Chasmothecia of Erysiphe macleayae on Chelidonium majus confirm species identification

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Chasmothecia of Erysiphe macleayae on Chelidonium majus confirm species identification Wentao Jiang a, Shuyan Liu a,b,*, Baoning An a, Lilan Wang a, Yu Li a, Susumu Takamatsu c, Uwe Braun d a

Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, Changchun 130118, Jilin Province, PR China b Section of Plant Pathology, College of Agronomy, Jilin Agricultural University, Changchun 130118, Jilin Province, PR China c Department of Plant Pathology, Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan d Martin-Luther-Universita¨t, Institut fu¨r Biologie, Bereich Geobotanik, Herbarium, Neuwerk 21, D-06099 Halle/S, Germany

article info

abstract

Article history:

The asexual stage of a powdery mildew on Chelidonium majus has been reported from

Received 7 February 2014

various countries, but the corresponding sexual stage of this fungus has not yet been re-

Received in revised form

ported. In Oct 2011, many chasmothecia of this powdery mildew was found on C. majus

22 April 2014

plants growing in the campus of Jilin Agricultrual University in Changchun, China. The

Accepted 23 April 2014

morphological characteristics of the asexual and sexual stages revealed this fungus as

Available online 15 June 2014

Erysiphe macleayae. A sequence of the ribosomal DNA internal transcribed spacer region including the 5.8S rRNA gene, amplified from DNA of several chasmothecia, was 99.8e100%

Keywords:

identical to that of E. macleayae records on GenBank. This is the first report of the tele-

Greater celandine

omorph of E. macleayae on C. majus.

Powdery mildew

ª 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

Teleomorph

Greater celandine (Chelidonium majus L.) is a perennial herb used in Chinese medicine due to its rich alkaloids. An anamorphic powdery mildew has been known on this plant from Europe and East Asia since the 1960s, in countries such as Spain, France, Italy (Amano 1986), Poland (Dynowska et al. 1999), Hungary (Jankovics 2007), Germany (Schmidt and Scholler 2011), China (Zheng and Chen 1981; Liu 2010) and Korea (Shin 2000). Because the teleomorph has not been observed, the fungus has been tentatively assigned to Erysiphe

cruciferarum Opiz ex L. Junell, E. macleayae R. Y. Zheng & G. Q. Chen and to other species, but the identification remained dubious and could not even be solved with DNA data which have been available for the fungus on C. majus for several years. In powdery mildews, in contrast to many other plant parasitic fungi, several species cannot be identified based solely on the combination of host identity, the anamorph morphology, and sequences of the nuclear ribosomal internal transcribed spacer (ITS) region. The reasons are that both

* Corresponding author. Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, Changchun 130118, Jilin Province, PR China. Tel.: þ86 15943062088. E-mail address: [email protected] (S. Liu). http://dx.doi.org/10.1016/j.myc.2014.04.008 1340-3540/ª 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

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anamorph morphology and ITS sequences of many species of powdery mildews are not available for comparison and host specificity is not as simple and narrow as believed previously. Only the morphology of the teleomorph can provide the definitive data for resolving such issues, along with DNAbased identification to provide dual morphological and molecular support and facilitate subsequent attempts at identification. In Oct 2011, typical powdery mildew symptoms were observed on leaves of celandine and chasmothecia were found on infected leaves located at Jilin Agricultural University, Changchun, China (Fig. 1). The disease incidence was about 80e100% on leaves of several plants. Voucher specimens have been deposited in the Herbarium of Mycology of Jilin Agricultural University (HMJAU01275) and the Herbarium of Martin-Luther-Universita¨t, Halle (HAL 2608 F), Germany to represent the holotype. Another specimen from Macleaya cordata (Willd.) R. Br. also used for the phylogenetic analysis was deposited as HMJAU00624. Hyphae, conidiophores and conidia from fresh samples were examined in wet mounts using a light microscope (Nikon YS100, Nikon, Tokyo, Japan) and a digital microscope (VHX600, Keyence, Osaka, Japan), with at least 30 replicates to assess morphological features. Chasmothecia from fresh leaves were transferred onto a glass slides and observed at 100 and 400 magnification. The following information was noted during the examination: size and shape of ascomata, asci, and ascospores; characteristics of appendages, e.g. number, length, color, and shape of the apex; and number of asci per chasmothecium and ascospores per ascus. The powdery mildew formed whitish colonies over the entire surface of leaves of C. majus. Mycelia were amphigenous, mostly epiphyllous, also on stems, white with lobed or nipple-shaped hyphal appressoria (Fig. 2A). Conidia formed singly, oval to ellipsoidal, 21.5e52.5 mm long and 13.5e29.0 mm wide (Fig. 2B). Germ tubes were of the Pseudoidium type, formed subapically (Fig. 2C). Conidiophores arose from superficial hyphae, on top of the mother cell and mostly towards

Fig. 1 e Symptoms of powdery mildew (Erysiphe macleayae) on Chelidonium majus.

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one end. They are erect, straight, hyaline, 54.5e95.0 mm long and 8.5e9.5 mm wide, somewhat attenuated toward the base. Foot-cells were cylindrical followed by 1 or 2 shorter cells (Fig. 2D). Chasmothecia were scattered to gregarious, partly immersed in the mycelial mass, 66.5e109.0 mm in diameter, on average 87.0 mm, dark brown, and subglobose (Fig. 2E). Appendages (6e25 per chasmothecium) arose from the lower half closest to the plant tissue, and were 4e8 times as long as the chasmothecial diameter. There were 2e5 asci per chasmothecium, short-stalked, 46.5e64.5  30.5e47.0 mm, 2e5spored, and most often 3-spored (Fig. 2E). Ascospores were ellipsoid-ovoid, colorless, and 15.0e31.0  11.5e18.0 mm. The fungus was identified as E. macleayae R.Y. Zheng & G.Q. Chen based on the anamorphic and teleomorphic characteristics as outlined by Braun and Cook (2012). For molecular characterization of the fungus, genomic DNA was extracted from chasmothecia by the Chelex method (Walsh et al. 1991). The nuclear rRNA gene including the ITS regions (ITS1 and ITS2) and the 5.8S rRNA gene were amplified by polymerase chain reaction (PCR) using primers ITS5 (White et al. 1990) and P3 (Kusaba and Tsuge 1995) in seminested PCR. From this first reaction mixture, 1 ml was used for the second amplification with the primers ITS5 and ITS4 (White et al. 1990). The resulting 553 bp sequence (GenBank accession No. KF856294) was 100% identical to those of Oidium sp. on C. majus (HQ286645 and HQ286646) and 99.8% identical to those of E. macleayae on Macleaya microcarpa (AB016048) and M. cordata (JQ681217), with a difference of only one nucleotide. In order to further analyze the relationship of this fungus on C. majus with related species, 22 ITS sequences were retrieved from GenBank to construct a phylogenetic tree. Erysiphe adunca (Wallr. : Fr.) Fr. was used as an outgroup taxon. Of the remaining 21 sequences, seven were from C. majus powdery mildew, and the remaining 14 sequences were from Erysiphe powdery mildew of various hosts. Another powdery mildew sequence obtained in this research from M. cordata (KJ160268) was also used to construct the phylogenetic tree. The sequences were initially aligned using the Clustal X package (Thompson et al. 1997). The alignments were manually edited using MEGA 6 (Tamura et al. 2013), and deposited in TreeBASE (http://www.treebase. org/) under the accession number S15273. Phylogenetic trees were obtained from the data by the Maximum-Parsimony method using the heuristic search option in the program PAUP* 4.0b8 (Swofford 2001). This search was repeated 100 times with different random starting points, using the stepwise addition option to increase the likelihood of finding the most parsimonious tree. Transversions and transitions were treated with equal weight. All sites were treated as unordered, with gaps treated as missing data. The branchswapping algorithm was TBR, the MULPARS option was in effect, and zero length branches were collapsed. The strength of the internal branches from the resulting trees was tested by bootstrap analysis using 1000 replications. The alignment data matrix consisted of 24 taxa and 564 characters, of which 518 characters were constant, 23 sites were variable, and 23 sites were phylogenetically informative for parsimony analysis. A total of eight equally parsimonious trees with 158 steps (CI ¼ 0.879, RI ¼ 0.914, RC ¼ 0.805) were generated by the parsimony analysis. The MP tree having the

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Fig. 2 e The morphology of Erysiphe macleayae on Chelidonium majus. A: Appressorium. B: Conidia. C: Germ tube. D: Conidiophore. E: Chasmothecium. F: Ascus and ascospores. Bars: 20 mm.

highest likelihood score (elnL ¼ 1066.96602) was regarded as the best tree (Fig. 3). The phylogenetic analysis revealed that the fungus on C. majus belongs in a clade with E. macleayae, with very strong bootstrap support (99%).

Erysiphe macleayae was first reported on M. cordata and Papaver nudicaule L. from China by Zheng and Chen (1981). This fungal species is well characterized by its small chasmothecia with very long appendages and unusually large ascospores

Fig. 3 e Maximum parsimony tree of Erysiphe spp. inferred from the nucleotide sequences of the ITS region. Numerals on the branches show bootstrap support values from 1000 replicates. The black spot indicates sequence from Chelidonium majus, and the black triangle indicates the sequence from Macleaya cordata obtained in this research respectively.

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(Braun and Cook 2012). Anamorphic powdery mildew on C. majus has been reported from many countries. In Germany, the fungus was assigned to E. macleayae by Schmidt and Scholler (2011), but based on the characteristics of conidia and conidiophores. In Korea it was reported as E. cruciferarum (Shin 2000). Collections from Italy (Amano 1986), Poland (Dynowska et al. 1999) and China (Liu 2010) were simply referred to as Oidium sp., and Pseudoidium sp. was the a´kova´ and Pastirc a´k (2013) for denomination used by Pastirc specimens collected in the Czech Republic and Slovakia. The present Chinese collection of chasmothecia on C. majus, which is the first record ever of chasmothecia on this host, helped to identify powdery mildew on this plant as E. macleayae, and this finding was fully supported by ITS sequencing and phylogenetic analysis. Both Macleaya and Chelidonium belong to the family Papaveraceae, and occurrence of the same powdery mildew on these two related hosts seems reasonable.

Acknowledgments We wish to honestly thank Dr. Tom Hsiang for critical reading of the manuscript and helpful comments. This work was supported by National Natural Science Foundation of China (No. 31170013) and “The Twelfth Five Year Plan” of Science and Technology Project of Jilin Provincial Education Department ([2012]No.36).

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