First records of powdery mildew fungi (Erysiphales) on medicinal plants in Taiwan

Background Production of medicinal plants in Taiwan is not only hampered by international market competition, but also lack of knowledge of their pathogens, such as powdery mildew fungi (Erysiphales, Ascomycota). Records of these fungi in Taiwan originate from few researchers for the last one hundred years and are still incomplete. Since powdery mildews in tropical/subtropical environments rarely develop the sexual stages with morphologically diagnostic characteristics, internal transcribed spacer sequences (ITS) of the ribosomal RNA genes obtained from the asexual stages have become important modern tools for species identification. Results Powdery mildews on medicinal plants from educational and ornamental plantations in Taiwan were identified based on the anamorph morphology and ITS sequences. Four powdery mildews on medicinal plants are new records for Taiwan, Arthrocladiella mougeotii on Lycium chinense, Erysiphe glycines on Pueraria lobata, Erysiphe lespedezae on Bauhinia sp., Desmodium caudatum, and Uraria crinita, and E. lonicerae on Lonicera japonica. Eryngium foetidum is a new host for Erysiphe heraclei hitherto known on other host plants in Taiwan. Eryngium foetidum and Uraria crinita are new host plants for powdery mildews worldwide. Only specific field collection of the pathogens yielded the new records, not checking plant specimens in a phanerogam herbarium. The pathogens did not cause death of the host plants, but appeared to enhance stress by infection of mature leaves. Conclusions Taxonomic study of powdery mildews in Taiwan results into new host records of economically important medicinal plants in Taiwan with potential consequences for plant production and quarantine and also shows that host records are quite incomplete worldwide. Although ITS sequences were useful for species identification, the lack of data for several species on the same host genus on the one hand and the low variation between closely related species on the other indicate the need for further study.


Background
For enhancing the domestic market as well as export of agricultural products from Taiwan, the Council of Agriculture of Taiwan (COA) has established the Good Agricultural Practices (GAP) and the COA-certified organic labeling system (https ://eng.coa.gov.tw). Particularly for trading products based on medicinal plants, the GAP system is an important competitive advantage compared to cheaper products from outside Taiwan. Lycium chinense is one of the twenty most important medicinal plants covered by the GAP system, but the domestic production is scarce compared to the import from mainland China (Taiwan Institute of Economic Research 2017, http:// www.biota iwan.org.tw/downl oad/struc ture4 /%E5%8A% 89%E4%BE%9D%E8%93%81/107/%E6%88%91%E5%9C %8B%E4%B8%AD%E8%8D%89%E8%97%A5%E7%94%9 F%E7%94%A2%E5%8F%8A%E9%80%B2%E5%8F%A3%E 6%A6%82%E6%B3%81%20(20171 2).pdf ). An important requirement in the COA/GAP certification is the transparent and reduced usage of pesticides. The COA also promotes the development of recreational farms and forests for tourism where medicinal plants and other crops Open Access *Correspondence: hhy88961@gmail.com; kirschner@ntu.edu.tw † Yu-Wei Yeh and Pei-Yi Chou contributed equally to this work 1 School of Forestry & Resource Conservation, National Taiwan University, Taipei, Taiwan 2 Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei, Taiwan are produced for demonstration and local distribution rather than for mass production. In these environments, pesticides are usually much more rarely used than in industrial agriculture. Under these conditions, pathogens may spread more easily so that knowledge of their occurrence is the first prerequisite for estimating potential risks and for control measures.
Powdery mildew fungi (Erysiphales) are obligate biotrophic plant pathogens which form conspicuous white mycelia on leaves, stems and fruits of specific angiosperm host plants (Braun and Cook 2012). In addition to the mycelium and anamorph features causing the symptoms, sexually formed ascomata provide morphological clues for species identification. Under tropical/ subtropical conditions, however, instead of rarely formed ascomata, asexual structures such as mycelial appressoria, conidiophores and conidia of the anamorph stage provide the single morphological features which are less diagnostic for morphological species identification (Piepenbring et al. 2011). Under such conditions, sequences of the internal transcribed spacer (ITS) of the ribosomal RNA genes have become an important tool for species identification (Takamatsu et al. 2015a, b, Meeboon and Takamatsu 2016, 2017a, b, 2020Khodaparast et al. 2019). Identification and documentation of Erysiphales in Taiwan started over one hundred years ago, followed by occasional records of few other mycologists and plant pathologists as compiled by Kuo (1998) and subsequent publications (Kirschner and Chen 2008;Kirschner and Liu 2014;Chen and Kirschner 2018;Yeh et al. 2018;Kirschner et al. 2020).
On some medicinal plants grown in a botanical garden, recreation and educational farms and as ornamentals, we found powdery mildews hitherto unrecorded for these plants worldwide or in Taiwan. The aim of this study was to identify and document the fungal species on their hosts, based on fresh field collection as well as on dried plant herbarium specimens.

Sampling and morphology
Plant specimens with powdery mildew symptoms were collected in educational and tourist gardens or farms as well as a university campus and kept at ca. 8 °C before study. For light microscopical study, fresh specimens were removed from the leaf surface with transparent tape, mounted in 5-10% KOH and observed at 1000× magnification. For presenting sizes, length and width, 30 structures were measured (if not stated otherwise) and given in the descriptions as mean value ± standard deviation with extreme values in brackets. Drawings were made free hand using scaled paper. Specimens were dried by pressing plant samples between paper and subsequent final drying on an electrical dryer. Representative specimens were deposited at the National Museum of Natural Science, Taichung, Taiwan (TNM). For evaluating the presence of powdery mildews on plant herbarium specimens, we checked 7 specimens of Eryngium foetidum, more than 30 of Lonicera japonica, 9 of Lycium chinense, and over 50 of U. crinita, all collected in Taiwan between the years 1917 and 2004 and kept in the herbarium of National Taiwan University (TAI). In cases where colonies of powdery mildew were assumed to be present on leaves, they were removed with a transparent tape and transferred to a lactophenol/cotton blue mounting for microscopical observation.

Molecular identification
For molecular identification with internal transcribed spacer (ITS) sequences of the nuclear ribosomal RNA genes (ITS 1, 5.8S rDNA, ITS2 and bordering short fragments of the 18S and 28S rDNA), total genomic DNA was isolated from freshly collected mycelium, conidiophores and conidia; the ITS was amplified, sequenced, and edited as in Wei and Kirschner (2017). ITS sequences were used for megaBLAST searches at GenBank and deposited in GenBank. Sequences from BLAST searches and Meeboon and Takamatus (2017a) were selected for confirming the megaBLAST identification of mildews on Fabaceae also with phylogenetic analyses. A phylogenetic estimate for the powdery mildew specimens from Fabaceae was based on an alignment made in MEGA7 with the default options of MUSCLE (Kumar et al. 2016). Except for trimming the ends of the alignment block, no further manipulations were done. The alignment was analyzed with the Maximum Likelihood method with the Kimura-2 parameter model (gamma-distributed) as best model and 1000 bootstrap replicates (Kumar et al. 2016). GenBank numbers and host names are given in Fig. 1.

Results
Five species of powdery mildews were found only in the anamorph stage on mature leaves of the host plants. The ITS sequences obtained in this study ranged between 570 and 680 bp. Based on anamorph morphology, host specificity, and identification with BLAST as well as ML analysis, four powdery mildews on medicinal plants are new records for Taiwan, Arthrocladiella mougeotii on Lycium chinense, Erysiphe glycines on Pueraria lobata, E. lespedezae on Bauhinia sp., Desmodium caudatum, and Uraria crinita, and E. lonicerae on Lonicera japonica. Eryngium foetidum is a new host for Erysiphe heraclei hitherto known on other host plants in Taiwan. Eryngium foetidum and Uraria crinita are new host plants for powdery mildews worldwide. The phylogenetic analysis of the powdery mildews on Fabaceae identified in this study confirmed the species identification, but also indicated well supported subclades in E. glycines and E. lespedezae (Fig. 1). The pathogens did not cause death of the host plants, but appeared to enhance stress by colonizing mature leaves. The fungi were only found on specifically mycologically collected specimens in the field, but not among over 80 plant herbarium specimens collected during the last 100 years in Taiwan. A documentation of the morphological and molecular identification of the five powdery mildew species is given below.
Notes The ITS sequence was 100% identical to over ten published ones exceeding 590 bp of Arthrocladiella mougeotii in GenBank. Arthrocladiella mougeotii is the single species of its genus and is almost globally distributed and limited to Lycium hosts (Braun and Cook 2012). In contrast to Farr and Rossman (2020) who list several powdery mildew species for Lycium species, only A. mougeotii and Phyllactinia chubutiana Havryl., S. Takam. & U. Braun are accepted for Lycium hosts in Braun and Cook (2012). Both fungi can be distinguished not only by different ITS sequences, but also the solitary conidia in Ph. chubutiana and catenate conidia in A. mougeotii. Fig. 3 Colonies amphigenous on leaves Hyphae smooth, 4-6 μm wide. Hyphal appressoria solitary or in opposite pairs, nipple-shaped or lobed. Conidiophores arising solitarily from middle of hyphal mother cell, slightly rough at base, composed of foot cell and 1-4 distal cells. Foot cell with basal septum at same level as upper surface of supporting hypha, straight or curved, (16-)20-30(-34) × (7-)8-10(-11) μm. Conidia solitary, ellipsoid-ovoid to doliiform, smooth-walled, (27- Notes When ITS sequences exceeding 600 bp were compared, there was 99% identity with 6 different bp between our specimen and five published specimens labeled as E. glycines in GenBank. Besides there are another four published specimens also labeled as E. glycines with 94% identity. In E. glycines, the sequences on Pueraria lobata form a well-supported subclade, which may represent E. puerariae R.Y. Zheng & G.Q. Chen, hitherto only known from P. lobata in mainland China for which hitherto no anamorph or DNA data are available.

Erysiphe heraclei DC. Fig. 4
Colonies amphigenous on leaves Hyphae smooth, 4-7 μm wide. Hyphal appressoria solitary or in opposite pairs, nipple-shaped or lobed. Conidiophores Notes There was 99% ITS sequence identity with 1 to 6 different bp between our specimen and over ten other published specimens labeled as E. heraclei in GenBank. Presently, it is not possible to distinguish between E. betae (Vaňha) Weltzien, E. heraclei, and E. malvae V.P. Heluta based on rDNA sequences (Takamatsu et al. 2015a). The three species are hardly distinguishable also in morphology (Braun and Cook 2012). Future study may reveal whether the species can be separated with additional markers or need to be put into synonymy. In this case, E. heraclei would have priority since it is the oldest available name.
Specimens Notes When ITS sequences exceeding 600 bp were compared, there was 98% to 100% identity with 1 to 7 Fig. 5 Erysiphe lespedezae on Bauhinia sp. (AKII 0055) a Powdery mildew symptoms on leaves. b Hyphae with nipple-shaped appressoria and conidiophores with curved foot cell. c Conidia. Scale bars = 10 μm different bp between our specimens and published specimens labeled as E. lespedezae in GenBank, whereas the identity with other species was 96% or lower, with more than 4 bp different. In a previous study (Liu and Kirschner 2013), we identified a specimen on B. variegata as Pseudoidium caesalpiniacearum (Hosag. & U. Braun) U. Braun & R.T.A. Cook because of the long foot cell of the conidiophore. Following the species concept of E. lespedezae on Bauhinia and other hosts in Meeboon and Takamatus (2017a), however, distinction between these species based on the foot cell can no longer be upheld. The sizes of the foot cells varied also considerably between our specimens on different hosts (see below).

Erysiphe lespedezae R.Y. Zheng & U. Braun on Desmodium caudatum Fig. 6
Colonies amphigenous on leaves Hyphae smooth, 2-5 μm wide. Hyphal appressoria solitary or in opposite pairs, nipple-shaped or lobed. Conidiophores arising solitarily from middle of hyphal mother cell, slightly curved and rough at base, composed of foot cell and 1-3 distal cells. Foot cell with basal septum at same level as upper surface of supporting hypha, straight or curved, Notes A powdery mildew was reported on D. caudatum in Taiwan, but not further identified (Hung 2016). The fungus was identified based on our previous collection from Yingge, New Taipei City (Wen 2019), but the material was not preserved. Fig. 7 Colonies amphigenous on leaves Hyphae smooth, 3-6 μm wide. Hyphal appressoria mostly solitary, nipple-shaped or lobed. Conidiophores arising solitarily from middle of hyphal mother cell, slightly curved and rough at base, composed of foot cell and 1-4 distal cells. Foot cell with basal septum at same level as upper surface of supporting  ship, Lavender Cottage, ca. 24.587072, 120.896818, ca. 80 m, 09 Nov 2018, AKII 0005 (TNM), ITS sequence GenBank MT703848; Taipei City, Shilin District, Medicinal Botanical Garden, 25.115751, 121.576885, ca. 220 m, 12 Nov 2018, AKII 0006 (TNM).

Erysiphe lespedezae R.Y. Zheng & U. Braun on Uraria crinita
Notes Uraria species have not yet been recorded as hosts of powdery mildews worldwide (Braun and Cook 2012;Farr and Rossman 2020). The foot cell of conidiophores was longer [(35-)45-78(-115) μm] than reported in Braun and Cook (2012): about 20-40 μm long), but Meeboon and Takamatus (2017a)  Erysiphe lonicerae DC. Fig. 8 Colonies amphigenous on green leaves Hyphae verruculose, 3-6 μm wide. Hyphal appressoria mostly single, rarely in opposite pairs, nipple-shaped, lobed or elongated. Conidiophores arising solitarily from middle of hyphal mother cell, straight or curved at base (only in one case curvature formed in cell distal to foot cell), cylindrico-clavate, verruculose, composed of foot cell and Notes When ITS sequences exceeding 600 bp were compared, there was 99% identity with 1 to 4 different bp between our specimen and seven published specimens labeled as E. lonicerae in GenBank, whereas the identity with other species was 98% or lower, with more than 10 different bp. Erysiphe lonicerae is known on different Lonicera species in Europe and East Asia (mainland China, Korea, Japan) and is a new record for Taiwan (Farr and Rossman 2020). Information about conidiophore morphology and DNA sequences do not exist, however, for two other Erysiphe species on Lonicera hosts in East Asia, namely E. lonicerae-ramosissimae (Tanda) U. Braun & Takam. and E. miurae (U. Braun) U. Braun & Takam. These data are also lacking for E. magnusii (S. Blumer) U. Braun & Takam. reported from Europe and Central Asia, and for E. caprifoliacearum (U. Braun) U. Braun & Takam. in America (Braun and Cook 2012).

Advances and limitations of molecular species recognition
Identification of powdery mildew fungi to species can be based on the traditional combination of host specificity and fungus morphology, particularly of the sexual stage, when sufficient data are available in a given area. Since the sexual stage is rarely found in the tropical/ subtropical environment in Taiwan and data are far from being sufficient for Erysiphales in Taiwan, ITS sequence-based identification is an important requirement for species identification of the powdery mildews. For obtaining DNA from powdery mildews, careful selection of clean, fresh specimens is necessary, since powdery mildews cannot be cultivated in vitro, are often contaminated by other microscopic fungi and DNA deteriorates quickly during drying specimens. In Erysiphales, the ITS is the single available barcode for species identification particularly through the accomplishments of Takamatsu (Takamatsu et al. 2015a, b;Meeboon and Takamatus 2016, 2017a, b, 2020. Five species of powdery mildews were identified here based primarily on ITS sequence data. The fungus recorded by Sawada (1959) as E. pisi DC. on Lespedeza bicolor in Taiwan, may actually be E. lespedezae. This is an example for using ITS sequence data for progress in former problematic identifications. In Erysiphales, the barcode gap of ITS sequences is often very narrow, which was also confirmed in our study with 99 to 100% sequence identities and a variation of 0 to 6 different bp found in the same species. The variation depends on the available data as well as the species, since, for example 6 different bp were still within the range of variability in E. heraclei, whereas for E. lespedezae the same number of different bp would indicate an overlap with other, closely related species. Because of the high identities between ITS sequences of related species, even in phylogenetic analyses it is sometimes to not possible to resolve the species which then still remain provisionally based on the traditional combination of host occurrence and fungal morphology (Chen and Kirschner 2018 Braun and Cook (2012). The two fairly well supported subclades in each of both clades indicate the presence of further two more species (Fig. 1). In E. glycines, the well-supported subclade formed by the two specimens on Pueraria lobata may represent E. puerariae, which is hitherto only known from P. lobata in mainland China and for which hitherto no anamorph or DNA data are available.
Since in the clade of E. lespedezae we could presently not find a correlation between distinct morphological characteristics such as the length of foot cell of the conidiophore or host specificity (e.g. Bauhinia hosts occur in both subclades), we preliminarily adopt the present broad species concept of E. lespedezae (Meeboon and Takamatsu 2017). In the future, perhaps one of the anamorph names for powdery mildews on Bauhinia hosts (see above) may reveal to be applicable to one of those subclades. In the example of E. lonicerae, we demonstrate that for several species on the same host genus, information of DNA data and anamorph morphology is lacking. Erysiphe heraclei is an example for illustrating the conflict between the ITS phylogeny and traditional taxonomy. Most recent new attempts to obtain DNA from herbarium specimens of Erysiphales and to develop alternative barcodes for this group of fungi are very promising for resolving these problems (Ellingham et al. 2019;Bradshaw and Tobin 2020).

Origin of host plants and detection of associated fungi
Except for L. chinense, the medicinal plants of this study are also valued as ornamentals (Hsueh and Yang 2011, 2016. Among these plants cultivated in Taiwan, D. caudatum, Lonicera japonica and U. crinita are native (Ohashi and Iokawa 2007). The parasitic fungi on these hosts may be native, but have escaped attention due to limited research of fungi. Lycium chinense was introduced from mainland China ca. 300 years ago (Wu et al. 2010), Arthrocladiella mougeotii has been introduced on its Lycium hosts from Eurasia to other countries and continents (Braun and Cook 2012) and also most likely from mainland China to Taiwan. Eryngium foetidum was introduced to Taiwan in 1926(Wu et al. 2010. Bauhinia species were also introduced in the 20th century for ornamental purposes (Wu et al. 2010). Bauhinia spp., L. chinense and E. foetidum have been classified as medicinal plants as well as potential weeds (Kumar and Chandrashekar 2011, Singh et al. 2019, Wu et al. 2010). The kudzu (P. lobata) is a native weed, which has become invasive in other countries so that pathogens may provide some potential for biological control . In contrast to another pathogen on kudzu leaves, Marasmius puerariae R. Kirschner , the powdery mildew E. glycines in its present broad species concept (see above) is not specific to kudzu, but can also infect economically valuable crops among the Fabaceae. Even if this powdery mildew on kudzu may be classified as more kudzu-specific species, the potential for control of kudzu would be low because of the rarity of the pathogen on kudzu.
In some plant parasitic fungi, such as smut fungi (Basidiomycota, Ustilaginomycotina), it is possible to retrieve new information of fungal distribution from phanerogam herbarium specimens (Piepenbring 2001). Our scanning of over 80 herbarium specimens of the four host plants in TAI for the accidental presence of powdery mildews, however, did not retrieve any fungi except for few saprobic molds. It is, therefore, not possible to conclude from plant specimens whether associated powdery mildews have escaped from attention in the past or have been introduced from abroad more recently. This result also shows the importance of specific mycological field study in order to complement the insufficient data of plant pathogenic fungi as well as of building up and curating professional mycological specimen collections.

Conclusions
Five species of powdery mildews on medicinal plants as new hosts from Taiwan were identified here based primarily on ITS sequence data. Our new records and the lack of DNA data as well as anamorph characteristic in several Erysiphe species show that knowledge of Erysiphales is far from being complete. Compared to plant biogeography, mycological exploration apparently suffers from more limitations concerning funding and numbers of researchers. Such study is relevant for estimating risks for quarantine and for growing medicinal plants under particular strategies of crop marketing.