Erysiphe ruyongzhengiana sp. nov., a new powdery mildew species on Aristolochia debilis, belonging to the Erysiphe aquilegiae clade

Abstract

Powdery mildew was found on Aristolochia debilis (Aristolochiaceae) in Jiangsu Province and Shandong Province, China. This fungus is characterized by having long conidiophore foot-cells which are straight or curved at the base, and chasmothecia with numerous appendages. Phylogenetic analysis using internal transcribed spacer sequences showed that five sequences on A. debilis determined in this study and two sequences retrieved from Erysiphe sp. on A. debilis formed an independent cluster within the Erysiphe aquilegiae clade with 58% bootstrap support. This powdery mildew differs from allied species of the E. aquilegiae clade in producing longer conidia and conidiophores with longer foot-cells, which are often curved at the base. Morphological observations and molecular phylogenetic analysis revealed a new powdery mildew species, described as Erysiphe ruyongzhengiana.

The family Aristolochiaceae comprises eight genera and about 600 species, mainly distributed in tropical, subtropical or temperate regions (Kelly & González, 2003). There are about 350 species of the genus Aristolochia in the world (Flora of China Editorial Committee, 2001). Stems, leaves, fruits and roots from plants of this genus are used in traditional Chinese medicine (Yu, Huang, & Cheng, 1995). They take effect as medicine relieving pain, cough and asthma, lowering blood pressure, and so on (Wang, 2018). The Aristolochia species contain aristolochic acid, which may cause severe renal failure (Bogusz & Maciej, 2008). Due to its various therapeutic effects in traditional Chinese medicine, Aristolochia species attract great attention in China.

There are only a few records of powdery mildew species on Aristolochia spp. Erysiphe aristolochiae (Y.N. Yu) U. Braun & S. Takam., belonging to Erysiphe sect. Microsphaera (Braun & Cook, 2012), is characterized by having appendages with tightly and regularly branched apices, with obtuse, truncated and recurved tips. This species is known to be endemic to China and recorded only on Aristolochia heterophylla Hemsl. (Yu & Lai, 1981). The record of Leveillula taurica (Lév.) G. Arnaud (s. lat.) on A. bracteata Retz. from Sudan is unclear and in need to be re-examined (Amano, 1986). The conidiophores of L. taurica usually emerge through stomata (previously assigned to the anamorph genus Oidiopsis), and the chasmothecial appendages of the very large chasmothecia are mycelioid, simple or often irregularly branched, and relatively very short (Braun & Cook, 2012). Zhu, Ji, Zhao, Chai and Li (2020) described the asexual characteristics of a powdery mildew on A. debilis Siebold & Zucc.(chasmothecia were not found). The morphological characteristics showed that the fungus concerned belongs to Pseudoidium (asexual morph of the genus Erysiphe). The ITS sequence of this fungus is 99 % similar to other sequences of taxa of the E. aquilegiae DC. clade. This powdery mildew has been tentatively identified as Erysiphe sp.

Leaves of A. debilis with obvious powdery mildew symptoms have recently been collected in China at different places in the Jiangsu Province and Shandong Province. Microscopical observations and molecular phylogenetic analysis using internal transcribed spacer (ITS) revealed that this fungus is an undescribed species belonging to the genus Erysiphe, introduced herein as E. ruyongzhengiana.

In this study, five powdery mildew samples on A. debilis were collected in Lianyungang city, Xuzhou city of Jiangsu Province and Tai'an city of Shandong Province. These collections are deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS) and the Herbarium of Mycology of Jilin Agricultural University (HMJAU). Details for these collections are listed in Table1. The fungi were detached from the infected leaves with transparent adhesive tape and mounted in a few drops of distilled water on a glass slide for light microscopy. The examined structures of the anamorph include hyphae, conidiophores, conidia and germ tubes. The chasmothecia were stripped off from the infected leaves with a clean needle and mounted in 3 % NaOH solution on a glass slide. In order to characterize the teleomorph, the diameters of chasmothecia, size of peridium cells, number, size and shape of appendages, asci and ascospores were examined. Thirty measurements for all structures of taxonomical value were performed.

Table 1 Details of the specimens of Erysiphe ruyongzhengiana on Aristolochia debilis used in this study

Vouchera	Location	Longitude/ Latitude	Collection Date	Collector	Accession No.
HMAS0351194	Hancheng Scenic Area, Pei County, Xuzhou city, Jiangsu Province	34.730481N 116.939072E	18 Oct 2020	Shou-Rong Yu	OM103691
HMAS0249832 (Holotype)	Chaoyang Street, Development district, Lianyungang City, Jiangsu Province	34.60465N 119.170958E	4 Nov 2020	Shou-Rong Yu	OM103692
HMAS0351363	Yunlong mountain scenic spot, Quanshan district, Xuzhou city, Jiangsu Province	34.232218N 117.168960E	28 Sep 2017	Shou-Rong Yu	OM103693
HMJAU-PM92061	Puzhao temple, Tai'an City, Shandong Province	36.209330N 117.110772E	13 Aug2018	Feng-Yun Zhao, Jing Feng	OM103694
HMJAU-PM92062 (Paratype)	Chaoyang Street, Development district, Lianyungang City, Jiangsu Province	34.60465N 119.170958E	26 Oct 2021	Shou-Rong Yu	OM103695

^a HMAS=the Herbarium Mycologicum Academiae Sinicae; HMJAU=the Herbarium of Mycology of Jilin Agricultural University

Genomic DNA was extracted from mycelia and chasmothecia using the chelex-100 method (Walsh, Metzger, & Higuchi, 1991) as previously described (Hirata & Takamatsu, 1996). PCR and DNA sequencing were conducted according to the procedures described in Liu et al. (2020), except for the primers that were used. PM10/ITS4 were used to amplify internal transcribed spacer (ITS) including the 5.8S rDNA (White, Bruns, Lee, & Taylor, 1990; Bradshaw & Tobin, 2020). Sequences obtained in this study were deposited in GenBank. The sequences were aligned with closely related sequences of other Erysiphe species retrieved from GenBank using MUSCLE implemented in the MEGA7 program (Kumar, Stecher, & Tamura, 2016). Alignments were further manually refined and deposited in TreeBASE (https://www.treebase.org/) under the accession number 29181. Phylogenetic trees were obtained from the data using the maximum-parsimony (MP) method in PAUP4.0 (Swofford, 2002) with heuristic search option using the tree-bisection-reconstruction (TBR) algorithm with 100 random sequence additions to find the global optimum tree. Gaps were treated as missing data. All characters were unordered and of equal weight. The strength of internal branches of resulting trees was tested with bootstrap analysis (1000 replications) (Felsenstein, 1985). Bootstrap (BS) values of 50 % or higher are indicated in the tree (Fig. 4). Tree scores, including tree length, consistency index (CI), retention index (RI) and rescaled consistency index (RC) were also calculated.

Erysiphe ruyongzhengiana S. R. Yu & S. Y. Liu, sp. nov. Figs 1, 2, 3.

MycoBank no.: MB842390

Fig. 1 -Symptoms and asexual morph of Erysiphe ruyongzhengiana on Aristolochia debilis (HMJAU-PM92062). A: Symptoms on Aristolochia debilis. B: Mycelia almost cover the whole upper leaf surface. C: Mycelia on the lower leaf surface. D: Numerous chasmothecia formed on the lower leaf surface. E: Close-up view of chasmothecia. F-I: Hyphal appressoria solitary, lobed. J: Hyphal appressoria in opposite pairs, nipple-shaped. K-N: Conidiophores with sinuous foot-cells. O: Conidiophores with straight foot-cells. P-T: Conidia. U-X: Germ tubes. Bars: F-X 20 μm.

Fig. 2 -Sexual morph of Erysiphe ruyongzhengiana on Aristolochia debilis (HMAS0249832). A: Chasmothecium. B: Peridium cells. C-F: Appendages. G: Asci with two ascospores. H-K: Asci with three ascospores. L-N: Asci with four ascospores. O-Q:Asci with five ascospores. R: Asci with six ascospores. Bars: A 100 μm, B, G-R 20 μm, C-F 50 μm.

Fig. 3 - Erysiphe ruyongzhengiana on Aristolochia debilis (HMAS0249832). A: Hyphal appressoria. B: Conidiophores. C: Conidia. D: Chasmothecium. E: Appendages. F: Asci.

Fig. 4 - Phylogenetic analysis of Erysiphe ruyongzhengiana based on the internal transcribed spacer (ITS) sequences. This is the tree with the highest likelihood value of the 100 equally parsimonious trees, 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 (>50%) values by the maximum parsimony (MP) are shown on the respective branches. The sequences of E. ruyongzhengiana are indicated in the shadow.

Diagnosis: Distinguished from E. aquilegiae and E. macleayae, two morphologically similar species of the E. aquilegiae clade, by differences in the asexual and sexual morphs, viz. very long, often curved-sinuous conidiophore foot cells (vs. much shorter and usually straight foot-cells in E. aquilegiae) and larger chasmothecia (vs. smaller chasmothecia, on average < 100 µm diam in E. macleayae).

Type: CHINA, Chaoyang Street, Lianyungang Development district, Jiangsu Province, on Aristolochia debilis (Aristolochiaceae), 4 Nov 2020, Shou-Rong Yu (holotype, HMAS249832); 26 Oct 2021, Shou-Rong Yu (paratype, HMJAU-PM92062).

Gene sequences ex-holotype: OM103692 (ITS)

Etymology: The new species is named in honor of Dr. Ruyong Zheng, who has considerably contributed to the study of the biodiversity of powdery mildew.

Description: Mycelium amphigenous, mostly epiphyllous, at first forming white circular patches, later covering entire leaves, thin, persistent (Fig. 1A-C); hyphae smooth, hyaline, thin-walled, 3-7 µm wide; hyphal appressoria lobed, solitary or in opposite pairs (Fig. 1F-J); conidiophores arising from the upper surface of mother cells, erect, foot-cells cylindrical, hyaline, smooth, width uniform throughout, straight or curved-sinuous, 28.5-60.0 × 7.0-9.0 µm, followed by 1-2 shorter cells, forming conidia singly (Fig. 1K-O); conidia cylindrical, oblong-ellipsoid, ellipsoid, 30-65(-70) × 13-20 µm, length/width ratio 1.7-4.5 (Fig. 1P-T); germ tubes almost terminal, short to moderately long, conidial appressoria simple to somewhat swollen or distinctly lobed (Fig. 1U-X). Chasmothecia scattered to gregarious, brown to dark brown(Fig. 1D-E), depressed globose to subglobose, 60-125 µm diam (Fig. 2A); peridium cells irregularly polygonal to nearly square, 5-32 µm diam (Fig. 2B); appendages 10-55, mycelioid, unbranched, occasionally irregularly branched, (0.2-)1-7(-9) times as long as the chasmothecial diam (about (22-)75-655 µm long), stiff, sinuous, geniculate, flexuous, curved, sometimes forming nodules, width uniform throughout or becoming gradually narrower upwards, 4-8.5 µm wide below, 3-7 µm at the tip, 0-6-septate, mostly 2-4-septate, slightly brown to distinctly brown at the base, becoming paler towards the tip, sometimes brown throughout, walls thin above, thick towards the base, smooth(Fig. 2C-F); asci 2-8, broad obovate, obovoid, ellipsoid, 45.0-67.5 × 29.0-61.5 µm, short-stalked, 3-6-spored, mostly 4-spored (Fig. 2G-R); ascospores ellipsoid, ovoid, oblong ellipsoid, 21.0-35.0 × 11.0-17.5 µm.

Host range and distribution: On Aristolochia debilis (China, endemic).

We constructed a phylogenetic tree using a data set of ITS sequences to investigate the phylogenetic placement of E. ruyongzhengiana in the genus Erysiphe (Microsphaera lineage). A sequence of E. glycines F. L. Tai was used as outgroup. The data set consisted of 45 sequences. Of the 566 characters, 51 characters were variable and 59 characters were informative for parsimony analysis. A total of 100 equally parsimonious trees were constructed by the MP analysis. Tree topologies were almost consistent among the trees, except for branching orders of the terminal groups and branch lengths. One of the trees with the highest likelihood value is shown in Fig. 4. Erysiphe ruyongzhengiana forms an independent cluster with Erysiphe sp. (MN701000, MN700999) with moderate BS support (58%).

Phylogenetic analyses of collections from A. debilis (from Jiangsu Province and Shandong Province) revealed that the powdery mildew belong to the E. aquilegiae clade (Takamatsu et al., 2015; Shin, Meeboon, Takamatsu, Adhikari, & Braun, 2019; Bradshaw et al., 2021). The sequence (GenBank no.: OM103692) from holotype is identical to OM103693, OM103695, and differs one base to OM103694 which is identical to two sequences (MN700999, MN701000) on A. debilis published by Zhu et al. (2020). OM103691 showed both C and G peaks at the same site. The sequences from A. debilis formed a separate cluster with a moderate BS support (58%). Erysiphe sp. reported on A. debilis by Zhu et al. (2020) can be identified as E. ruyongzhengiana on the basis of molecular sequence analyses and a morphological comparison of the asexual morphs, which widens the distribution area of this species. E. aquilegiae clade (defined by Takamatsu et al., 2015) is a homogeneous clade composted of sequences retrieved from numerous Erysiphe species, including E. aquilegiae, E. catalpae S. Simonyan, E. euphorbiae Peck, E. macleayae R. Y. Zheng & G. Q. Chen, E. knautiae Duby, E. coriariae (Y. Nomura) U. Braun & S. Takam., E. circaeae L. Junell, E. sedi U. Braun, E. hommae U. Braun, E. chloranthi (Golovin & Bunkina) U. Braun, E. pileae (Jacz.) Bunkina, E. takamatsui Y. Nomura, Pseudoidium neolycopersici (L. Kiss) L. Kiss, Ps. hortensiae (Jørst.) U. Braun & R.T.A. Cook and Ps. boroniae (Crooks) U. Braun & R.T.A. Cook (Takamatsu et al., 2015; Shin et al., 2019; Bradshaw et al. 2021). These species, which parasitize on different plant family, have identical or rather similar rDNA sequences. The E. aquilegiae clade (complex) has recently been discussed in detail by Shin et al. (2019) and Bradshaw et al. (2021), including a survey of previous papers dealing with taxa of this complex, such as Cunnington, Lawrie and Pascoe (2004), Liberato and Cunnington (2006), Pastirčáková, Jankovics, Komáromi, Pintye and Pastirčák (2016), and Cho, Zhao, Choi and Shin (2017). The crux of the problem around the E. aquilegiae clade lies in that there is insufficient resolution with ITS and 28S rDNA trees and thus a multilocus approach is needed in future work in this complex. For the time being, Bradshaw et al. (2021) recommended to maintain the described species, based on Braun and Cook (2012), and to be cautious with the introduction of new species, above all in instances when only asexual morphs are known or only a single holomorph specimen. Descriptions based on infections of exotic host plants outside of their natural ranges should also be avoided.

However, the present case of Erysiphe on A. debilis is in accordance with the recommendations in Bradshaw et al. (2021): (1) There are several sequenced holomorph collections with abundant fruiting bodies (chasmothecia). (2) A. debilis is an Asian species known from China and Japan (Flora of China Editorial Committee, 2001), i.e., the new species is from the natural range of this host species. (3) Aristolochia (Aristolochiaceae, Piperales) is not closely allied to the hosts of the most closely allied Erysiphe species relatives, viz., E. aquilegiae on Ranunculaceae (Ranunculales) and E. macleayae on Papaveraceae (Ranunculales). (4) There are morphological differences to the morphologically and genetically most closely related species (conidiophores longer and frequently curved-sinuous compared to shorter, usually straight conidiophore foot-cells in E. aquilegiae; chasmothecia larger in comparison to E. macleayae with smaller chasmothecia, on average <100 µm diam, see Table 2).

Table 2 Morphological comparison of Erysiphe ruyongzhengiana and closely related species in Braun and Cook (2012)

Species	Conidia (µm)	Foot cells (µm)	Diameter of Chasmothecia (µm)	Numbers of appendages	Asci		Ascospores
					Number	Size (µm)	Number	Size (µm)
E. ruyongzhengiana	30-65(-70) × 13-20	28.5-60.0 × 7.0-9.0	60-125	10-55	3-8	45.0-67.5 × 29.0-61.5	3-6	21.0-35.0 × 11.0-17.5
E. aquilegiae	(25-)28-50 × (12-)16-22(-24)	(15-)20-40 × 7-11	(65-)75-115(- 125)	(4-)10- 30(-50)	(2-)3-8(-12)	40-70(-80) × 25-45(-55)	(2-)3-5(-6)	16-25.5 × 9-15
E. macleayae	25-50(-60) × (10-)11.5-20(-29)	25-120 × 6-10.5	70-95(-100)	5-25	2-5	40-75 × 25-60	(2-)3-4(-5)	18-38 × 12-18
E. sedi	(20-)25-50 × 11.5-24	(15-)25-50 (-70) × 7-11(-12)	80-150	5-20	(2-)4-9(-12),	(45-)50-85 × (30-)35-55(-60)	2-5-	(17.5-)20-26 (-30) × (10-)11.5-17(-20)
E. pileae	25-50(-55) × 12-8	20-55 × 7-10	80-120 (-130)	few to numerous	3-10	40-65 × 25-45(-50)	2-5	15-25 × 7-14
E. hommae	(20-)25-40 × 9-23	20-50 × 6-11(-14)	(60-)85-130(-150)	numerous	(2-)4-8(-15)	40-70(-80) × 30-50	(2-)3-6(-8)	15-26(-30) × 7.5-18
E. takamatsui	35-45 × 15-22	50-80 × 7-10	80-140	-	2-6	40-70 × 25-65	2-5	(13-)17.5-35 × (8-)10-17.5
E. chloranthi	25-45 × 13-20	15-30 × 7-10	75-120	5-18,	3-13	40-65(-70) × (20-)25-50	(2-)3-5	(14-)16-32 × (6.5-)10-18
E. circaeae	30-46 × 14-20	25-35 × 6-9	70-105	not very numerous	(2-)3-5(-6)	45-65 × 30-40	3-5	18-23 × 10-12
E.knautiae	(20-)25-45(-50) × (12-)15-20(-25)	(20-)30-45(-65) × 7-11(-13)	(70-)80-120(-125)	5-15	(2-)3-6(-8)	40-75(-85) × 30-50	(2-)3-5	(18-)20-30.5 × (8.5-)11-17 (-19)
E. euphorbiae	22.5-37.5(-45) × 10-16	15-37.5 × 7.5-10	65-105	4-28	2-5	35-75 × 25-55	3-4(-5)-	16-27.5(-32.5) × 10-15
E. catalpae	(17-)22-40 × (10-)14-18(-21)	16-45 × 7-12	(70-)90-125(-160)	4-19	3-8	(45-)60-85 × 20-40(-47)	3-5	17-27× (10-)14-18

Disclosure

The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.

Acknowledgments

This research was supported by the National Natural Science Foundation of China (No.31970019, No.U21A20177). The authors sincerely acknowledge Prof. Uwe Braun for reading the full text, for critical comments and for corrections. The authors are very grateful to the Herbarium Mycologicum Academiae Sinicae for providing specimens.

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