Erysiphales , Ascomycota ) , another East Asian powdery mildew fungus introduced to Ukraine

In 2015, a powdery mildew caused by a fungus belonging to Erysiphe sect. Uncinula was recorded on two species of ash, Fraxinus excelsior and F. pennsylvanica (Oleaceae), from Ukraine (Kyiv, two localities). Based on the comparative morphological analysis of Ukrainian specimens with samples of Erysiphe  fraxinicola and E. salmonii collected in Japan and the Far East of Russia, the fungus was identified as E. salmonii. This identification was confirmed using molecular phylogenetic analysis. This is the first report of E. salmonii not only in Ukraine but also in Europe. It is suggested that the records of E. fraxinicola from Belarus and Russia could have been misidentified and should be corrected to E. salmonii. In 2016, the fungus was found not only in Kyiv but also outside the city. The development of the fungus had symptoms of a potential epiphytotic disease. Thus, it may become invasive in Ukraine and spread to Western Europe in the near future.


Introduction
Species of ash (Fraxinus L.) are quite decorative and grown as ornamental plants in parks, botanical gardens and green spaces.For example, twenty species of this genus are cultivated in Ukraine (Kokhno, 1986), of which eight species are native to Ukraine (Mosyakin, Fedoronchuk, 1999).Of these, F. excelsior L. is the most common species in natural forests and artificial plantations of Ukraine.Some Fraxinus species are hosts of powdery mildews.Phyllactinia fraxini (DC.)Fuss was reported here on F. americana L., F. excelsior, F. ornus L., F. oxycarpa Willd., and F. sogdiana Bunge (Heluta et al., 1987;Heluta, 1989;Andrianova et al., 2006).This fungus is a semi-endophytic parasite, since it forms both endophytic and superficial mycelia.Fruit bodies and conidial sporulation of the fungus are confined to lower surfaces of leaves.Thus, whilst the fungus does not cause significant damage to host plants and does not immediately reduce their ornamental value, it does lead to premature defoliation.Until now, only this powdery mildew was recorded on Fraxinus species in Ukraine, while outside the country other powdery mildews of ash have been described by Braun and Cook (2012), e.g.: Erysiphe fraxinicola U. Braun & S. Takam. and E. salmonii (Syd.)U. Braun & S. Takam.photographed in a drop of distilled water under a light microscope "Primo Star" (Carl Zeiss, Germany) with the camera "Canon A 300" and the software "AxioVision 4.7".To restore shape and size of dried appendages, chasmothecia were put in a droplet of 40% lactic acid solution on a microscope slide, covered with a cover glass, gently heated to boiling point, and then examined under the light microscope.

Morphological study
A careful examination of all Erysiphe specimens collected in Ukraine on Fraxinus spp.showed that both asexual and sexual morphs were uniform and clearly belonged to the same species.They were compared with the descriptions of E. fraxinicola and E. salmonii given in the monograph by Braun and Cook (2012), and with specimens collected in Japan and the Far East of Russia.The asexual morphs of the Ukrainian material were very similar to those of E. salmonii and clearly distinct from those reported for E. fraxinicola.They had much shorter conidiophores (50-66 μm long compared to up to 120 μm for E. fraxinicola).As shown in the table below, the quantitative characteristics of the sexual morphs mostly overlapped.However, chasmothecia of Ukranian origin had appendages gradually increasing in width towards the tip, with uncinate to tightly circinate apices (Fig. 1, f), whereas E. fraxinicola had mostly longer appendages of the same width throughout (Fig. 1, h) and a larger number of spores in the ascus.Based on both the asexual and sexual characters, Ukrainian material fitted much better with E. salmonii, than with E. fraxinicola.This conclusion was consistent with the results of the phylogenetic analysis given below.Detailed morphological characteristics of the fungus are also provided in the taxonomic description below.

Molecular phylogenetic analysis
We used a combined data set of ITS and 28S rDNA sequences to investigate phylogenetic relationships of the eight sequences from Fraxinus powdery mildews.The ITS + 28S rDNA combined data set consisted of 9 sequences and 1417 characters, of which 191 (13.5%) characters were variable and 33 (0.3%) were informative for parsimony analysis.A total of three equally parsimonious trees with 209 steps were constructed by the MP analysis.Tree topologies were almost consistent among the trees, except for branching orders of the terminal branches.One of the trees with the highest likelihood value is shown in Fig. 2. ML analysis

Molecular phylogenetic analysis
The nucleotide sequences of the 5'-end of the nuc 28S rDNA (including domains D1 and D2) and the nuc rDNA ITS1-5.8S-ITS2(ITS) were determined by the procedure described by Takamatsu et al. (2013).The sequences determined in this study were deposited in DNA Data Base of Japan (DDBJ) under the accession numbers LC259500-LC259502.They were aligned with other sequences of the genus Erysiphe retrieved from DNA databases using MUSCLE (Edgar, 2004) implemented in MEGA 6 (Tamura et al., 2013).The alignment was further manually refined using the MEGA6 program and deposited in TreeBASE (http:// www.treebase.org/)under the accession number S20912.Phylogenetic trees were obtained from the data with maximum parsimony (MP) and maximum likelihood (ML) analyses.MP analyses were conducted by PAUP 4.0a152 (Swofford, 2002) with the heuristic search option using 'tree bisection-reconstruction' (TBR) algorithm with 100 random sequence additions to find the global optimum tree.All sites were treated as unordered and unweighted, with gaps treated as missing data.Tree scores, including tree length, consistency index (CI), retention index (RI), and rescaled consistency index (RC) were also calculated.The strength of internal branches of the resulting trees was tested with bootstrap (BS) analysis (Felsenstein, 1985) using 1000 replications with the step-wise addition option set as simple.The ML analysis was done with raxmlGUI (Silvestro, Michalak, 2012) under a GTRGAMMA model.The BS supports and trees were obtained by running rapid bootstrap analysis of 1000 pseudo replicates followed by a query for the tree with the highest likelihood.BS supports of 70% or higher are shown.

Morphological study
Leaves of Fraxinus spp. with powdery mildew symptoms were collected from F. excelsior and F. pennsylvanica in Ukraine, from F. rhynchophylla in the Far East of Russia and from F. longicuspis in Japan.They were dried between paper at 22-24 °С and deposited in the National Herbarium of the M.G.Kholodny Institute of Botany of the National Academy of Sciences of Ukraine (KW-M) and partly in Mie University Mycological Herbarium (TSU-MUMH).The detailed sample list is shown after the species description.The mycelium, conidiophores and conidia were removed from the surface of infected leaves by transparent adhesive tape, observed and

Erysiphe fraxinicola
On Fraxinus longicuspis Siebold & Zukk.: Japan: Niigata Pref., Yahiko-mura, Mt. Yahiko, 18.10.1996, S. Takamatsu (TSU-MUMH 211).belonging to the genera Fraxinus L. and Syringa L. (Chen et al., 1987;Braun, Cook, 2012).However, the same species was also recorded by one of the authors of this paper in the Far East of the Russian Federation in September 1989 (unpublished data).Obviously, some of the specimens collected there by Bunkina (1991) also belong to this species, and not to E. fraxinicola mentioned by the author as Uncinula fraxini.It is possible that 'U.fraxini' from South Korea also belongs to E. salmonii, as the appendages of Korean specimens depicted by Shin (Shin, 2000, fig. 94F) are increasing towards the tip.Unfortunately, reports on the findings of E. fraxinicola in Belarus and the European part of Russia were not accompanied by morphological descriptions.Thus it is possible that they actually belonged to E. salmonii taking into account the finding of this species in Ukraine.Either way, we can say that at least one more East Asian species of powdery mildew fungi has been introduced into Europe.
Note that E. salmonii was found only in two localities in Kyiv in 2015, viz. in the center of the city and on a residential area Pivdenna (South) Borshchahivka, but no mature plants, only 2-3-year-old ash seedlings, were infected at this time.However, next year E. salmonii was recorded in the central part of the city (but in different localities) and elsewhere in South Borshchahivka and on the outskirts of Kyiv.Although only young plants were infected, the monitoring of the development of E. salmonii in and around Kyiv demonstrated the significant invasive potential of this ash parasite.Therefore, it is highly likely that the fungus will soon reach Western Europe.

Fig. 2 .
Fig.2.Phylogenetic analysis of rDNA ITS region and 28S rDNA (including D1/D2 domains) for eight sequences from Fraxinus powdery mildews.This tree is one of the three equally parsimonious trees with 209 steps, which were found using a heuristic search.Horizontal branch lengths are proportional to the number of substitutions that were inferred to have occurred along a particular branch of the tree.BS (≥ 70%) values by the maximum parsimony (MP) and maximum likelihood (ML) methods were shown on the respective branches Schlechtendalia 4: 23.2000 (Fig. 1, a-g, i-o) Syn.Uncinula salmonii Syd.& P. Syd. [as 'salmoni'], Annls mycol.11(2): 114.1913.