New Records of Powdery Mildews from Taiwan: Erysiphe ipomoeae comb. nov., E. aff. betae on Buckwheat, and E. neolycopersici comb. nov. on Cardiospermum halicacabum

Abstract

Erysiphe is the largest genus of powdery mildews (PMs), a group of obligate plant pathogenic fungi. Exploration of biodiversity generally relies on regional surveys and our aim is to investigate the PMs in Taiwan. Collections of the fungi on five plant species around agricultural environments were subjected to morphological and molecular characterization, using both internal transcribed spacer (ITS) and β-tubulin gene (TUB2) regions for the phylogenetic analyses. Erysiphe ipomoeae comb. nov., a species able to infect Ipomoea obscura and I. aquatica demonstrated by pathogenicity tests, has been neotypified. The two buckwheat species, Fagopyrum esculentum and F. tataricum, are found to be hosts of E. aff. betae. These results suggest that hosts in some plant families can be infected by more than one Erysiphe pathogen, e.g., Convolvulaceae by E. ipomoeae and E. convolvuli and Polygonaceae by E. polygoni and E. aff. betae, respectively. In addition, phylogenetic analyses of PMs on Cardiospermum halicacabum and tomato belonging to the E. aquilegiae complex are allocated under E. neolycopersici comb. nov. This extends the potential host range of E. aquilegiae complex to the plant family Sapindaceae. We conclude that awareness of the host associations of PMs can potentially benefit crop disease management.

1. Introduction

Powdery mildews (PMs) are a group of obligate biotrophic plant pathogenic fungi in Erysiphaceae within Helotiales, Leotiomycetes, and Ascomycota [1]. There are about 900 known species worldwide [2,3,4] and the estimated diversity of PMs would be far more than that number [5]. This group of fungi can affect around 10,000 species of angiosperms, including numerous economically important plants, such as crops, cereals, vegetables, fruits, and ornamentals [2,6]. The symptoms of the disease appear as conspicuous white, powdery mycelial and spore masses on the above-ground parts of the plants, e.g., the leaves, buds, stems, flowers, and fruits. Severe infections can cause leaf drop, withering, reduction in growth, and even death of plants, resulting in substantial economic losses [7,8].

Erysiphe is the largest genus comprising about half of all the powdery mildew species [2,9,10]. This genus has been classified into five practical morphological sections [2] and further research has divided Erysiphe into phylogenetic lineages, such as Microsphaera and Uncinula lineage [9,10]. The basic morphological characteristics of Erysiphe are the anamorph (asexual morph) of Pseudoidium-type conidiogenesis (conidia formed singly) and the teleomorph (sexual morph) of polyascal chasmothecia [2,11]. The anamorphic genus name Pseudoidium is still in use where the teleomorph had not been found, however, these species could also be assigned under its teleomorphic genus name, Erysiphe [12].

Many investigations about Erysiphe have been carried out in different geographic regions. Limited by the obligatory parasitic, nonculturable nature of these fungi, exploration of the biodiversity of PMs in a region generally relies on local surveys. The first compilations of PMs in Taiwan are in the pioneer works of Sawada, Kaneyoshi [13,14]. Two pieces of literature in the 1910s recorded the currently known Erysiphe spp. on at least 15 plant species, e.g., wheat, pea, buckwheat, tomato, and grape, that revealed the earliest Erysiphe specimens in Taiwan, collected by Suzuki, Rikiji, could be dated back to 1907 [13,14]. Subsequent studies related to this group of PMs in Taiwan can be referred in Kuo [15], Braun and Cook [2], and the List of Plant Diseases in Taiwan [16]. Additional investigations in recent years are those in Kirschner and Liu [17], Kirschner [18], Wang et al. [19,20,21], Kirschner et al. [22], Yeh et al. [23], and Xiao et al. [24].

Presently, using both morphological and phylogenetic approaches is routine for accurate fungal identification and in taxonomic studies. The ribosomal DNA internal transcribed spacer (ITS) region is a standard sequence identifier for PMs [25,26,27]. However, the ITS marker might be insufficient for species discrimination [9,26]. In this study, specimens of Erysiphe in Taiwan were collected to clarify the species identities and phylogeny of PMs on the plant genus Ipomoea, Fagopyrum, and Cardiospermum. We obtained not only the ribosomal DNA sequences including the ITS and large subunit rRNA gene (28S rDNA) region but also the β-tubulin gene region (TUB2), another protein-coding gene region potentially useful in the phylogenetic analyses [26]. Our initial objective was to clarify the taxonomy of Pseudoidium ipomoeae (J.M. Yen and Chin C. Wang) U. Braun and R.T.A. Cook, a powdery mildew on obscure morning glory (Ipomoea obscura (L.) Ker Gawl.) occurring in Taiwan and Thailand [2,28,29], and resolve the taxonomic confusion associated with PMs on Convolvulaceae [27]. In addition, powdery mildew infections on buckwheats and herbaceous plants were found at high incidences (>50%) in an agricultural environment near the Taichung District Agricultural Research and Extension Station (TDARES) in Changhua County, Taiwan. These PMs were identified based on the morphology and molecular analyses. Pathogenicity tests were also conducted. Moreover, the host ranges and the implication on crop production were discussed in this study.

2. Materials and Methods

2.1. Sampling and Morphology

Diseased leaves bearing powdery mildew symptoms on obscure morning glory (I. obscura), water spinach (I. aquatica Forssk.), common buckwheat (Fagopyrum esculentum Moench, cv. Taichung No. 5), Tartary buckwheat (F. tataricum (L.) Gaertn., cv. Taichung No. 2) [30], and lesser balloon vine (Cardiospermum halicacabum L.) were collected in central Taiwan between 2021 and 2022. The samples were temporarily kept at 6 °C until morphological observations, extraction of nucleic acids, and pathogenicity tests were carried out. Voucher specimens were deposited at the herbarium of the National Museum of Natural Science (TNM) in Taichung, Taiwan. For light microscopical examination, fungal structures from the fresh leaf surfaces were mounted in distilled water and observed using a Leica DM2500 light microscope (Leica Microsystems, Wetzlar, Germany) under 400× magnification. At least 10 conidiophores, foot cells, and 50 conidia were measured for each of the samples whenever possible. Photographs were taken using a Leica MC190 HD camera (Leica Microsystems, Wetzlar, Germany) equipped on the microscope.

2.2. Molecular Phylogeny

For molecular analysis, the total genomic DNA of the fungi was extracted from the mycelia with a Plant Genomic DNA Extraction Miniprep Kit (Viogene, New Taipei, Taiwan) following the manufacturer’s instructions. The sequences of ITS, 28S rDNA, and TUB2 were amplified using primer pairs ITS1/PM6 and PM5/ITS4 [31,32] for ITS, PM3/TW14 [31,33] for 3′ half of ITS and 28S rDNA, and BtubF5/BtubR7a or BtubF5b/BtubR7a [26] for TUB2. The PCR conditions had an initial denaturing step at 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 sec, 55 °C for 30 sec, and 72 °C for 2 min, and a final step at 72 °C for 5 min. Alternative PCR conditions were used while the initial PCR amplification failed: denaturation at 95 °C for 5 min, followed by 37 cycles of 95 °C for 30 sec, 55 °C for 1 min, and 72 °C for 1.5 min, and final extension at 72 °C for 5 min. The amplicons were sequenced in both directions using the same primer sets at Tri-I Biotech, Inc., New Taipei, Taiwan. Additional sequences of the powdery mildew on tomato, other newly collected Erysiphe species, and those from our previous collections [24] were also obtained in this study. The resulting ITS and 28S rDNA sequences were combined (ITS + 28S), then the ITS + 28S and TUB2 sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 28 January 2022).

The sequences of the PMs used in this study are listed in

Table 1. List of powdery mildew isolates used in the phylogenetic analyses.

Species	Host Family	Host Species	Voucher 1	Accession Number 2		Reference
				ITS	TUB2
Erysiphe alphitoides	Convolvulaceae	Ipomoea obscura	HNIO-18	MN186769	n.a.	Pan et al. [34]
	Fabaceae	Wisteria sinensis	OE2014PM13	KY660754	KY786697	Ellingham et al. [26]
	Fagaceae	Quercus robur	OE2014PM103CS	KY660753	KY786753	Ellingham et al. [26]
	Fagaceae	Q. robur	MUMH1442	AB257430 *	n.a.	Takamatsu et al. [35]
	Fagaceae	Q. robur	MUMH631(t)	AB292708	n.a.	Takamatsu et al. [36]
E. aquilegiae	Ranunculaceae	Aquilegia sp.	BCRU00359	LC009883 *	n.a	Takamatsu et al. [9]
	Ranunculaceae	A. vulgaris	OE2014PM147CS	KY653191	KY786773	Ellingham et al. [26]
	Ranunculaceae	Clematis apiifolia	MUMH277	LC009938 *	n.a	Takamatsu et al. [9]
	Ranunculaceae	Ranunculus repens	OE2014PM109	KY653197	KY786756	Ellingham et al. [26]
E. betae	Amaranthaceae	Beta vulgaris	KUS F29140	KX574674	n.a.	Joa et al. [37]
	Amaranthaceae	B. vulgaris	WW-14	DQ164433	n.a.	Francis et al. [38]
	Amaranthaceae	B. vulgaris subsp. cicla	SE1R	KY399969	n.a.	Vakalounakis and Kavroulakis [39]
	Amaranthaceae	Dysphania ambrosioides	MUMH0395	LC009946 *	n.a.	Takamatsu et al. [9]
E. aff. betae	Polygonaceae	Fagopyrum esculentum	TNM F0034604	OM033347 *	OM056697	This study
	Polygonaceae	F. esculentum	TNM F0035414	OM368494 *	OM423597	This study
	Polygonaceae	F. esculentum	TNM F0035415	OM368495 *	OM423598	This study
	Polygonaceae	F. tataricum	TNM F0034605	OM033348 *	OM056698	This study
E. convolvuli	Convolvulaceae	Calystegia sepium	MUMH7001	LC270835 *	n.a.	Abasova et al. [11]
	Convolvulaceae	Ca. sepium	OE2014PM62CS	KY660794	KY786726	Ellingham et al. [26]
	Convolvulaceae	Ca. silvatica	OE2014PM48CS	KY660793	KY786719	Ellingham et al. [26]
	Convolvulaceae	Convolvulus arvensis	UC1512307	AF011298	n.a.	Saenz and Taylor [40]
	Convolvulaceae	C. arvensis	VPRI 20227	AF154327	n.a.	Cunnington et al. [25]
	Convolvulaceae	I. aquatica	HMUT1009256	KJ885178	n.a.	Abasova et al. [11]
	Convolvulaceae	I. tricolor	n.a.	EU621391	n.a.	Takamatsu et al. [9]
E. convolvuli var. convolvuli	Convolvulaceae	C. arvensis	MUMH7048	LC328325	n.a.	Abasova et al. [11]
E. heraclei	Apiaceae	Heracleum sphondylium	OE2014PM52	KY660792	KY786720	Ellingham et al. [26]
	Apiaceae	H. sphondylium	OE2014PM65CS	KY660830	KY786728	Ellingham et al. [26]
E. ipomoeae	Convolvulaceae	I. obscura	TNM F0035015(t)	OM033351 *	OM056701	This study
	Convolvulaceae	I. obscura	TNM F0035013	OM033349 *	OM056699	This study
	Convolvulaceae	I. obscura	TNM F0035014	OM033350 *	OM056700	This study
	Convolvulaceae	I. obscura	TNM F0035018	OM033354 *	OM056704	This study
	Convolvulaceae	I. obscura	MUMH2978	LC163910	n.a.	Meeboon and Takamatsu [29]
	Convolvulaceae	I. aquatica	TNM F0034602	OM033346 *	OM056696	This study
	Convolvulaceae	I. aquatica	TNM F0035410	OM368491 *	OM423594	This study
E. lespedezae	Fabaceae	Bauhinia variegata	TNM F0033672	MT471985	OM056688	Xiao et al. [24] and this study
	Fabaceae	B. variegata	TNM F0033677	MT471988	OM056693	Xiao et al. [24] and this study
	Fabaceae	B. variegata	TNM F0033678	MT471989	OM056694	Xiao et al. [24] and this study
	Fabaceae	B. blakeana	TNM F0033679	MT471990	OM056695	Xiao et al. [24] and this study
	Fabaceae	Desmodium caudatum	TNM F0033671	MT471984	OM056687	Xiao et al. [24] and this study
	Fabaceae	D. caudatum	TNM F0033675	MT471986	OM056691	Xiao et al. [24] and this study
	Fabaceae	D. caudatum	TNM F0033676	MT471987	OM056692	Xiao et al. [24] and this study
E. neolycopersici	Caricaceae	Carica papaya	n.a.	GU358451	n.a.	Tsay et al. [41]
	Sapindaceae	Cardiospermum halicacabum	TNM F0035016	OM033352 *	OM056702	This study
	Sapindaceae	C. halicacabum	TNM F0035412	OM368492 *	OM423595	This study
	Sapindaceae	C. halicacabum	TNM F0035413	OM368493 *	OM423596	This study
	Solanaceae	Solanum lycopersicum	Et-1(t)	AF229019	n.a.	Kiss et al. [42]
	Solanaceae	S. lycopersicum	UMSG2	KX776199 *	MCFK01008520	Wu et al. [43]
	Solanaceae	S. lycopersicum	MUMH66	LC009912 *	n.a.	Takamatsu et al. [9]
	Solanaceae	S. lycopersicum	TNM F0035404	OM368490 *	OM423593	This study
E. pedaliacearum	Pedaliacea	Sesamum indicum	KUS F30128	LC342963 *	n.a.	Shin et al. [44]
E. polygoni	Polygonaceae	Antigonon leptopus	R. Kirschner 4701	MK685172	n.a.	Wang et al. [19]
	Polygonaceae	F. esculentum	n.a.	KP076437	n.a.	Lu et al. [45]
	Polygonaceae	Polygonum sp.	OE2014PM85CS	KY660828	KY786741	Ellingham et al. [26]
	Polygonaceae	P. aviculare	OE2014PM120CS	KY660829	KY786760	Ellingham et al. [26]
	Polygonaceae	P. aviculare	MUMH7036	LC328322	n.a.	Abasova et al. [11]
E. quercicola	Anacardiaceae	Mangifera indica	TNM F0033673	OM033344 *	OM056689	This study
	Fagaceae	Q. phillyraeoides	MUMH124	AB193591	n.a.	Limkaisang et al. [46]
	Lauraceae	Cinnamomum camphora	TNM F0033674	OM033345 *	OM056690	This study
	Sapindaceae	Nephelium lappaceum	MUMH6769	MN081591 *	n.a.	Meeboon and Takamatsu [47]
E. sedi	Crassulaceae	Sedum aizoon	MUMH2575	LC010045 *	n.a.	Takamatsu et al. [9]
E. takamatsui	Nelumbonaceae	Nelumbo nucifera	TNS F-52102	AB916688 *	n.a.	Meeboon and Takamatsu [48]
G. cichoracearum	Asteraceae	Sonchus oleraceus	UMSG1	HM449077	MCBS01024998	Wu et al. [43]

¹ Type specimens are marked with (t) after the voucher names. ² Sequences obtained in this study are shown in bold. Sequences of ITS + 28S are marked with asterisks.

[9,11,19,24,25,26,29,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48]. To determine the phylogenetic relationships using ITS and concatenated sequences of ITS and TUB2, the sequences were aligned using the MAFFT v.7 online version (https://mafft.cbrc.jp/alignment/software/, accessed on 28 January 2022) with the L-INS-i strategy [49]. The best evolutionary models were estimated under the Akaike Information Criteria (AIC) by jModelTest v. 2.1.10 [50,51]. The phylogenetic trees were generated by the maximum-likelihood (ML) method. The ML analyses were performed by raxmlGUI v.2.0.6 [52] under general time-reversible model with gamma distribution plus invariant sites (GTR + G + I) for ITS and combined ITS and TUB2 sequences with 1000 bootstrap replications. ML bootstrap values ≥ than 70% were given at each node. Posterior probabilities (PPs) of Bayesian inference were determined by Markov Chain Monte Carlo sampling in MrBayes 3.2.7a [53]. Six chains with a temperature setting of 0.15 were run for 30 million generations, with trees being sampled every 1000 generations. The first 20% of the trees were discarded as part of the burn-in procedure. PPs ≥ than 0.95 were indicated at each node.

2.3. Pathogenicity Tests

Pathogenicity tests were conducted by gently pressing each of the fresh, powdery mildew-infected leaves onto the leaves of approximately two-week-old healthy plants that had been sprayed with water. (1) Four plants of I. obscura were inoculated with an infected leaf of I. obscura. (2) Six plants of I. aquatica were inoculated with an infected leaf of I. obscura. (3) Ten plants of I. aquatica were inoculated with an infected leaf of I. aquatica. (4) Two pots of F. esculentum cv. Taichung No. 5 and two pots of F. tataricum cv. Taichung No. 2, containing at least fifteen plants in each pot, were inoculated with an infected leaf of F. esculentum. (5) Two plants of C. halicacabum were inoculated with an infected leaf of C. halicacabum. The same number of non-inoculated plants in each pathogenicity test served as controls, except that controls of F. esculentum and F. tataricum were one pot for each. The inoculation tests were performed in a greenhouse of the TDARES between March and June 2021 (test 1–4) and in January 2022 (test 5), with average temperatures of 29.4 °C, 25.2 °C, 21.1 °C, 22.9 °C, and 20.0 °C, respectively. A repeated experiment of test 1 was conducted with the same method under an average temperature of 23.7 °C in November 2021. The plants were inspected about every three days to note the dates when the symptoms appeared after the inoculations. While the symptoms of the powdery mildew clearly appeared on the leaf surfaces, the leaves were collected for morphological observations and subjected to ITS sequence characterization as previously mentioned.

3. Results

3.1. Morphology

Morphological characteristics of the PMs on the plants of Convolvulaceae, Polygonaceae, and Sapindaceae in this study were consistent with those of the anamorph in the genus Erysiphe [2,54]. The conidia, formed singly (Pseudoidium type) on conidiophores [2,54], were ellipsoid–ovoid to cylindrical in shape. Chasmothecia were absent in the collections. Voucher specimens were deposited under TNM numbers F0035013-15, F0035018, F0035401-3, and F0035409 (on I. obscura), F0034602 and F0035410 (on I. aquatica), F0034604 and F0035414-5 (on F. esculentum), F0034605 (on F. tataricum), and F0035016 and F0035412-3 (on C. halicacabum). Some details of the vouchers are shown in Table 1.

3.2. Phylogentic Analyses

The nucleotide sequences of the ITS + 28S and TUB2 gene regions were determined for six specimens on the plant genus Ipomoea, four specimens on Fagopyrum, and three specimens on C. halicacabum. An additional three ITS sequences and ten TUB2 sequences were obtained from the other Erysiphe specimens (Table 1). They were further aligned with the sequences retrieved from the DNA database. Sequences of Golovinomyces cichoracearum (DC.) V.P. Heluta [43] were used as outgroups.

A best-scoring ML tree of ITS with a final optimization likelihood of −1646.264627 is shown in Figure 1. The PMs on I. obscura and I. aquatica from Taiwan were clustered with P. ipomoeae on I. obscura (MUMH2978) from Thailand [29]. Our samples on F. esculentum and F. tataricum were grouped in a clade with E. betae (Vaňha) Weltzien on other host plants belonging to Amaranthaceae [9,37], distinguished from those in the clade of E. heraclei DC. and E. polygoni DC. [11,19,26,45]. Besides, the PMs on C. halicacabum were grouped with species in the E. aquilegiae complex [9,44,48].

On the other hand, a best scoring ML tree inferred by the concatenated sequences of ITS and TUB2 with a final optimization likelihood of −3606.639810 is provided in Figure 2. In the phylogram, the PMs on Ipomoea in Taiwan were phylogenetically close to E. alphitoides (Griffon and Maubl.) U. Braun and S. Takam. and they were in a clade sister to E. quercicola S. Takam. and U. Braun; those on Fagopyrum clustered into a well-supported clade, clearly distinct from E. heraclei and E. polygoni; and those on C. halicacabum were placed within a clade comprising P. neolycopersici (L. Kiss) L. Kiss (Oidium neolycopersici L. Kiss) on tomato.

3.3. Pathogenicity Tests

PMs on I. obscura were successfully inoculated on I. obscura and I. aquatica, producing powdery mildew symptoms. White fungal colonies appeared on an inoculated leaf of I. obscura after three weeks post inoculation (wpi) (Figure 3l), whereas the mycelia were produced on leaves of all the treated I. aquatica at 1 wpi (Figure 4e). Using the powdery mildew on I. aquatica as an inoculum, white fungal patches were also produced on the leaves of I. aquatica at 2 wpi (Figure 4f). In the pathogenicity test of the powdery mildew on Fagopyrum, white colonies on inoculated leaves of F. esculentum and F. tataricum were observed on the fifth day post inoculation, as shown in Figure 5g,h. After 2 wpi, the incidences in all the inoculated pots exceeded 85% and the controls were symptomless. In the experiment of C. halicacabum, white colonies on inoculated leaves appeared after 1 wpi (Figure 6g). In the repeated I. obscura experiment under a cooler temperature, mycelial colonies appeared on the inoculated leaves at 2 wpi. The morphological and molecular characteristics of the pathogen on inoculated plants were consistent with those of the originals. All the control plants in each pathogenicity test remained symptomless during the experimental period.

3.4. Taxonomy

Erysiphe ipomoeae (J.M. Yen and Chin C. Wang) H. Y. Hsiao and Y. M. Shen, comb. nov.Figure 3 and Figure 4.

MycoBank: MB842410.

Basionym:Oidium erysiphoides f. ipomoeae J.M. Yen and Chin C. Wang, Rev. Mycol. 37(3): 138, [“1972”] 1973.

≡ Oidium ipomoeae (J.M. Yen and Chin C. Wang) U. Braun, Mycotaxon 25: 268, 1986.

≡ Pseudoidium ipomoeae (J.M. Yen and Chin C. Wang) U. Braun and R.T.A. Cook, CBS Biodiversity Series 11: 607, 2012.

Typification: (Holotype: TAIWAN, Fengshan, on Ipomoea obscura (L.) Ker Gawl., 5 May 1972, Yen and Wang (not preserved)) Neotype (designated here, MycoBank: MBT 10006013): Taiwan, Nantou, N23.921944, E120.697333, 90 m altitude, on Ipomoea obscura, 28 March 2021, H. Y. Hsiao and Y. M. Shen (TNM F0035015). Ex-neotype reference sequences: OM033351 (ITS); OM056701 (TUB2).

Description: (on I. obscura, TNM F0035015).

Mycelium amphigenous, mainly epiphyllous, white, effuse or in patches. Hyphae 4.3–8.8 μm wide. Hyphal appressoria mainly lobed to multilobed, sometimes nipple-shaped, solitary or in opposite pairs. Conidiophores 50.0–75.0 μm long, erect. Foot cells 27.5–40.0 × 6.3–10.0 μm, cylindrical, straight, followed by 1–2 shorter cells, forming conidia singly. Conidia 33.8–60.0 × 12.5–25.0 μm, ellipsoid–ovoid–subcylindrical, with a length-to-width ratio of 1.5–3.9. Chasmothecia absent.

Description: (on I. aquatica, TNM F0034602).

Mycelium amphigenous, mainly epiphyllous, white, effuse or in patches. Hyphae 4.8–9.5 μm wide. Hyphal appressoria mainly lobed to multilobed, sometimes nipple-shaped, solitary or in opposite pairs. Conidiophores 52.5–82.5 μm long, straight. Foot cells 28.8–45.0 × 8.8–12.5 μm, cylindrical, followed by 1–2 shorter cells, forming conidia singly. Conidia 30.0–58.8 × 12.5–25.0 μm, ellipsoid–ovoid–subcylindrical, with a length-to-width ratio of 1.6–3.4. Chasmothecia absent.

Material Examined: Taiwan, Nantou, on I. obscura, 28 March 2021, TNM F0035015 (neotype), GenBank: OM033351 (ITS), OM056701 (TUB2); Taiwan, Changhua, on I. obscura, 24 March 2021, TNM F0035013, GenBank: OM033349 (ITS), OM056699 (TUB2); Taiwan, Nantou, on I. obscura, 28 March 2021, TNM F0035014, GenBank: OM033350 (ITS), OM056700 (TUB2); Taiwan, Changhua, on I. obscura, 26 May 2021, TNM F0035018, GenBank: OM033354 (ITS), OM056704 (TUB2); Taiwan, Nantou, on I. obscura, 11 November 2021, TNM F0035401; Taiwan, Nantou, on I. obscura, 11 November 2021, TNM F0035402; Taiwan, Taichung, on I. obscura, 1 December 2021, TNM F0035403; Taiwan, Nantou, on I. obscura, 17 December 2021, TNM F0035409; Taiwan, Changhua, on I. aquatica, 22 February 2021, TNM F0034602, GenBank: OM033346 (ITS), OM056696 (TUB2); Taiwan, Nantou, on I. aquatica, 28 December 2021, TNM F0035410, GenBank: OM368491 (ITS), OM423594 (TUB2).

Known Distribution: Taiwan, Thailand [29], and India [2,55].

Notes: The powdery mildew on I. obscura was first discovered at Fengshan, Taiwan, on 5 May 1972 by Yen and Wang [28]. It was introduced under the name of Oidium erysiphoides f. ipomoeae J.M. Yen and Chin C. Wang and later replaced by the names O. ipomoeae (J.M. Yen and Chin C. Wang) U. Braun [56] and P. ipomoeae in Braun and Cook [2]. The morphology of our collections is generally in agreement with previous descriptions [2,28]. The teleomorph of the powdery mildew has never been found so far. Interestingly, Meeboon and Takamatsu [29] reported P. ipomoeae on two host plants, I. obscura and I. aquatica, in Thailand. A DNA sequence covering the ITS region of P. ipomoeae on I. obscura was determined (LC163910) [29] but that of the pathogen on I. aquatica was not available. The P. ipomoeae sequence from Thailand clustered with sequences from the PMs on I. obscura and I. aquatica in Taiwan in the phylogenetic analysis (Figure 1). Evidently, the newly obtained PMs on Ipomoea in this study are conspecific with P. ipomoeae, and moreover, the molecular phylogeny suggests that this powdery mildew should be allocated under Erysiphe. To the best of our knowledge, the holotype material may not be extent ([28] and personal communication with Uwe Braun) so that a neotype is needed. The powdery mildew is here neotypified by the specimen collected near the type locality with the name Erysiphe ipomoeae, and this may resolve the taxonomic confusion of Erysiphe on Ipomoea. In addition, the DNA sequences of E. ipomoeae on I. aquatica were generated in this study for the first time and this is the first report of the powdery mildew E. ipomoeae on I. aquatica in Taiwan.

Erysiphe aff. betae Figure 5.

Description: (on F. esculentum, TNM F0034604).

Mycelium amphigenous, often covering the entire surface of the leaves, effuse or in patches. Hyphae 3.3–7.9 μm wide. Hyphal appressoria lobed, solitary or in opposite pairs. Conidiophores 43.8–105.0 μm long, straight. Foot cells 27.5–46.3 × 6.3–8.8 μm, cylindrical, occasionally slightly curved–sinuous, followed by a cell of approximately the same length or 1–2 shorter cells, forming conidia singly. Conidia 36.3–58.8 × 16.3–22.5 μm, cylindrical to ovoid, with a length-to-width ratio of 1.7–3.6. Chasmothecia not observed.

Description: (on F. tatarcium, TNM F0034605).

Mycelium amphigenous, mainly hypophyllous, effuse or in patches. Hyphae 3.6–8.6 μm wide. Hyphal appressoria lobed, solitary or in opposite pairs. Conidiophores 42.5–118.8 μm long, straight. Foot cells 31.3–55.0 × 6.3–10.0 μm, cylindrical, occasionally slightly curved–sinuous, followed by 1–2 shorter cells, or one cell of equal or longer length, forming conidia singly. Conidia 37.5–55.0 × 17.5–27.5 μm, cylindrical to ovoid, with a length-to-width ratio of 1.6–3.1. Chasmothecia not observed.

Material Examined: Taiwan, Changhua, on F. esculentum, 3 March 2021, TNM F0034604, GenBank: OM033347 (ITS), OM056697 (TUB2); Taiwan, Changhua, on F. esculentum, 12 January 2022, TNM F0035414, GenBank: OM368494 (ITS), OM423597 (TUB2); Taiwan, Changhua, on F. esculentum, 12 January 2022, TNM F0035415, GenBank: OM368495 (ITS), OM423598 (TUB2); Taiwan, Changhua, on F. tataricum, 3 March 2021, TNM F0034605, GenBank: OM033348 (ITS), OM056698 (TUB2).

Notes: PMs on buckwheats in Taiwan were reported on F. esculentum and F. tatarcium as Microsphaera polygoni (DC.) Sawada [13,14] and Ischnochaeta polygoni (DC.) Sawada [57], respectively, and the fungal names have been changed to E. polygoni. Although the morphology of the PMs on Fagopyrum in this study was similar to previous descriptions of E. polygoni, the morphological and molecular characteristics showed a close relationship between these recent collections and E. betae. E. betae in Taiwan, first recorded under the anamorphic name O. cylindricum Sawada, was found on Dysphania ambrosioides (L.) Mosyakin and Clemants (Chenopodium ambrosioides L.) in the northern part of the island [57]. Our newly obtained ITS + 28S sequences of the PMs on Fagopyrum shared more than 99.7% sequence identities over a 1356 bp alignment to that of E. betae MUMH0395 (LC009946) on D. ambrosioides from Japan [9]. However, the ITS analyses may not be sufficient to resolve the complex around E. betae and E. heraclei and TUB2 sequences for E. betae on Beta spp. are currently not available, therefore, the powdery mildew on Fagopyrum in this study is herein named as E. aff. betae. Analogous to the case to compare the ITS sequences of sugar-beet PMs previously known as E. betae and E. polygoni and given the conclusion that they are supposed to be E. betae [38], the result of the phylogenetic analyses (Figure 1 and Figure 2) indicated that the powdery mildew diseases of buckwheats in this study were caused by Erysiphe species closely related to E. betae. To the best of our knowledge, this is the first report of the powdery mildew E. aff. betae on F. esculentum and F. tatarcium in Taiwan.

Erysiphe neolycopersici (L. Kiss) H. Y. Hsiao and Y. M. Shen, comb. nov. Figure 6.

MycoBank: MB843164.

Basionym:Oidium neolycopersici L. Kiss, Mycological Research 105 (6): 695, 2001.

(Typus: France, Aigue Mortes, on Lycopersicon esculentum, 1989, P. C. Nicot Et1 (BPI 747013-holotypus, HAL-isotypus). Living material maintained at INRA, Unité de Pathologie Végétale, Avignon, France.)

≡ Pseudoidium neolycopersici (L. Kiss) L. Kiss, CBS Biodiversity Series 11: 612, 2012.

Description: (on C. halicacabum, TNM F0035016, TNM F0035412-3).

Mycelium amphigenous, also on stems and capsules, effuse or in patches. Hyphae 4.3–8.3 μm wide. Hyphal appressoria nipple-shaped to lobed, solitary or in opposite pairs. Conidiophores 38.8–72.6 μm long, straight. Foot cells 21.3–50.0 × 7.1–12.5 μm, cylindrical, followed by 1–2 shorter cells, or one cell of equal or longer length, forming conidia singly. Conidia 25.0–50.0 × 12.5–25.0 μm, ellipsoid–ovoid–subcylindrical, with a length-to-width ratio of 1.5–3.6. Chasmothecia absent.

Material Examined: Taiwan, Changhua, on C. halicacabum, 14 April 2021, TNM F0035016, GenBank: OM033352 (ITS), OM056702 (TUB2); Taiwan, Nantou, on C. halicacabum, 1 January 2022, TNM F0035412, GenBank: OM368492 (ITS), OM423595 (TUB2); Taiwan, Nantou, on C. halicacabum, 2 January 2022, TNM F0035413, GenBank: OM368493 (ITS), OM423596 (TUB2); Taiwan, Nantou, on Solanum lycopersicum, 9 December 2021, TNM F0035404, GenBank: OM368490 (ITS), OM423593 (TUB2).

Notes: Phylogenetic analyses of the PMs on C. halicacabum and tomato revealed that they should be allocated to Erysiphe. They are assignable to E. neolycopersici, which has priority over the previous anamorph-typified names. The morphological characteristics of the powdery mildew on C. halicacabum generally matched those of the powdery mildew on tomato (P. neolycopersici) [2]. The phylogenetic relationships inferred by the ITS sequences showed the powdery mildew on C. halicacabum nested together with PMs on tomato in the clade of E. aquilegiae complex [9] (Figure 1) and differed by only one base from the type sequence (AF229019) of O. neolycopersici Et1 [42]. In addition, the ITS and TUB2 sequences of the powdery mildew on C. halicacabum showed high sequence identities with those of PMs on tomato (differ by only two bases in the aligned dataset) and they formed a monophyletic clade in the phylogenetic tree (Figure 2). Consequently, the powdery mildew on C. halicacabum is here identified as E. neolycopersici. E. neolycopersici was originally found on leaves and stems of tomato [2,42], further recorded on other solanaceous plants [9,58,59,60,61], and on hosts in a variety of plant families such as Caricaceae [41,62], Euphorbiaceae [63,64], Moringaceae [65], and Fabaceae [66]. There are only a few records of PMs on the family Sapindaceae, mainly on woody plants [67]; and the recorded PMs on the herbaceous plant genus Cardiospermum were Oidium spp. on C. microcarpum Kunthand and C. velutinum Hook. and Arn. (synonym of C. grandiflorum Sw.) in Jamaica and Argentina, respectively [6]. To the best of our knowledge, this is the first report of C. halicacabum as the host of E. neolycopersici.

4. Discussion

4.1. Erysiphe ipomoeae and E. convolvuli, the Two Distinct Species Causing Powdery Mildews on Convolvulaceae

In addition to E. ipomoeae (previously Pseudoidium ipomoeae), another species in the same genus, E. convolvuli DC., is commonly known to cause the powdery mildew disease on the plant family Convolvulaceae [2,11,27,54]. Bolay et al. [27] observed E. convolvuli on I. lobata (Cerv.) Thell. in Switzerland and doubted that P. ipomoeae and E. convolvuli may be conspecific. Probably due to the unclear relationships of the anamorph of PMs on Ipomoea [2], this study further noted that sequence analyses of PMs on Ipomoea are urgently necessary [27]. In this perspective, the new powdery mildew collections on I. obscura and I. aquatica in Taiwan, where the fungus on I. obscura was first discovered, provided valuable information to this issue.

Our phylogenetic analyses confirmed that E. ipomoeae is obviously distinguished from E. convolvuli in the phylogenetic trees (Figure 1 and Figure 2). The results revealed that at least two Erysiphe spp., E. ipomoeae and E. convolvuli, were able to infect Ipomoea in the plant family Convolvulaceae, supporting the speculation that more than one powdery mildew species may occur on Ipomoea spp. [27]. Recently, a study reported the powdery mildew E. alphitoides on I. obscura in China [34] but the sequence of their sample (HNIO-18) was closer to E. ipomoeae (Figure 1). It appears that E. ipomoeae may occupy a wider geographic range in East and Southeast Asia. In addition, other Erysiphe spp. have been reported to occur on plants of the Convolvulaceae, such as E. cruciferarum Opiz ex L. Junell and E. heraclei [6,25,68], and identifications of other collections on convolvulaceous hosts remained unclear and could only be referred to as Erysiphe sp. or Oidium sp. [69].

Erysiphe ipomoeae has already been confirmed on I. obscura and I. aquatica in Taiwan and Thailand [29], but it may well be that this species also occurs on other hosts in Convolvulaceae in India, Myanmar, and other parts of Asia [2,55,70]. On the other hand, E. convolvuli has hitherto been reported on the plant genus Calystegia, Convolvulus, and Ipomoea of Convolvulaceae in Asia, Africa, Europe, and America [2,27,54,67,71,72,73]. In regards to the geographic ranges of the main convolvulaceous plant-infecting PMs, E. ipomoeae is known to be distributed in tropical to subtropical regions in Asia, whereas E. convolvuli has a worldwide distribution, especially in the temperate regions.

4.2. Powdery Mildews on Buckwheat and the Host Range of Erysiphe betae

The present study reports the first occurrence of E. aff. betae associated with the powdery mildew diseases of F. esculentum and F. tatarcium. Previously, PMs recorded on buckwheats were referred to as E. polygoni (including E. polygoni var. fagopyri, E. polygoni var. kailashi, and E. polygoni var. rumicis) and Oidium sp. [2,54,67]. The identifications were mainly based on morphological traits and the host association, however, Lu et al. [45] acquired an ITS fragment of E. polygoni on F. esculentum, confirming the pathogen–host relationship molecularly. That ITS sequence (KP076437) was grouped with those of E. polygoni on other polygonaceous hosts, whereas the collections of PMs on buckwheats in this study are apparently separated from this group (Figure 1). The result revealed that in the case of Fagopyrum, more than one powdery mildew species of Erysiphe may infect species of this genus, and this scenario is analogous to those on Ipomoea (host of E. ipomoeae and E. convolvuli), Bauhinia (host of E. lespedezae R.Y. Zheng and U. Braun and E. quercicola) [24,74,75], Quercus and Mangifera (host of E. quercicola, E. alphitoides, and other species) [2,67,76,77], etc. The discovery of E. aff. betae on buckwheats raised the question whether some of the previous regional records of E. polygoni on Fagopyrum have to be reidentified as powdery mildew species other than E. polygoni, similar to that in Francis et al. [38]; and if E. betae and E. polygoni could occupy an overlapping niche on the same host in a limited spatial range, however, what is the proportion of each of the species? The occurrence frequency of E. quercicola and E. alphitoides on infected mango [76] and oak [78] provided examples of the possible distribution of the PMs. Various proportions of powdery mildew species may be detected on the same host according to different locations, sampling dates, infected organs, host developmental stages, and environmental factors at multi-scales [76,78]. It is, therefore, worthwhile to obtain more samples of PMs on buckwheats of a wider range for detailed characterization in future research.

The recorded host range of E. betae covers the plant genera Beta [2,37,38,54,79], Dysphania (=Chenopodium p.p.) [2,80,81], and Spinacia [2,81] in Amaranthaceae, Euphorbia of Euphorbiaceae, Phaseolus, Pisum, and Vigna of Fabaceae [82,83]. However, the latter host references are doubtful and not confirmed by sequence analyses. The finding of E. aff. betae on Fagopyrum of Polygonaceae lends support to the idea that E. betae might have a wider host range than that of our previous understanding. Thus, the host range of E. betae requires further validation through additional molecular analyses and cross-inoculation studies. It is likely that the host–range breadths of the PMs could ideally fit in the pattern of modularity (but not strict nestedness) in the infection matrices in Morris and Moury [84].

4.3. Erysiphe aquilegiae Complex Needs Further Exploration

The phylogenetic analyses in this study verify that the powdery mildew on C. halicacabum in Taiwan is a member of the E. aquilegiae complex, a group comprising many closely allied species that are indistinguishable by the ITS data [9,44,69]. The type hosts of E. aquilegiae DC. (var. aquilegiae) and E. aquilegiae var. ranunculi (Grev.) R.Y. Zheng and G.Q. Chen are the ranunculaceous species Aquilegia vulgaris L. and Ranunculus cf. muricatus L., respectively [2], whereas the E. aquilegiae clade [9] encompasses many species, i.e., E. neolycopersici, P. hortensiae (Jørst. ex S. Blumer) U. Braun and R.T.A. Cook, E. sedi R.Y. Zheng and G.Q. Chen, E. pileae U. Braun, E. hommae U. Braun, E. takamatsui Y. Nomura, E. chloranthi (Golovin and Bunkina) U. Braun, E. macleayae R.Y. Zheng and G.Q. Chen 1981, E. circaeae L. Junell, E. knautiae Duby, E. euphorbiae Peck, P. boroniae (Crooks) U. Braun and R.T.A. Cook, and E. catalpae Simonyan, on a wide range of hosts. More recently, additions to species belonging to the E. aquilegiae complex, such as E. pedaliacearum (H.D. Shin) H.D. Shin on sesame [44], E. asclepiadis U. Braun and V. Kumm. on Asclepias tuberosa L., E. tortilis (Wallr.) Link on Cornus spp., and two Erysiphe spp. on Astilbe × arendsii H.Hara and Calystegia sp. [69], provided insight into the discussions of this group. One of the viewpoints that an increasing number of new collections which pertain to the E. aquilegiae complex could be found [69] is compatible with our finding of E. neolycopersici on C. halicacabum in Taiwan. This extends the understanding of the potential host range of E. neolycopersici and the E. aquilegiae complex to the plant family Sapindaceae. Previous studies have demonstrated that the powdery mildew on tomato (E. neolycopersici) could cause infections on solanaceous hosts and alternative hosts within the other 12 plant families [85] and Sedum alboroseum Baker (synonym of Hylotelephium erythrostictum (Miq.) H. Ohba) (Crassulaceae) [86] through artificial inoculation. Well-designed cross-inoculation tests should be carried out to validate the pathogen–host relationship of this polyphagous powdery mildew. Beside the biological approach, using additional phylogenetic markers may provide better resolutions in the phylogenetic trees [44,69]. Our result supported the use of TUB2, a DNA marker offering additional diagnostic utility to powdery mildew [26], to distinguish species within the E. aquilegiae clade. However, it also revealed that TUB2 sequences had limitations in resolving the E. alphitoides complex [36] (Figure 2). It is suggested that the increasing availability of multiple independent DNA barcodes [26] and the announcement of genome resources [43,87,88] will help the species delimitation and facilitate the exploration of the evolutionary history of the PMs.

4.4. Perennation of Powdery Mildews in the Subtropics and the Impact on Crop Production

This study highlights three PMs on several herbaceous wild plants and crops around the agricultural environment in the subtropical region of Taiwan. The existence of the pathogens on the alternative hosts may play a key role in perennation, the process of surviving from one season to another, in this group of obligatory parasites. Perennation of PMs under unfavorable conditions such as cold winter and hot summer temperatures may occur by the sexual reproduction of chasmothecia, infection in dormant buds, and persistence as mycelia [89]. Examples include overwintering of hop and cucurbit PMs through bud perennation [90,91] and chasmothecia [92], respectively, and over summering of wheat powdery mildew through chasmothecia [93]. In the present study, the occurrence periods of the PMs in the winter and spring imply they prefer mild weather instead of excessively high temperature (up to 35 °C) in summer, and it is suggested that the PMs in the subtropical regions may stay in a quiescent state in their hosts during hot summers. However, chasmothecia are rarely formed in subtropical and tropical environments [23,47,77,94]. Thus, the reservoirs of the PMs on all their possible hosts as mycelia or other structures are crucial for their survival and for the infection in the next season.

Considering the crop disease management related to the PMs in this study, if there exist essential reservoirs of the pathogen for each of the powdery mildews, the presence of alternative hosts in the vicinity may pose a significant impact on the disease development of the target crops. Taking water spinach as an example, the vegetable is fast growing [95], can be cultivated year round (up to 12–13 times a year), and the growing period is 2–4 weeks in Taiwan [96]. The harvest of the vegetable in short terms may interrupt the buildup of E. ipomoeae populations on the leaves, and this may explain why the powdery mildew disease on water spinach was not noticed in Taiwan [16]. However, an overlooked flourish of I. obscura nearby the vegetable field may serve as additional reservoir of the inocula according to our study results. The effect of lengthening the presence time of hosts is comparable with lengthening the growing season for plants, which would promote severe powdery mildew infections [97]. The conclusion is also in line with Mulpuri et al. [98], who state that continuous cultivation of crop, and even the existence of collateral hosts and weeds, will lead to increased inoculum load of PMs. It is suggested that more comprehensive knowledge of the PMs with a wide host range would be beneficial to management of crop diseases, e.g., awareness of the potential host associations of E. ipomoeae, E. betae, and E. neolycopersici may bring about improvements in cultural practices to reduce the inoculum. Finally, accurate recognition of the pathogen species and their hosts could minimize the risk of cryptic invasion [78] of PMs in a regional scale and in agricultural systems.