1 Data of the samples collected in the present study
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In those cases when chasmothecia were found on the leaves the genomic DNA was extracted from a single chasmothecium following a described protocol (Pintye et al. 2020). In the case of the other samples, a piece of cellotape was used for the collection of the mycelium, afterwards the cellotape was incubated in 200 μl TE buffer (Lonza) at 97 °C for 10 min (Pintye et al. 2023).

The ITS region was amplified using general (ITS4 and ITS5; White et al. 1990) and powdery mildew specific primers (PM5 and PM6; Kiss et al. 2001; Takamatsu and Kano 2001). All PCR amplifications were performed in a final volume of 20 µL. Reaction components included 1 µL of 10 µM forward and reverse primers (Thermo Fisher Scientific Inc), 1 µL DNA template and 10 µL Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific). The cycling times and temperatures for both primer pairs (PM5-ITS4 and ITS5-PM6) were as follows: 98 °C for 2 min, followed by 35 cycles of 10 s at 98 °C, 20 s at 58 °C and 21 s at 72 °C, and a final extension step at 72 °C for 5 min. The obtained sequences were compared with the accessions in the National Center for Biotechnology Information database (NCBI, http://www.ncbi.nlm.nih.gov/Blast.cgi) using the BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al. 1990). The resulting sequences were deposited in GenBank (Table 1).

Two of the newly obtained sequences were aligned with 30 other sequences (Table 2) retrieved from GenBank using the online version of MAFFT 7 (Katoh and Standley 2013) with the E-INS-i method. The alignments were examined and edited using MEGA 7 (Kumar et al. 2016). The dataset consisted of 32 sequences and 462 characters; Erysiphe aphananthes was used as outgroup. Maximum likelihood (ML) phylogenetic analysis was carried out with the raxmlGUI 1.5 implementation (Silvestro and Michalak 2012; Stamatakis 2014). A GTR + G nucleotide substitution model was used with ML estimation of base frequencies. Maximum likelihood bootstrap (BS) analysis with 1000 replicates was used to test the support of the branches. Phylogenetic tree was visualized and edited in TreeGraph (Stöver and Müller 2010).

Table 2 List of powdery mildew sequences obtained from GenBank and used for phylogenetic analysis
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Results

Numerous chasmothecia and only a few conidiophores were found on samples PM265 from F. ornus and PM339 from F. excelsior, while only mycelium and a few conidia were observed on other samples. The morphological features of the specimens were identical to those of Erysiphe salmonii described by Braun and Cook (2012). Chasmothecia measured 83–120 µm in diameter, had 10–30 appendages, 103–147 µm long, straight or curved. The apices of the appendages were uncinated, spirally curved (Fig. 1a). Chasmothecia did not contain asci. Mycelium was epiphytic with moderately lobed hyphal appressoria (Fig. 1b, c). The conidiophores were straight and produced single conidia, hyaline, ellipsoid-ovoid, measuring 25–33 × 9–13 µm, without fibrosin bodies. Germ tubes were subterminal and the conidial appressoria were lobate and multilobate (Fig. 1d).

Fig. 1
figure 1

Erysiphe salmonii on Fraxinus ornus. a Chasmothecium with uncinated, spirally curved appendages. b, c Moderately lobed, hyphal appressoria. d Germinating conidium with multilobed appressorium. Bars: a 100 µm; b–d 12 µm

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The sequences of the Hungarian specimens were identical, and showed 100% identity to sequences of E. salmonii. The phylogenetic analysis revealed (Fig. 2) that the Hungarian samples grouped with the epitype of E. salmonii (MUMH4167; accession no. LC577619), other specimens from Japan (LC028981), Ukraine (LC259501), Romania (MW633028), Austria (OK383397), and from Switzerland (MW265935) infecting F. mandshurica, F. rhynchophylla, F. excelsior, F. pennsylvanica, F. sieboldiana and F. ornus. Grouping was supported by high bootstrap value (96). Thus, both morphological examination and phylogenetic analysis have clearly shown that the newly occurring species of powdery mildew that infects ash trees is E. salmonii.

Fig. 2
figure 2

Maximum likelihood tree based on nrDNA internal transcribed spacer (ITS) sequences of Erysiphe species infecting ash trees. The ITS sequence of E. aphananthes served as outgroup. The bootstrap values presented as percentages, below 70% are not shown. The data set comprised 462 characters. Samples collected in this work are shown in boldface. Bar indicates 0.01 expected change per site per branch. The country of origin is provided with two-letter code. (HT: ex holotype; ET: ex epitype)

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Discussion

Only Phyllactinia species were reported to infect ash trees (Heluta et al. 2017; Yamaguchi et al. 2021) in Europe until 2015 (Heluta et al. 2017). After this first report (Heluta et al. 2017), a rapid spread of E. salmonii through the Eastern and Central regions of Europe was described (e.g. Beenken and Brodtbeck 2020; Chinan and Dascălu 2022). A similar pattern of spread of a PM fungus originating also from Asia was observed in the case of hazel powdery mildew in Europe. An epiphytic species, E. corylacearum appeared alongside a widespread hemiendophytic species (P. guttata) commonly visible on the lower side of leaves on the same host plant (Heluta et al. 2019; Sezer et al. 2017). This species, E. corylacearum was also recently introduced to Hungary (Kalmár et al. 2023). E. salmonii is presumably an invasive species, which has recently migrated to Eastern Europe (Heluta et al. 2017), and the source of inoculum could be the chasmothecia accumulated on goods, people’s clothes or imported plants as it was assumed for E. kenjiana by Heluta et al. (2009).

The present study represents the first report of E. salmonii infecting ash trees in Hungary, and provides another example for the geographical expansion potential of PM fungi.