Powdery Mildews on Trees and Shrubs in Botanical Gardens, Parks and Urban Green Areas in the Czech Republic

: A total of 103 tree and shrub samples infected with powdery mildew were collected during 2002–2019 from locations within parks, botanical gardens and urban green areas within the Czech Republic and the powdery mildews were morphologically analyzed and identiﬁed. The most frequently represented genera were: Erysiphe (27, including former genera Microsphaera and Uncinula ), Podosphaera (11, including former genus Sphaerotheca ), Phyllactinia (3), Sawadaea (2) and Arthrocladiella (1). New records for the Czech Republic were: E. (U.) arcuata , E. (M.) deutziae , E. (M.) euonymicola , E. (U.) ﬂexuosa , E. (M.) platani , E. (M.) symphoricarpi , E. (M.) vanbruntiana var. sambuci-racemosae , E. (U.) ulmi , Po. amelanchieris , Po. ( Sph. ) pruinosa and Po. ( Sph. ) spiraeae . The results were compared with the spectrum of powdery mildew species in the surrounding countries (Slovakia; Hungary; Poland and Germany (Bavaria)).


Introduction
Recently, the recognition of the importance of trees and shrubs in parks and urban green areas within cities has been growing. Urban green areas play a crucial role in helping to keep the air clean and improving community well-being. Shrubs and trees are able to remove both harmful gases and particles from the air, whilst promoting a better quality of health and well-being [1]. Diseases on these trees and shrubs can reduce their durability, and in the most extreme cases totally destroy them (e.g., Pseudomonas syringae pv. aesculi on horse chestnut).
Powdery mildews are biotrophic, highly specialized pathogens, attacking nearly 10,000 species of vascular plants [2]. Somewhat confounding is the fact that more than one powdery mildew species can parasitize a single host plant. A deeper understanding of the taxonomy of this group has brought about an ever increasing number of powdery mildew species described, which in 2012 stood at 873, including 794 holomorphs; i.e., those with described asexual and sexual morphs [2]. The taxonomy of this group, after many revisions, is now greatly different, thus complicating some comparisons with historical data. Until the 1990s, the taxonomy and identification of powdery mildew fungi were based primarily on the characteristics of the teleomorph, such as the shape of the appendages on the chasmothecium (cleistothecium), or on the number of asci in the ascocarps [3]. However, studies under the scanning electron microscope showed an increased importance of the anamorphic forms in the

Field Survey
Between 2002 and 2019, samples of leaf materials infected with powdery mildew were collected from trees and shrubs in parks, botanical gardens and urban green areas in the Czech Republic. Figure 1 shows the locations of the collected samples that are analysed microscopically. All the samples are deposited in the collections of Department of Botany, Faculty of Science, Palacký University in Olomouc.
Forests 2020, 11, x FOR PEER REVIEW 3 of 20 Between 2002 and 2019, samples of leaf materials infected with powdery mildew were collected from trees and shrubs in parks, botanical gardens and urban green areas in the Czech Republic. Figure 1 shows the locations of the collected samples that are analysed microscopically. All the samples are deposited in the collections of Department of Botany, Faculty of Science, Palacký University in Olomouc. The Czech survey was compared with check-lists from the following three neighbouring countries: Slovakia by Paulech [21], Poland by Dynowska et al. [27], Germany/Bavaria by Bresinsky [28] and one close non-adjoining country, Hungary by Nagy and Kiss [29], as well as some later recordings in these countries.

Microscopic Analysis of Morphological Characteristics
Pieces (ca. 20 × 20 mm) of severely infected leaves were used for evaluation by light microscopy (Olympus BX60, Japan). The pathogen was not separated from the host tissue, and the microscopy was done on leaf segments fixed in glacial acetic acid (acetic acid 99%; Lach-Ner, Neratovice, Czech Republic) for 48 h, and stored in glycerol (glycerolum 85%; Tamda, Olomouc, Czech Republic). The conidia and conidiophores, mostly on the upper leaf surfaces, were microscopically examined after staining with cotton blue (Methyl Blue, Sigma-Aldrich, Prague, Czech Republic) [30]. The presence of fibrosin bodies in the conidia were assessed by mounting fresh conidia in 3% KOH (diluted from potassium hydroxide 90%, Fichema, Czech Republic) [31]. The sexual morph (chasmothecia) was inspected without any staining. In cases where dried leaf samples were analyzed, a modified method of Shin [32] was used, i.e., the heating of herbariumised tissues in fuchsine (Sigma-Aldrich, Prague, Czech Republic) in lactic acid (80% Lach-Ner, Neratovice, Czech Republic). For statistical analyses (means, standard deviations and range), 30 measurements of each characteristic (where possible) were calculated using MS Excel (2010).

Taxonomic Determination
The names of the host plants were used according to Kubát et al. [33] and Biolib (www.biolib.cz) and names of the powdery mildews according to the Taxonomic Manual of the Erysiphales [2], Mycobank (www.mycobank.org), and in some cases according to recent taxonomic literature. The Czech survey was compared with check-lists from the following three neighbouring countries: Slovakia by Paulech [21], Poland by Dynowska et al. [27], Germany/Bavaria by Bresinsky [28] and one close non-adjoining country, Hungary by Nagy and Kiss [29], as well as some later recordings in these countries.

Microscopic Analysis of Morphological Characteristics
Pieces (ca. 20 × 20 mm) of severely infected leaves were used for evaluation by light microscopy (Olympus BX60, Japan). The pathogen was not separated from the host tissue, and the microscopy was done on leaf segments fixed in glacial acetic acid (acetic acid 99%; Lach-Ner, Neratovice, Czech Republic) for 48 h, and stored in glycerol (glycerolum 85%; Tamda, Olomouc, Czech Republic). The conidia and conidiophores, mostly on the upper leaf surfaces, were microscopically examined after staining with cotton blue (Methyl Blue, Sigma-Aldrich, Prague, Czech Republic) [30]. The presence of fibrosin bodies in the conidia were assessed by mounting fresh conidia in 3% KOH (diluted from potassium hydroxide 90%, Fichema, Czech Republic) [31]. The sexual morph (chasmothecia) was inspected without any staining. In cases where dried leaf samples were analyzed, a modified method of Shin [32] was used, i.e., the heating of herbariumised tissues in fuchsine (Sigma-Aldrich, Prague, Czech Republic) in lactic acid (80% Lach-Ner, Neratovice, Czech Republic). For statistical analyses (means, standard deviations and range), 30 measurements of each characteristic (where possible) were calculated using MS Excel (2010).

Taxonomic Determination
The names of the host plants were used according to Kubát et al. [33] and Biolib (www.biolib.cz) and names of the powdery mildews according to the Taxonomic Manual of the Erysiphales [2], Mycobank (www.mycobank.org), and in some cases according to recent taxonomic literature.

Results
In total, during 2002-2019, 103 samples of trees and shrubs infected by powdery mildew were collected. A list of the host plants complemented with recently valid names of the powdery mildews according to Braun and Cook [2], along with their basic morphological characteristics are listed in Table 1. From our survey, it was clear that the upper sides of the leaves were most frequently colonised. However, the chasmothecia were frequently found on the lower sides of leaves. Powdery mildew anamorphs occurred most frequently in spring and summer, while teleomorphs were found mostly in autumn. On trees and shrubs in the Czech Republic our survey confirmed: one species of the genus Arthrocladiella, 27 species of Erysiphe, three species of Phyllactinia, 11 species of Podosphaera and two species of Sawadaea as detailed in Table 2. For simplicity, Table 3 indicates the additional species in each region compared with the Czech Republic. (Thus, species that were recorded in both another country and the Czech Republic do not appear in this Table). Figure 2 shows the leaves of the trees and shrubs with symptoms of powdery mildew, while Figure 3 shows the variability in the shapes of appendages of the chasmothecia of selected powdery mildew species.
Although the older descriptions of powdery mildews were very broadly based and involved wide host ranges, most of the species described in Klika [14] were re-confirmed in our survey as displayed in Table 2. On the other hand, there were several differences as detailed below.

Results
In total, during 2002-2019, 103 samples of trees and shrubs infected by powdery mildew were collected. A list of the host plants complemented with recently valid names of the powdery mildews according to Braun and Cook [2], along with their basic morphological characteristics are listed in Table 1. From our survey, it was clear that the upper sides of the leaves were most frequently colonised. However, the chasmothecia were frequently found on the lower sides of leaves. Powdery mildew anamorphs occurred most frequently in spring and summer, while teleomorphs were found mostly in autumn. On trees and shrubs in the Czech Republic our survey confirmed: one species of the genus Arthrocladiella, 27 species of Erysiphe, three species of Phyllactinia, 11 species of Podosphaera and two species of Sawadaea as detailed in Table 2. For simplicity, Table 3 indicates the additional species in each region compared with the Czech Republic. (Thus, species that were recorded in both another country and the Czech Republic do not appear in this Table). Figure 2 shows the leaves of the trees and shrubs with symptoms of powdery mildew, while Figure 3 shows the variability in the shapes of appendages of the chasmothecia of selected powdery mildew species.

Taxonomic Name of Powdery Mildew Species According to Braun and Cook [2] Unless Otherwise Indicated in Footnotes
Relative Length (Multiples of Chasmothecia Diameter) and Shape of Appendages.

Type of Conidiogenesis/ Number of Distal Cells
Ribes uva-crispa L.

Taxonomic Name of Powdery Mildew Species According to Braun and Cook [2] Unless Otherwise Indicated in Footnotes
Relative Length (Multiples of Chasmothecia Diameter) and Shape of Appendages.

Type of Conidiogenesis/ Number of Distal Cells
Sorbus aucuparia L.

Sawadaea and Arthrocladiella
In our survey we distinguished two Sawadaea taxa on Acer as the now well-defined species of S. bicornis and S. tulasnei. These two taxa were described by Klika [14] by the older names of Uncinula aceris and its var. tulasnei. In our collections, the sexual state was not always present with S. bicornis, but in these cases it was identified on the basis of symptoms. These species were confirmed in the four nearby countries, apart from Poland that recorded only S. tulasnei [27]. In Slovakia S. bicornis was more frequent [21].

Phyllactinia Species
Only three Phyllactinia species were found in our survey, namely Ph. guttata, Ph. fraxini and Ph. orbicularis, on respectively, Corylus, Fraxinus and Fagus. The differences with our neighbours, see Table 3, show that Slovakia [21] had two additional species, Ph. mali and Ph. roboris, but not Ph. orbicularis. Hungary [29] had exactly the same results as Slovakia. Poland [27] recorded an additional three species, Ph. berberidis, Ph. mali and Ph. roboris, but not Ph. fraxini or Ph. orbicularis. A very different situation was found in Bavaria [28], which had seven additional species namely: Ph. berberidis, Ph. betulae, Ph. carpini, Ph. corni, Ph. mali, Ph. marissalii and Ph. populi.

Erysiphe Species
This group gave rise to most problems in identification, in particular, those genera involving recent name changes. In our survey 27 species of Erysiphe were recorded on trees. Klika [14] described the following genera that are now mostly classed as Erysiphe emend; four Uncinula spp. (one of them Sawadaea on Acer), 4 Microsphaera spp. (one now Arthrocladiella on Lycium) and 2 Trichocladia spp. Compared with Klika [14], some additional recordings of powdery mildew species (including invasive ones) were detected. Indeed, it is an interesting fact that during the last few decades several new powdery mildew species were described in Europe, frequently due to their introduction from Asia or America. This is well illustrated with Erysiphe species whose hosts, origins and present locations are listed below. New records for the Czech Republic are in bold:

Discussion
This was the first detailed study since Klika's monograph [14], that was focussed on the spectrum of powdery mildew species on a selected group of plants in the Czech Republic. Questions had logically arisen as to whether the spectrum had changed and if so, how it had changed, and what were the reasons for the change.
By comparing our results with Klika's monograph [14], it is clear that most of the powdery mildew species on trees and shrubs in our country have occurred here for a long time. Although Klika [14] did not distinguish Sawadaea, Arthrocladiella and other genera, this does not mean that these have not occurred previously in our country. This can be due to the historical changes in the taxonomy of powdery mildews, e.g., Arthrocladiella being recorded by the old name of Microsphaera mougeotii. Braun [9] placed it in Erysiphe, which was later divided into the three aforementioned genera: Erysiphe, Golovinomyces and Arthrocladiella. Now Arthrocladiella is a monotypic genus containing a single species (A. mougeotii), with Lycium (Solanaceae) as the sole host genus.
The situation with Sawadaea is similar, being recorded as Uncinula aceris in older literature [14]. Later, the name Sawadaea was introduced [52], and its status was proven by molecular sequence analysis [7].
Concerning Phyllactinia, there is also the problem of older publications having a very wide definition of some species, e.g., Ph. guttata (= Ph. corylea) has a wide host range in Klika's monograph [14]. At the end of the last century, Ph. guttata was recognized as a species complex, consisting of several biological species [3]. Then, based on a phylogenetic study by Takamatsu et al. [53], Braun and Cook [2] confined Ph. guttata to Corylus hosts and accepted separate species on single hosts, e.g., the Phyllactinia spp. alnicola, betulae and carpini that they said were present in all or almost all Europe. Indeed, Ph. betulae and Ph. carpini were found in Bavaria [28], but Ph. alnicola was not found in our part of Europe. We all did, however, find the genuine Ph. guttata on Corylus spp. Species of Phyllactinia probably prefer much colder regions. For instance, in Wales, Chater and Woods [54] recorded 12 Phyllactinia species. Although Scholler et al. [55] had described how P. fraxinicola on Fraxinus was introduced into Europe from Eastern Asia, neither this nor any other recently introduced species of Phyllactinia were recorded in our four country surveys.
As explained above, earlier workers recognised the sections of Podosphaera (sects Podosphaera and Sphaerotheca) as full genera. For instance, on trees and shrubs in the Czech Republic, Klika [14] described two Sphaerotheca and three Podosphaera species whilst Slovakia [21] had four Sphaerotheca and four Podosphaera species. By comparison with Klika's survey [14], the probably newly occurring species in our republic were: Po. amelanchieris, Po. (Sph.) pruinosa and Po. (Sph.) spiraeae and they were also found in Bavaria by Bresinsky [28]. The discrepancy with Klika's list is not surprising, because the hosts (respectively Amelanchier, Rhus and Spiraea) are grown here as ornamentals, and would not have been present in Klika's time. All three species are missing in both the Hungarian [29], and Polish check-lists [27]. Later however, Piątek [34] confirmed Po. spiraeae in Poland.
The most complicated situation is with the genus Erysiphe. As mentioned above, the previous genera Microsphaera and Uncinula were recently made part of the genus Erysiphe; while another problem is the ever-narrowing definitions of particular species. On trees Klika [14] described four Uncinula spp. (one of them is now Sawadaea on Acer), 4 Microsphaera spp. (one is now Arthrocladiella on Lycium) and 2 Trichocladia spp. (now Erysiphe spp.).
A complicated situation arises when more than one powdery mildew species can infect one host plant. This is the case with Salix (with E. adunca and E. capreae), Quercus (with E. alphitoides and E. hypophylla), Acer (with S. bicornis and S. tulasnei), Catalpa (with Erysiphe catalpae, E. elevata and Neoerysiphe galeopsidis), Cornus (with Erysiphe pulchra and E. tortilis) and Syringa (with E. syringae and E. syringae-japonicae). Accurate and experienced interpretations of microscopical, as well as molecular analyses, are needed to fully appreciate the results of the valuable research on these species. For instance, the powdery mildews of Erysiphe genus on legumes represent one of the taxonomically most complicated groups within this genus as highlighted by the revision of Braun et al. [56].

Conclusions
Despite the problems with the naming of powdery mildews on so wide a range of host species, our study both confirmed previously described species and revealed some new records for our country, i.e., Podosphaera (sect.