Short Communication
The asexual morph and molecular phylogeny of endemic Phyllactinia verruculosa on Indigofera tinctoria
2022 年 63 巻 4 号 p. 165-168
詳細
2022 年 63 巻 4 号 p. 165-168
Phyllactinia verruculosa is a powdery mildew species (Erysiphaceae, tribe Phyllactinieae) so far only known from its type material collected in China on Indigofera scabrida in 1992, which only comprised the sexual morph. Two asexual morph samples were observed, one was collected on I. tinctoria on the campus of Guizhou University, Guiyang, China, and another one, on I. scabrida, was borrowed from Kunming Institute of Botany, Chinese Academy of Sciences. The anamorphic characters were observed, described and illustrated. The phylogenetic analysis of the combination of internal transcribed spacer regions (ITS) and 28S rDNA sequences showed that Ph. verruculosa is phylogenetically distantly related from other Phyllactinia species. To our knowledge, this is the first record of the asexual morph and first ITS+28S sequences for Ph. verruculosa, and I. tinctoria is a new host record for this species.
Indigofera (Fabaceae) is a large genus comprising about 750 species of shrubs, shrublets, perennial herbs, or rarely annual herbs or small trees, which are widely distributed throughout tropical and subtropical regions worldwide (Gao & Schrire, 2010). Indigofera tinctoria L. is enriched with antitoxic, hemostatic and sedative properties and used in the treatment of cancer, piles, chronic bronchitis, asthma, healing of ulcers and dropsy (Vijayan, Joseph, & Mathew, 2018).
Phyllactinia verruculosa D. Z. Xie was described on I. scabrida Dunn by Xie (1992) only based on the sexual morph. Since type material or any other collections of this species were not available, the treatment of this species in Braun and Cook (2012) was just based on Xie’s original description. Twelve additional powdery mildew species have been recorded on Indigofera spp., viz., Erysiphe communis (Wallr.) Schltdl., E. diffusa (Cooke & Peck) U. Braun & S. Takam., E. indigoferae (H. D. Shin & Y. J. La) U. Braun & S. Takam., E. pisi DC., E. polygoni DC., E. trifoliorum (Wallr.) U. Braun, E. robiniae Grev., Leveillula sp., Oidiopsis sp., Oidium indigoferae J. M. Yen, Oidium sp. and Phyllactinia guttata (Wallr.) Lév. (Amano, 1986; Braun & Cook, 2012; Farr & Rossman, 2021). The asexual morph and the phylogenetic position of Ph. verruculosa are so far unknown.
In Nov 2019, severe powdery mildew infections were observed and collected on I. tinctoria on the campus of Guizhou University (106.39°E, 26.48°N), Guiyang, China, and deposited in the Herbarium of Mycology of Jilin Agricultural University (HMJAU) under the accession number HMJAU-PM91867. In addition, a specimen of Phyllactina on I. scabrida (KUN 1323049) in Nanjian County (100.26°E, 24.51°N), Yunnan, China was borrowed to observe the morphology. This study was conducted (1) to examine the anamorphic characteristics of Ph. verruculosa and (2) to retrieve DNA sequences from this species to clarify the phylogenetic status within Phyllactinia.
The fresh powdery mildew sample and herbarium specimen were used for morphological examinations. Hyphae, conidiophores and conidia were photographed using a light microscope (ZEISS Scope A1, Göttingen, Germany), with at least 30 replicates to assess morphological features and to quantify size ranges.
Genomic DNA was extracted from mycelia using the Chelex-100 method (Walsh, Metzger, & Higuchi, 1991). The nucleotide sequences of internal transcribed spacer regions (ITS) and the 5’- end of the 28S rRNA gene (including domains D1and D2) were amplified by polymerase chain reaction (PCR) using the respective primer pairs: ITS5/Ph8 (Meeboon, Kokaew, & Takamatsu, 2018) for ITS1, and ITS4/Ph7 (Meeboon et al., 2018) for ITS2, and PM3 (Takamatsu & Kano, 2001)/NLP2 (Mori, Sato, & Takamatsu, 2000) for 28S rRNA gene. PCR reactions were conducted under the following thermal cycling conditions: an initial denaturation step of 5 min at 95 °C, 35 cycles of 1 min at 94 °C, followed by 30 s at 56 °C for annealing, and 30 s at 72 °C for extension, and a final extension for 8 min at 72 °C. Negative control lacking template DNA was included in each set of reactions. PCR products were sequenced using primer pairs of Ph7 and Ph8 for ITS, PM3 and NLP2 for 28S rDNA by Sangon Biotech (Shanghai). The whole procedure above was repeated three times to confirm the reliability of this study.
The new sequences were initially aligned with Phyllactinia species retrieved from GenBank, using MUSCLE (Edgar, 2004) implemented in MEGAX (Kumar, Stecher, Li, Knyaz, & Tamura, 2018). The alignments were further manually edited in MEGAX and deposited in TreeBASE (http://treebase.org/treebase-web/) under the accession number S28695. Phylogenetic trees were obtained from the data using the maximum parsimony (MP) method in PAUP＊ 4.0 (Swofford, 2002) with heuristic search option using tree bisection reconnection (TBR) algorithm with 100 random sequence additions to find the global optimum tree. All sites were treated as unordered and unweighted, with gaps treated as missing data. The strength of the internal branches of the resulting tree was tested with bootstrap analysis using 1000 replications (Felsenstein, 1985). The tree scores of length, consistency index (CI), retention index (RI) and rescaled consistency index (RC) were also calculated.
The asexual morph of Ph. verruculosa is characterized as follows: Forming distinct white colonies on both sides of leaves (Fig. 1A). Mycelium internal and external, superficial mycelium amphigenous, mainly hypophyllous, effuse or in irregular patches. Hyphae flexuous to straight, branched, septate, hyaline, thin-walled, smooth, 3.7-7.4(-9.0) μm wide (mean 5.2 μm). Hyphal appressoria well-developed, nipple-shaped, rod-shaped to hooked, forked, occasionally lobed, solitary or in opposite pairs (Fig. 1B, C). Conidiophores arising from external hyphae, on the upper surface of mother cells, straight or slightly curved, hyaline, (62.3-)72.4-186.7 × 5.1-7.0 μm (mean 131.4 × 6.1 μm), basal septum elevated up to 16.0 μm, forming conidia singly. Foot-cells sinuous to twisted at the base, 31.9-132.4(-157.9) × 4.5-7.5 μm (mean 75.2 × 6.1 μm), followed by 1-3(-5) shorter cells (Fig. 1D, E). Conidia clavate, apex rounded to slightly narrowed, but not papillate, spathulate, slightly curved, subcylindrical, somewhat wider towards the apex, 47.8-83.7 × 10.1-20.4 μm (mean 68.3 × 14.6 μm) (Fig. 1F, H). Germ tubes of the Ovulariopsis type, close to either the apex or the base, straight to sinuous, apex without conidial appressorium or occasionally with short branchlets or lobate (Fig. 1I, J, K).
ITS and 28S rDNA sequences were retrieved from specimen HMJAU-PM91867 with three repetitions and deposited in GenBank, under the accession numbers MN970683, MN970684 and MN970685 for ITS, MN970686, MN970687 and MN970688 for 28S rDNA. Three ITS sequences were identical among each other, also the three 28S rDNA sequences were identical. The maximum sequence similarities were 86.06% (469/545) with Ph. fraxini (DC.) Fuss (AB080551, AB080552) for ITS and 97.88% (784/801) with Ph. actinidiae-latifoliae Sawada (AB985506) for 28S rDNA. This fungus belongs to Phyllactinia by the anamorph morphology and BLAST identities (Braun & Cook, 2012). A phylogenetic tree was constructed based on the combination of ITS and 28S rDNA sequences. Sixty-one sequences of Phyllactinia, Leveillula and Pleochaeta species were retrieved from DNA databases and Pleochaeta shiraiana (Henn.) Kimbr. & Korf was used as outgroup taxon. In the combined dataset, including complete ITS and partial 28S rDNA, and the overlap part was first trimmed off when the two sequences were combined. This combined data set consisted of 64 sequences and 952 characters, of which 65 (6.8%) characters were variable but not informative, and 187 (19.6%) characters were informative for parsimony analysis. One most parsimonious tree (Fig. 2) was generated from the MP analysis (TL = 733, CI = 0.516, RI = 0.796, RC = 0.411, HI = 0.484). The phylogenetic tree showed that Ph. verruculosa forms an isolated clade and nests within the “Core Phyllactinia group” as defined by Takamatsu, Siahaan, Moreno-Rico, Cabrera de Álvarez, & Braun (2016). In addition, Leveillula is nested within the “Basal Phyllactinia group” with the same finding by Takamatsu et al. (2016), and Marmolejo, Siahaan, Takamatsu, and Braun (2018).
Phyllactinia verruculosa is, as far as known, endemic to China. This species was so far only known from the original description on I. scabrida that only comprised the teleomorph (Xie, 1992). This species obviously differs from other Phyllactinia spp. in having appendages with obtuse apices and irregular outline, above all coarsely verrucose walls in the upper part (Braun & Cook, 2012). The examined asexual morphs (chasmothecia not observed) on I. scabrida and I. tinctoria were similar. The asexual morph of Ph. verruculosa differs from Ph. guttata and most other core Phyllactinia species in having sinuous to twisted foot-cells at the base, which is similar to the asexual morphs of Ph. fraxinicola U. Braun & H. D. Shin, distributed in Asia on Fraxinus spp. (Oleaceae), but the conidiophores of Ph. verruculosa are much shorter, (62.3-)72.4-186.7 µm vs. 200-350 μm long in Ph. fraxinicola (Braun & Cook, 2012). Based on the molecular analysis of ITS and partial 28S rDNA sequences, the low sequence similarities confirm that Ph. verruculosa represents a separate species. This species is phylogenetically distantly related from other Phyllactinia species. Indigofera tinctoria is a new host of Ph. verruculosa.
The authors declare no conflicts of interest. All experiments undertaken in this study comply with the current laws of the country where they were performed.
We are much obliged to Prof. Uwe Braun for the critical review of the text and helpful comments, PhD Zhi-Yuan Zhang for collecting samples. The curators of the Herbarium, Kunming Institute of Botany (KUN), Chinese Academy of Sciences are acknowledged for allowing access to specimens for this study. This work was supported by National Natural Science Foundation of China (No. 31970019 and 31670022).
