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Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America
Mycoscience xxx (2018) 1e6
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Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America a María G. Cabrera a, Roberto E. Alvarez , Susumu Takamatsu b, * a b
Facultad de Ciencias Agrarias, Universidad Nacional del Nordeste, Sargento Cabral 2131 C.P. (3400) Corrientes, Argentina Graduate School of Bioresources, Mie University, 1577 Kurima-machiya, Tsu, Mie 514-8507, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 January 2018 Received in revised form 4 April 2018 Accepted 10 April 2018 Available online xxx
Brasiliomyces, comprising only four species, is a unique powdery mildew genus characterized by having small, semitransparent chasmothecia with a thin, single peridium layer. Brasiliomyces malachrae, a type species, is distributed in Central and South America and the morphology of this species, especially its asexual morph, is poorly known. This study was performed to describe the sexual and asexual morphs of B. malachrae on Malvastrum coromandelianum in detail, and to clarify the phylogenetic placement of this fungus. The asexual morph of this species is characterized by having hyaline ectophytic mycelium with lobed hyphal appressoria and catenescent conidia. Molecular phylogenetic analyses based on 18S, 5.8S, and 28S rRNA gene sequences revealed that B. malachrae is a sister to the genus Erysiphe and situated at the boundary between tribes Golovinomyceteae and Erysipheae, which supports its unique phylogenetic position. The evolutionary relationships among the tribes Golovinomyceteae, Erysipheae, and Phyllactinieae are discussed on the basis of the present phylogenetic analyses. © 2018 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Keywords: Asexual morph Brasiliomyces malvastri DNA sequence Erysiphales Malvastrum coromandelianum
1. Introduction Powdery mildew fungi is a fungal group comprising 18 genera and ca. 900 species worldwide (Braun & Cook, 2012; Marmolejo, Siahaan, Takamatsu, & Braun, 2018). All of them are exclusively obligate biotrophs of plants having ca. 10,000 host species in angiosperms (Amano, 1986). Some members of these fungi are known to be important plant pathogens because numerous economically important cultivated plants are included as hosts and decreasing of yields and deteriorations of the quality of the plants concerned are caused by these pathogens. Chasmothecia, i.e., the fruiting bodies of powdery mildews, are considered as overwintering organs. They are composed mainly of several peridium cell layers of thickwalled, melanized, densely arranged cells with sparse protoplasm. Ascospores, the sexual spores of ascomycetous fungi, are kept in a sac-like organ called ascus (pl. asci) within the chasmothecium during winter season and function a primary inoculum in the next year. The genus Brasiliomyces is unique in the Erysiphales in having thin semitransparent chasmothecial peridia composed of only a single conspicuous cell layer.
* Corresponding author. E-mail address: [email protected] (S. Takamatsu).
gas Brasiliomyces has a complicated taxonomic history. Vie (1944) proposed the new genus Brasiliomyces and its type species gas for a fungus found on Malvastrum coromandeB. malvastri Vie lianum (L.) Garcke in Brazil based on chasmothecia without appendage. Braun (1981) introduced the new genus Californiomyces e based on asci with for Erysiphe trina Harkn. on Quercus agrifolia Ne two large ascopores and chasmothecia with thin, single-layered peridia, and proposed the combination C. trinus (Harkn.) U. Braun. Zheng (1984) considered Californiomyces as a synonym of Brasiliomyces, based on the assumption that the number and size of ascospores being unreliable to distinguish powdery mildew genera, and since chasmothecia of the type species of Brasiliomyces are also equipped with short mycelioid appendages similar to those of C. trinus. She also emphasized the importance of thin, singlelayered chasmothecial peridia as a basic character for Brasiliomyces, and thus four Brasiliomyces species, viz., B. entadae Marasas & Rabie, B. malachrae (Seaver) Boesew. (syn. B. malvastri), B. setosus Hodges, and B. trinus (Harkn.) R. Y. Zheng, were recognized in Braun (1987). Brasiliomyces species are confined to narrow subtropical and tropical areas. Obtaining specimens suitable for sequencing is often difficult. DNA sequences from only one Brasiliomyces species, B. trinus (now E. trina), has been deposited in GenBank until now. Our preliminary phylogenetic analysis using DNA sequences from
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Please cite this article in press as: Cabrera, M. G., et al., Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.04.003
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B. trinus, B. malachrae and B. chiangmaiensis To-anun & S. Takam. revealed that B. trinus is nested in a clade of the genus Erysiphe, but B. malachrae and B. chiangmaiensis formed an independent linage not belonging to any known genera (unpublished data), suggesting that thin, single-layered chasmothecial peridia evolved at least twice during the evolution of powdery mildews. Based on this result, Braun and Cook (2012) transferred Brasiliomyces species on Fagaceae to Erysiphe sect. Californiomyces and confined Brasiliomyces to species on hosts of other plant families, viz., B. chiangmaiensis, B. entadae, B. malachrae, and B. setosus. However, DNA sequences retrieved from these four Brasiliomyces species are not yet available. Thus, little is currently known about the phylogenetic placement of Brasiliomyces. The conidial formation is known so far only for B. malachrae, and regarded to be unknown or probably not produced at all in the gas (1944) and three other species (Braun & Cook, 2012). Vie ~ on, Cabrera de Alvarez, Mazzanti de Castan & Alvarez (1990) reported catenescent conidia for B. malachrae. On the other hand, this species is characterized by a combination of lobed hyphal appressoria and Reticuloidium-type conidial germ tube (Cichoracearum~ on et al., 1990), which is a type in Hirata (1955)) (Mazzanti de Castan very unusual combination quite different from the asexual morphs of any other powdery mildew genera, and led to the classification of the Brasiliomyces anamorph in Braun and Cook (2012) as “insufficiently known”. Therefore, the objective of this study was to describe the sexual and asexual morphs of B. malachrae on M. coromandelianum in detail, and to clarify the phylogenetic placement. 2. Materials and methods 2.1. Samples All samples of M. coromandelianum infected with powdery mildew were collected in the Northeast Argentinean Province Corrientes that is a part of the phytogeographical Paranaense Region (Cabrera, 1976). The specimens examined were deposited in CTES and a part of them also in Phytopathology Laboratory UNNE, Corrientes city and additional duplicates also in HAL and TSU. The taxonomy and geographical distribution of host plants were recorded according to Dimitri (1980) and Cabrera (1976). We also re-examined the holotype material of B. malachrae on Malachra capitata L. (NY 6488).
pieces of 5 mm2 with powdery mildew colonies were cut out and fixed for 48 h in FAA (formalin: acetic acid: 70% ethanol ¼ 5: 5: 90). These samples were washed, dehydrated, taken to drying at critical point, and metalized with gold according to the procedure described by D'Ambrogio de Argüeso (1986). These samples were observed by a JEOL 5800 LV model at the Electronic Microscopy Service of the Institute of Botany of the Northeast (IBONE).
2.3. Molecular phylogeny DNA extraction was conducted according to the procedure described by Meeboon and Takamatsu (2015) based on Hirata and Takamatsu (1996). 18S rRNA gene, internal transcribed spacer (ITS) including 5.8S rRNA gene, and 50 -end of 28S rRNA gene including D1/D2 domain were amplified by PCR, and sequenced according to the procedure of Mori, Sato, and Takamatsu (2000a). Newly determined sequences were deposited in the DNA Data Bank of Japan (DDBJ) under the accession numbers LC191217 and LC191218. These sequences were aligned with the sequences of the Erysiphales reported in Mori et al. (2000a, 2000b) using MUSCLE (Edgar, 2004) implemented in MEGA7 (Kumar, Stecher, & Tamura, 2016). Alignments were further manually refined using the MEGA7 program and were deposited in TreeBASE (http://www.treebase.org/) under the accession number S22155. Phylogenetic trees were obtained from the data using the maximum parsimony (MP) and maximum likelihood (ML) methods. MP analysis was performed in PAUP 4.0 (Swofford, 2002) with heuristic search option using the tree bisection reconnection (TBR) algorithm with 100 random sequence additions in order to find the global optimum tree. All sites were treated as unordered and unweighted, with gaps treated as missing data. The strength of internal branches of the resulting trees was tested with bootstrap (BS) analysis using 1000 replications with the step-wise addition option set as simple (Felsenstein, 1985). Tree scores, including tree length, consistency index (CI), retention index (RI), and rescaled consistency index (RC), were also calculated. The ML analysis was done using raxmlGUI (Silvestro & Michalak, 2012), under a GTRGAMMA model. The BS supports and trees were obtained by running rapid bootstrap analysis of 1000 pseudo-replicates followed by a search for the tree with the highest likelihood.
2.2. Morphology
3. Results
Observations of asexual morphs were conducted according to Boesewinkel (1980). Size and shape of fungal structures in fresh samples were studied according to Braun and Cook (2012), and those of dried specimens were restored in gently heated lactic acid as described in Shin and La (1993). In addition, the lacto-fuchsine boiling method was used to restore shrivelled structures in dried samples (Carmichael, 1955). The following data were recorded: size and shape of conidia, presence or absence of fibrosin bodies, characteristics of the conidiophores, size and shape of foot cells, position of the basal septum, shape of hyphal appressoria, position of germ tubes, and shape of appressoria on germ tubes of conidia (Boesewinkel, 1980; Braun, 1987). For the examination of sexual morphs, chasmothecia were scrapped off the leaf surfaces with moistened pointed dissection needles and mounted in water. Later, the specimens were stained with cotton blue in lacto-phenol for general morphological observations, and with Congo red in order to study the asci. Diameter of chasmothecia, number of appendages, asci per chasmothecium and ascospores per ascus were measured. For scanning electron microscopy (SEM) observations, small leaf
3.1. Field observations The powdery mildew affecting M. coromandelianum showed both asexual and sexual stages. The asexual morph of this fungus was characterized by having hyaline ectophytic mycelium and catenescent conidia. Mycelium was occasionally present on leaves, petioles and stems of the plants (Fig. 1A). The sexual morph was characterized by forming whitish ashen chasmothecia covering the surface of host leaves. Based on these characters, this fungus was identified as a Brasiliomyces species. The fungus occurred during the cold season of year in the Northeast Argentina. High relative humidity at night and low relative humidity during daytime with temperatures of 8e16 C (conditions prevailing in spring and fall) favor the occurrence of powdery mildews. Rapid drying of foliage is probably effective to reduce disease incidence. Because powdery mildews cause polycyclic diseases that impair the photosynthesis, cause stunt growth, and increases the rate of senescence of host tissues, the present fungus could be a potential biological control agent of M. coromandelianum.
Please cite this article in press as: Cabrera, M. G., et al., Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.04.003
M.G. Cabrera et al. / Mycoscience xxx (2018) 1e6
Fig. 1. Brasiliomyces malachrae on Malvastrum coromandelianum. A: Infected plant. B: Conidiophores with catenescent conidia. C: Scattered white chasmothecia on leaf surface. D: Conidiophores. E: Conidial germtubes. F: Broken chasmothecia with asci and ascospores. Bars: 25 mm.
3.2. Morphology Brasiliomyces malachrae (Seaver) Boesew., Bot. Rev. 46: 171. 1980. Figs. 1 and 2. ≡ Erysiphe malachrae Seaver, in Seaver & Gordon, Sci. Surv. Porto Rico & Virgin Islands 8(1): 27. 1926. ≡ Salmonia malachrae (Seaver) S. Blumer & E. Müll., Phytopathol. Z. 50: 328. 1964. gas, Bragantia 4: 17. 1944; type ¼ Brasiliomyces malvastri Vie host e Malvastrum coromandelianum. Asexual morph: Mycelium amphigenous, mycelial growth more
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extensive on the upper leaf surface, finally covering the entire leaf surface, sometimes forming irregular patches on the upper leaf surfaces with mycelium and later with minute, globose, white chasmothecia (Fig. 1C). Hyphae hyaline, thin walled, hyphal cells 30e57 2.5e7.5 mm, hyphal appressoria simply lobed (Fig. 2B). Conidiophores mostly straight, 45e100 9e12.5 mm (Fig. 1B, D, 2A). Foot-cells cylindrical, 25e65 7.5e12.5 mm, followed by one or two (rarely three) shorter cells. Conidia catenescent, i.e., developing in genuine chains of 3e5-conidia, ellipsoid to doliiform, hyaline, 25e37.5 15e20 mm, lacking conspicuous fibrosin bodies, length to width (l/w) ratio 2.0 or less. Conidia germination perihilar, Reticuloidium-type, germ tube 1.0e1.5 times as long as the conidial length (Fig. 1E). Sexual morph: Chasmothecia subgregarious to densely scattered, generally spherical to subglobose (Fig. 1B and C), white, subhyaline, smooth, thin walled (peridium composed of a single cell layer), 45e98 mm in diameter (Figs. 1C and 2B, C), having very few conspicuous, short appendages. Asci three per chasmothecium, subspherical, 30e80 26e60 mm, 4e6-spored (Figs. 1F and 2D). Ascospores ellipsoid to ovoid, hyaline, with granulate cytoplasm, 16e30 10e18 mm (Figs. 1F and 2D). Specimens examined: PUERTO RICO, on Malachra capitata, 11 Mar 1922, leg. N. L. Britton & F. S. Earle (NY 6488, holotype); ARGENTINA, Corrientes province, Corrientes Capital city, Capital Department, on Malvastrum coromandelianum, 5 Aug 2005, leg. M. G. Cabrera (Phytopathology Laboratory UNNE Herb. MC 0390); Bella Vista Department, leg. R. E. Alvarez, 18 Jul 2006 (Phytopathol. Lab. UNNE Herb. MC 0396); Department San Cosme, Ensenada Grande, leg. R. E. Alvarez, 14 Nov 2006 (Phytopathol. Lab. UNNE Herb. MC 0416; CTES 0566617); Dept. San Cosme, Santa Ana de los caraes, leg. R. E. Alvarez, 16 Nov 2006 (Phytopathol. Lab. UNNE Gua Herb. MC 0417); Dept. San Cosme, Ensenada Grande, leg. R. E. Alvarez, 18 Dec 2006 (Phytopathol. Lab. UNNE Herb. MC 0423); Corrientes Capital city, leg. R. E. Alvarez, 2 Aug 2007 (Phytopathol. Lab. UNNE Herb. MC 0477); Corrientes Capital city, leg. R. E. Alvarez, 10 Oct 2007 (Phytopathol. Lab. UNNE Herb. MC 0527); Dept. San Cosme, leg. M. G. Cabrera, 16 Nov 2007 (Phytopathol. Lab. UNNE Herb. MC 0532); Santa Ana, leg. R. E. Alvarez, 16 Nov 2007 (Phytopathol. Lab. UNNE Herb. MC 0533); Corrientes Capital city, leg. M. G. Cabrera, 22 Apr 2008 (Phytopathol. Lab. UNNE Herb. MC 0571); Corrientes, Dept. Capital, leg. M. G. Cabrera, 15 Aug 2008 (only anamorph, Phytopathol. Lab. UNNE Herb. MC 0678; CTES stoles, Colonia San 0566629); Misiones province, Department Apo , leg. M. G. Cabrera, 19 Dec 2008 (Phytopathol. Lab. UNNE Herb. Jose MC 0683); Santa Rosa, 31 Jul 2004, leg. S. Takamatsu (TSU-MUMH 3093); UNNE University Farm, 2 Aug 2004, leg. S. Takamatsu (TSUMUMH 3119). Note: The holotype specimen of B. malachrae (NY 6488) has also been re-examined. Although we could not find any trace of an asexual morph in this specimen, the sexual morph was well consistent with the specimens on M. coromandelianum from Argentina (Table 1). We did not try to sequencing of this specimen due to its too old origin and minute amount of fungal material. 3.3. Molecular phylogeny
Fig. 2. SEM micrograms of Brasiliomyces malachrae on Malvastrum coromandelianum. A: Conidiophores with catenescent conidia. B: Chasmothecia, conidia, and hyphal appressoria (shown by triangle). C: Chasmothecium. D: Broken chasmothecium showing asci and ascospores. Bars: 50 mm.
DNA sequences were determined for two Brasiliomyces samples, TSU-MUMH 3093 (asexual morph) and TSU-MUMH 3119 (sexual morph), collected in Corrientes (Northeastern Argentina). DNA sequences of 18S rRNA gene þ ITS region, and ITS region þ50 end of 28S rRNA gene including D1/D2 domains were determined for TSUMUMH 3093 and 3119, respectively. Both sequences were identical with each other in ITS region, indicating that the examined asexual morph is conspecific with the analysed sexual morph. We thus combined the two sequences into one sequence of 3104 nucleotide
Please cite this article in press as: Cabrera, M. G., et al., Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.04.003
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Table 1 Morphological characteristics of Brasiliomyces malachrae on M. coromandelianum and its holotype. Structure
This study
gas (1944) Vie
B. malachrae (holotype: NY 6488)
Hyphae Conidiophore Foot cell Conidium Chasmothecium Appendage number Ascus number Ascus dimension Ascospore number Ascospore dimension
30e57 2.5e7.5 mm 45e100 8.7e12.5 mm 25e65 7.5e12.5 mm 25e37.5 15e20 mm 45e98 mm 3e4 3 30e80 26e60 mm 4e6 16e30 10e18 mm
3e4 mm 30e50 3e5 mm NAa 24e35 10e13 mm 50e60 mm NA 3e4 36e40 28e30 mm 4 22e24 15e17 mm
NA NA NA NA 50e75 mm 4e6 3 30e40 mm 5 29 14 mm
a
NA: not available.
length. BLAST search using the ITS sequence as a query showed that powdery mildew species were listed as the highest score to this sequence, but the cover range of the alignments were less than 50%, and the highest similarity was only 88%. For example, powdery mildew taxa with highest score were Erysiphe adunca (LC028970, 88%), E. fernandoae (AB693962, 88%), E. glycines (LC028953, 88%), etc. This result indicates that B. malachrae belongs to the Erysiphaceae, but is distantly related to all other powdery mildew taxa. Because we were not able to make a reliable alignment in ITS sequences, we used a combined sequence of 18S, 5.8S and partial sequence of 28S rRNA gene region for the phylogenetic analyses. This DNA sequence was aligned with 35 sequences retrieved from DNA databases (Mori, Sato, & Takamatsu, 2000b, 2000a). Byssoascus striatosporus (AF062817) was used as outgroup according to Mori et al. (2000a). The alignment data matrix consisted of 36 sequences and 2742 characters, of which 424 (15.5%) characters were variable and 257 (9.4%) characters were informative for parsimony analysis. A total of nine equally parsimonious trees with 1122 steps were constructed by the MP analysis. Likelihood values were calculated for these trees by PAUP* and a tree with the highest likelihood value was given in Fig. 3. Brasiliomyces malachrae was sister to the Erysiphe clade. Although BS supports of this branch were not strong (MP ¼ 50%, ML ¼ 74%), this branch also appeared in a strict consensus tree. This Erysiphe þ Brasiliomyces clade was then sister to the combined clade of Microidium and Neo€ erysiphe. But this was not supported by BS. 4. Discussion
the asexual morph of B. malachrae. The tribes Erysipheae, Golovinomyceteae and Phyllactinieae form a large clade, but the branching order of these three tribes is still unknown (Mori et al., 2000a). Both Erysipheae and Phyllactinieae have conidia produced singly. Similarly, Golovinomyceteae and Phyllactinieae commonly have 2(e4)-spored asci, and Erysipheae and Golovinomyceteae are ecto-parasitic. Thus, the phylogenetic relationships among these three tribes are obscure just based on morphology. In the present phylogenetic analyses, the tribe Golovinomyceteae was divided into two groups, i.e., the Golovinomyces þ Arthrocladiella group and the Neo€ erysiphe group. The Neo€ erysiphe group further clustered with Microidium, and this combined clade formed a large clade together with the Erysiphe þ Brasiliomyces clade. Neo€ erysiphe and Microidium are distinguished from Golovinomyces and Arthrocladiella in having lobed hyphal appressoria and 2e8-spored asci (vs nipple-shape hyphal appressoria and 2(e4)-spored asci in the latter group). These characteristics of Neo€ erysiphe and Microidium are shared by the genus Erysiphe and B. malachrae. The result of the present phylogenetic analyses suggests evolutionary link among Neo€ erysiphe, Microidium, B. malachrae, and Erysiphe. The tribes Erysipheae and Phyllactinieae share conidia produced singly. Since these tribes do not form a common clade, the noncatenescent conidial formation may be evolved independently in the respective tribes. However, because only weak BS supports were obtained in the branching order among these three tribes, further phylogenetic analyses using other DNA regions are required to clarify the branching order among these three tribes.
4.1. Asexual morph and phylogeny
4.2. Brasiliomyces malachrae and B. malvastri
The catenescent conidial formation without distinct fibrosin bodies and the conidial germ tubes similar to Reticuloidium-type of B. malachrae are shared with the genus Golovinomyces. On the other hand, lobed hyphal appressoria are a character shared with the genus Erysiphe. Thus, it is difficult to discuss phylogenetic relationships of B. malachrae with other known powdery mildew genera just based on morphology (Braun & Cook, 2012). The phylogenetic analyses using 18S þ 5.8S þ 28S rRNA gene sequences showed that B. malachrae is situated at the boundary between the tribes Golovinomyceteae and Erysipheae, and sister to the Erysiphe clade. Brasiliomyces malachrae can be clearly distinguished from the genus Erysiphe in forming catenescent conidia (vs conidia formed singly in Erysiphe). The combined clade of Erysiphe þ Brasiliomyces was sister to the Microidium and Neo€ erysiphe clade although this was not supported by BS. The conidial formation in true chains and lobed hyphal appressoria of B. malachrae are shared by Microidium and Neo€ erysiphe. Thus, the result of the present phylogenetic analyses seems to be supported by the morphological characteristics of
Brasiliomyces malachrae was first described as Erysiphe malachrae based on a specimen on Malachra capitata (Seaver & Chardon, 1926). This species was then transferred to the newly introduced genus Salmonia as S. malachrae (Blumer & Müller, 1964). The genus Salmonia was then reduced to synonymy with the genus Brasiliomyces based on comparison of type specimens (Blumer, 1967), and E. malachrae was reallocated to Brasiliomyces (Boesewinkel, 1980). Zheng (1984) reduced B. malvastri to synonymy with B. malachrae also based on type specimens. However, Boesewinkel (1980) distinguished B. malvastri from B. malachrae on the basis of the size of conidia. Brasiliomyces malvastri and B. malachrae commonly infect host plants of the family Malvaceae, but the former species infects hosts of genus Malvastrum, and the latter occurs on Malachra species. Recent phylogenetic analyses often revealed that morphology-based species are actually species complex consisting of cryptic species confined to hosts of plant genera or species (Braun et al., 2006; Meeboon, Siahaan, Fujioka, & Takamatsu, 2017; Siahaan, Sakamoto, Shinoda, & Takamatsu, 2018; Takamatsu et al.,
Please cite this article in press as: Cabrera, M. G., et al., Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.04.003
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Fig. 3. Phylogenetic analysis of Brasiliomyces malachrae on Malvastrum coromandelianum based on the 18S, 5.8S, and 28S rRNA gene sequences. This tree is a phylogram with the highest likelihood value among the nine equally parsimonious trees with 1122 steps, 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 (70%) values by the maximum parsimony (MP) and maximum likelihood (ML) methods were shown on/under the respective branches.
2007). Thus, the synonymy of these two species should be reinvestigated by molecular analyses. 4.3. Phylogeny of Brasiliomyces DNA sequence data of Brasiliomyces spp. have not yet been deposited in DNA databases except for B. trinus, a species that was later transferred to the genus Erysiphe. Thus, the phylogenetic affinity of the genus Brasiliomyces has remained unknown for a long time. DNA sequences from other Brasiliomyces species are urgently required to clarify the phylogeny of this genus. Species of Erysiphe sect. Californiomyces, previously usually assigned to the genus Brasiliomyces, share chasmothecia with thin, single-layered peridia with true Brasiliomyces spp. Molecular phylogenetic analyses clearly showed that this character evolved at least twice independently along with the lineage leading to Brasiliomyces and Erysiphe
sect. Californiomyces. The four Brasiliomyces species described in Braun and Cook (2012) infect hosts of distantly related plant families, such as Malvaceae, Fabaceae, and Sapindaceae, and are confined to small subtropical or tropical distribution areas, such as Central & South America, Southeast Asia, South Africa, and Hawaii (Braun & Cook, 2012). Under these circumstances, it cannot be ruled out that the chasmothecia with single-layered peridia of Brasiliomyces constitute a polyphyletic character that evolved several times independently to adapt tropical or subtropical environments. Comprehensive phylogenetic analyses of this genus are urgently required. Disclosure The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the
Please cite this article in press as: Cabrera, M. G., et al., Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.04.003
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countries where they were performed. Voucher The specimens examined were deposited in CTES and a part of them also in Phytopathology Laboratory UNNE, Corrientes city and additional duplicates also in HAL and TSU. Acknowledgements The authors wish to acknowledge the kind assistance by Drs. G. Vobis and M. Havrylenko, CRUB Comahue University, especially for constructive comments, and Herbarium NY for providing holotype specimen of B. malachrae. We also thank Uwe Braun for critical reading the previous version of the manuscript. This work was financially supported in part by a Grant-in-Aid for Scientific Research (No. 16K07613 and 16F16097) from the Japan Society for the Promotion of Science to ST. References Amano, K. (1986). Host range and geographical distribution of the powdery mildew fungi. Tokyo: Japan Scientific Societies Press. Blumer, S. (1967). Echte Mehltaupilze (Erysiphaceae). Jena: G. Fischer Verlag. Blumer, S., & Müller, E. (1964). Über zwei Mehltauarten auf Baumwollein Peru (in German). Phytopathologische Zeitschrift, 50, 379e385. Boesewinkel, H. J. (1980). The morphology of imperfect states of powdery mildews (Erysiphaceae). The Botanical Review, 46, 167e224. Braun, U. (1981). Taxonomic studies in the genus Erysiphe I. Generic delimitation and position in the system of the Erysiphaceae. Nova Hedwigia, 34, 679e719. Braun, U. (1987). A monograph of the Erysiphales (powdery mildews). Beihefte zur Nova Hedwigia, 89, 1e700. Braun, U., & Cook, R. T. A. (2012). Taxonomic manual of the Erysiphales (powdery mildews). CBS Biodiversity Series No. 11. Utrecht: CBS-KNAW Fungal Biodiversity Centre. Braun, U., Takamatsu, S., Heluta, V., Limkaisang, S., Divarangkoon, R., Cook, R. T. A., et al. (2006). Phylogeny and taxonomy of powdery mildew fungi of Erysiphe sect. Uncinula on Carpinus species. Mycological Progress, 5, 139e153. https:// doi.org/10.1007/s11557-006-0509-6. ficas Argentinas. Enciclopedia Argentina de Cabrera, A. L. (1976). Regiones Fitogeogra Agricultura y Jardinería (2nd ed.). Buenos Aires: ACME. Tomo 2(1) (in Spanish). Carmichael, J. W. (1955). Lacto-fuchsine: A new medium for mounting fungi. Mycologia, 47, 611. D'Ambrogio de Argüeso, A. (1986). Manual de T ecnicas en Histología Vegetal. Buenos Aires: Hemisferio Sur S.A (in Spanish). Dimitri, M. J. (1980). Enciclopedia Argentina de Agricultura y Jardinería. (3rd. Ed.). Tomo 1 (Vol. 2). Buenos Aires: ACME SACI (in Spanish). Edgar, R. C. (2004). MUSCLE: Multiple sequence alignment with high accuracy and
high throughput. Nucleic Acids Research, 32, 1792e1797. https://doi.org/10.1093/ nar/gkh340. Felsenstein, J. (1985). Confidence limits on phylogenetics: An approach using the bootstrap. Evolution, 39, 783e791. https://doi.org/10.2307/2408678. Hirata, K. (1955). On the shape of the germ tubes of Erysipheae (II) (in Japanese). Bulletin of the Faculty of Agriculture, Niigata University, 7, 24e36. Hirata, T., & Takamatsu, S. (1996). Nucleotide sequence diversity of rDNA internal transcribed spacers extracted from conidia and cleistothecia of several powdery mildew fungi. Mycoscience, 37, 265e270. Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33, 1870e1874. https://doi.org/10.1093/molbev/msw054. Marmolejo, J., Siahaan, S. A. S., Takamatsu, S., & Braun, U. (2018). Three new records of powdery mildews found in Mexico with one genus and one new species proposed. Mycoscience, 59, 1e7. https://doi.org/10.1016/j.myc.2017.06.010. ~ on, M. A., Cabrera de Alvarez, Mazzanti de Castan M. G., & Alvarez, R. E. (1990). geno nuevo para la República Argentina (in Brasiliomyces malvastri, fitopato Spanish). Fitopatologia, 25, 60e67. Meeboon, J., Siahaan, S. A. S., Fujioka, K., & Takamatsu, S. (2017). Molecular phylogeny and taxonomy of Parauncinula (Erysiphales) and two new species P. polyspora and. P. uncinata. Mycoscience, 58, 361e368. https://doi.org/10.1016/ j.myc.2017.04.007. Meeboon, J., & Takamatsu, S. (2015). Erysiphe takamatsui, a powdery mildew of lotus: Rediscovery of teleomorph after 40 years, morphology and phylogeny. Mycoscience, 56, 159e167. https://doi.org/10.1016/j.myc.2014.05.002. Mori, Y., Sato, Y., & Takamatsu, S. (2000a). Evolutionary analysis of the powdery mildew fungi (Erysiphales) using nucleotide sequences of the nuclear ribosomal DNA. Mycologia, 92, 74e93. Mori, Y., Sato, Y., & Takamatsu, S. (2000b). Molecular phylogeny and radiation time of Erysiphales inferred from the nuclear ribosomal DNA sequences. Mycoscience, 41, 437e447. Seaver, F. J., & Chardon, C. E. (1926). Scientific survey of porto rico and the Virgin Islands, vol. VIII, Part 1. Botany of Porto Rico and the Virgin Islands. Mycology. New York: New York Academy of Sciences. Shin, H. D., & La, Y.-J. (1993). Morphology of edge lines of chained immature conidia on conidiophores in powdery mildews fungi and their taxonomic significance. Mycotaxon, 46, 445e451. Siahaan, S. A. S., Sakamoto, H., Shinoda, T., & Takamatsu, S. (2018). Morphophylogenetic study revealed that Erysiphe gracilis (powdery mildew of evergreen oaks, Erysiphales) is a species complex consisting of six different species. Mycoscience, 59, 124e136. https://doi.org/10.1016/j.myc.2017.08.011. Silvestro, D., & Michalak, I. (2012). raxmlGUI: a graphical front-end for RAxML. Organisms, Diversity and Evolution, 12, 335e337. https://doi.org/10.1007/s13127011-0056-0. Swofford, D. L. (2002). PAUP: Phylogenetic analysis using parsimony (and other methods) 4.0b10. Sunderland: Sinauer. Takamatsu, S., Braun, U., Limkaisang, S., Kom-un, S., Sato, Y., & Cunnington, J. H. (2007). Phylogeny and taxonomy of the oak powdery mildew Erysiphe alphitoides sensu lato. Mycological Research, 111, 809e826. https://doi.org/10.1016/ j.mycres.2007.05.013. gas, A. P. (1944). Alguns fungos do Brasil II. Ascomycetes (in Portuguese). BraVie gantia, 4, 1e392. Zheng, R. Y. (1984). The genus Brasiliomyces (Erysiphaceae). Mycotaxon, 19, 281e289.
Please cite this article in press as: Cabrera, M. G., et al., Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.04.003
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Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America a María G. Cabrera a, Roberto E. Alvarez , Susumu Takamatsu b, * a b
Facultad de Ciencias Agrarias, Universidad Nacional del Nordeste, Sargento Cabral 2131 C.P. (3400) Corrientes, Argentina Graduate School of Bioresources, Mie University, 1577 Kurima-machiya, Tsu, Mie 514-8507, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 January 2018 Received in revised form 4 April 2018 Accepted 10 April 2018 Available online xxx
Brasiliomyces, comprising only four species, is a unique powdery mildew genus characterized by having small, semitransparent chasmothecia with a thin, single peridium layer. Brasiliomyces malachrae, a type species, is distributed in Central and South America and the morphology of this species, especially its asexual morph, is poorly known. This study was performed to describe the sexual and asexual morphs of B. malachrae on Malvastrum coromandelianum in detail, and to clarify the phylogenetic placement of this fungus. The asexual morph of this species is characterized by having hyaline ectophytic mycelium with lobed hyphal appressoria and catenescent conidia. Molecular phylogenetic analyses based on 18S, 5.8S, and 28S rRNA gene sequences revealed that B. malachrae is a sister to the genus Erysiphe and situated at the boundary between tribes Golovinomyceteae and Erysipheae, which supports its unique phylogenetic position. The evolutionary relationships among the tribes Golovinomyceteae, Erysipheae, and Phyllactinieae are discussed on the basis of the present phylogenetic analyses. © 2018 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Keywords: Asexual morph Brasiliomyces malvastri DNA sequence Erysiphales Malvastrum coromandelianum
1. Introduction Powdery mildew fungi is a fungal group comprising 18 genera and ca. 900 species worldwide (Braun & Cook, 2012; Marmolejo, Siahaan, Takamatsu, & Braun, 2018). All of them are exclusively obligate biotrophs of plants having ca. 10,000 host species in angiosperms (Amano, 1986). Some members of these fungi are known to be important plant pathogens because numerous economically important cultivated plants are included as hosts and decreasing of yields and deteriorations of the quality of the plants concerned are caused by these pathogens. Chasmothecia, i.e., the fruiting bodies of powdery mildews, are considered as overwintering organs. They are composed mainly of several peridium cell layers of thickwalled, melanized, densely arranged cells with sparse protoplasm. Ascospores, the sexual spores of ascomycetous fungi, are kept in a sac-like organ called ascus (pl. asci) within the chasmothecium during winter season and function a primary inoculum in the next year. The genus Brasiliomyces is unique in the Erysiphales in having thin semitransparent chasmothecial peridia composed of only a single conspicuous cell layer.
* Corresponding author. E-mail address: [email protected] (S. Takamatsu).
gas Brasiliomyces has a complicated taxonomic history. Vie (1944) proposed the new genus Brasiliomyces and its type species gas for a fungus found on Malvastrum coromandeB. malvastri Vie lianum (L.) Garcke in Brazil based on chasmothecia without appendage. Braun (1981) introduced the new genus Californiomyces e based on asci with for Erysiphe trina Harkn. on Quercus agrifolia Ne two large ascopores and chasmothecia with thin, single-layered peridia, and proposed the combination C. trinus (Harkn.) U. Braun. Zheng (1984) considered Californiomyces as a synonym of Brasiliomyces, based on the assumption that the number and size of ascospores being unreliable to distinguish powdery mildew genera, and since chasmothecia of the type species of Brasiliomyces are also equipped with short mycelioid appendages similar to those of C. trinus. She also emphasized the importance of thin, singlelayered chasmothecial peridia as a basic character for Brasiliomyces, and thus four Brasiliomyces species, viz., B. entadae Marasas & Rabie, B. malachrae (Seaver) Boesew. (syn. B. malvastri), B. setosus Hodges, and B. trinus (Harkn.) R. Y. Zheng, were recognized in Braun (1987). Brasiliomyces species are confined to narrow subtropical and tropical areas. Obtaining specimens suitable for sequencing is often difficult. DNA sequences from only one Brasiliomyces species, B. trinus (now E. trina), has been deposited in GenBank until now. Our preliminary phylogenetic analysis using DNA sequences from
https://doi.org/10.1016/j.myc.2018.04.003 1340-3540/© 2018 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Cabrera, M. G., et al., Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.04.003
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B. trinus, B. malachrae and B. chiangmaiensis To-anun & S. Takam. revealed that B. trinus is nested in a clade of the genus Erysiphe, but B. malachrae and B. chiangmaiensis formed an independent linage not belonging to any known genera (unpublished data), suggesting that thin, single-layered chasmothecial peridia evolved at least twice during the evolution of powdery mildews. Based on this result, Braun and Cook (2012) transferred Brasiliomyces species on Fagaceae to Erysiphe sect. Californiomyces and confined Brasiliomyces to species on hosts of other plant families, viz., B. chiangmaiensis, B. entadae, B. malachrae, and B. setosus. However, DNA sequences retrieved from these four Brasiliomyces species are not yet available. Thus, little is currently known about the phylogenetic placement of Brasiliomyces. The conidial formation is known so far only for B. malachrae, and regarded to be unknown or probably not produced at all in the gas (1944) and three other species (Braun & Cook, 2012). Vie ~ on, Cabrera de Alvarez, Mazzanti de Castan & Alvarez (1990) reported catenescent conidia for B. malachrae. On the other hand, this species is characterized by a combination of lobed hyphal appressoria and Reticuloidium-type conidial germ tube (Cichoracearum~ on et al., 1990), which is a type in Hirata (1955)) (Mazzanti de Castan very unusual combination quite different from the asexual morphs of any other powdery mildew genera, and led to the classification of the Brasiliomyces anamorph in Braun and Cook (2012) as “insufficiently known”. Therefore, the objective of this study was to describe the sexual and asexual morphs of B. malachrae on M. coromandelianum in detail, and to clarify the phylogenetic placement. 2. Materials and methods 2.1. Samples All samples of M. coromandelianum infected with powdery mildew were collected in the Northeast Argentinean Province Corrientes that is a part of the phytogeographical Paranaense Region (Cabrera, 1976). The specimens examined were deposited in CTES and a part of them also in Phytopathology Laboratory UNNE, Corrientes city and additional duplicates also in HAL and TSU. The taxonomy and geographical distribution of host plants were recorded according to Dimitri (1980) and Cabrera (1976). We also re-examined the holotype material of B. malachrae on Malachra capitata L. (NY 6488).
pieces of 5 mm2 with powdery mildew colonies were cut out and fixed for 48 h in FAA (formalin: acetic acid: 70% ethanol ¼ 5: 5: 90). These samples were washed, dehydrated, taken to drying at critical point, and metalized with gold according to the procedure described by D'Ambrogio de Argüeso (1986). These samples were observed by a JEOL 5800 LV model at the Electronic Microscopy Service of the Institute of Botany of the Northeast (IBONE).
2.3. Molecular phylogeny DNA extraction was conducted according to the procedure described by Meeboon and Takamatsu (2015) based on Hirata and Takamatsu (1996). 18S rRNA gene, internal transcribed spacer (ITS) including 5.8S rRNA gene, and 50 -end of 28S rRNA gene including D1/D2 domain were amplified by PCR, and sequenced according to the procedure of Mori, Sato, and Takamatsu (2000a). Newly determined sequences were deposited in the DNA Data Bank of Japan (DDBJ) under the accession numbers LC191217 and LC191218. These sequences were aligned with the sequences of the Erysiphales reported in Mori et al. (2000a, 2000b) using MUSCLE (Edgar, 2004) implemented in MEGA7 (Kumar, Stecher, & Tamura, 2016). Alignments were further manually refined using the MEGA7 program and were deposited in TreeBASE (http://www.treebase.org/) under the accession number S22155. Phylogenetic trees were obtained from the data using the maximum parsimony (MP) and maximum likelihood (ML) methods. MP analysis was performed in PAUP 4.0 (Swofford, 2002) with heuristic search option using the tree bisection reconnection (TBR) algorithm with 100 random sequence additions in order to find the global optimum tree. All sites were treated as unordered and unweighted, with gaps treated as missing data. The strength of internal branches of the resulting trees was tested with bootstrap (BS) analysis using 1000 replications with the step-wise addition option set as simple (Felsenstein, 1985). Tree scores, including tree length, consistency index (CI), retention index (RI), and rescaled consistency index (RC), were also calculated. The ML analysis was done using raxmlGUI (Silvestro & Michalak, 2012), under a GTRGAMMA model. The BS supports and trees were obtained by running rapid bootstrap analysis of 1000 pseudo-replicates followed by a search for the tree with the highest likelihood.
2.2. Morphology
3. Results
Observations of asexual morphs were conducted according to Boesewinkel (1980). Size and shape of fungal structures in fresh samples were studied according to Braun and Cook (2012), and those of dried specimens were restored in gently heated lactic acid as described in Shin and La (1993). In addition, the lacto-fuchsine boiling method was used to restore shrivelled structures in dried samples (Carmichael, 1955). The following data were recorded: size and shape of conidia, presence or absence of fibrosin bodies, characteristics of the conidiophores, size and shape of foot cells, position of the basal septum, shape of hyphal appressoria, position of germ tubes, and shape of appressoria on germ tubes of conidia (Boesewinkel, 1980; Braun, 1987). For the examination of sexual morphs, chasmothecia were scrapped off the leaf surfaces with moistened pointed dissection needles and mounted in water. Later, the specimens were stained with cotton blue in lacto-phenol for general morphological observations, and with Congo red in order to study the asci. Diameter of chasmothecia, number of appendages, asci per chasmothecium and ascospores per ascus were measured. For scanning electron microscopy (SEM) observations, small leaf
3.1. Field observations The powdery mildew affecting M. coromandelianum showed both asexual and sexual stages. The asexual morph of this fungus was characterized by having hyaline ectophytic mycelium and catenescent conidia. Mycelium was occasionally present on leaves, petioles and stems of the plants (Fig. 1A). The sexual morph was characterized by forming whitish ashen chasmothecia covering the surface of host leaves. Based on these characters, this fungus was identified as a Brasiliomyces species. The fungus occurred during the cold season of year in the Northeast Argentina. High relative humidity at night and low relative humidity during daytime with temperatures of 8e16 C (conditions prevailing in spring and fall) favor the occurrence of powdery mildews. Rapid drying of foliage is probably effective to reduce disease incidence. Because powdery mildews cause polycyclic diseases that impair the photosynthesis, cause stunt growth, and increases the rate of senescence of host tissues, the present fungus could be a potential biological control agent of M. coromandelianum.
Please cite this article in press as: Cabrera, M. G., et al., Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.04.003
M.G. Cabrera et al. / Mycoscience xxx (2018) 1e6
Fig. 1. Brasiliomyces malachrae on Malvastrum coromandelianum. A: Infected plant. B: Conidiophores with catenescent conidia. C: Scattered white chasmothecia on leaf surface. D: Conidiophores. E: Conidial germtubes. F: Broken chasmothecia with asci and ascospores. Bars: 25 mm.
3.2. Morphology Brasiliomyces malachrae (Seaver) Boesew., Bot. Rev. 46: 171. 1980. Figs. 1 and 2. ≡ Erysiphe malachrae Seaver, in Seaver & Gordon, Sci. Surv. Porto Rico & Virgin Islands 8(1): 27. 1926. ≡ Salmonia malachrae (Seaver) S. Blumer & E. Müll., Phytopathol. Z. 50: 328. 1964. gas, Bragantia 4: 17. 1944; type ¼ Brasiliomyces malvastri Vie host e Malvastrum coromandelianum. Asexual morph: Mycelium amphigenous, mycelial growth more
3
extensive on the upper leaf surface, finally covering the entire leaf surface, sometimes forming irregular patches on the upper leaf surfaces with mycelium and later with minute, globose, white chasmothecia (Fig. 1C). Hyphae hyaline, thin walled, hyphal cells 30e57 2.5e7.5 mm, hyphal appressoria simply lobed (Fig. 2B). Conidiophores mostly straight, 45e100 9e12.5 mm (Fig. 1B, D, 2A). Foot-cells cylindrical, 25e65 7.5e12.5 mm, followed by one or two (rarely three) shorter cells. Conidia catenescent, i.e., developing in genuine chains of 3e5-conidia, ellipsoid to doliiform, hyaline, 25e37.5 15e20 mm, lacking conspicuous fibrosin bodies, length to width (l/w) ratio 2.0 or less. Conidia germination perihilar, Reticuloidium-type, germ tube 1.0e1.5 times as long as the conidial length (Fig. 1E). Sexual morph: Chasmothecia subgregarious to densely scattered, generally spherical to subglobose (Fig. 1B and C), white, subhyaline, smooth, thin walled (peridium composed of a single cell layer), 45e98 mm in diameter (Figs. 1C and 2B, C), having very few conspicuous, short appendages. Asci three per chasmothecium, subspherical, 30e80 26e60 mm, 4e6-spored (Figs. 1F and 2D). Ascospores ellipsoid to ovoid, hyaline, with granulate cytoplasm, 16e30 10e18 mm (Figs. 1F and 2D). Specimens examined: PUERTO RICO, on Malachra capitata, 11 Mar 1922, leg. N. L. Britton & F. S. Earle (NY 6488, holotype); ARGENTINA, Corrientes province, Corrientes Capital city, Capital Department, on Malvastrum coromandelianum, 5 Aug 2005, leg. M. G. Cabrera (Phytopathology Laboratory UNNE Herb. MC 0390); Bella Vista Department, leg. R. E. Alvarez, 18 Jul 2006 (Phytopathol. Lab. UNNE Herb. MC 0396); Department San Cosme, Ensenada Grande, leg. R. E. Alvarez, 14 Nov 2006 (Phytopathol. Lab. UNNE Herb. MC 0416; CTES 0566617); Dept. San Cosme, Santa Ana de los caraes, leg. R. E. Alvarez, 16 Nov 2006 (Phytopathol. Lab. UNNE Gua Herb. MC 0417); Dept. San Cosme, Ensenada Grande, leg. R. E. Alvarez, 18 Dec 2006 (Phytopathol. Lab. UNNE Herb. MC 0423); Corrientes Capital city, leg. R. E. Alvarez, 2 Aug 2007 (Phytopathol. Lab. UNNE Herb. MC 0477); Corrientes Capital city, leg. R. E. Alvarez, 10 Oct 2007 (Phytopathol. Lab. UNNE Herb. MC 0527); Dept. San Cosme, leg. M. G. Cabrera, 16 Nov 2007 (Phytopathol. Lab. UNNE Herb. MC 0532); Santa Ana, leg. R. E. Alvarez, 16 Nov 2007 (Phytopathol. Lab. UNNE Herb. MC 0533); Corrientes Capital city, leg. M. G. Cabrera, 22 Apr 2008 (Phytopathol. Lab. UNNE Herb. MC 0571); Corrientes, Dept. Capital, leg. M. G. Cabrera, 15 Aug 2008 (only anamorph, Phytopathol. Lab. UNNE Herb. MC 0678; CTES stoles, Colonia San 0566629); Misiones province, Department Apo , leg. M. G. Cabrera, 19 Dec 2008 (Phytopathol. Lab. UNNE Herb. Jose MC 0683); Santa Rosa, 31 Jul 2004, leg. S. Takamatsu (TSU-MUMH 3093); UNNE University Farm, 2 Aug 2004, leg. S. Takamatsu (TSUMUMH 3119). Note: The holotype specimen of B. malachrae (NY 6488) has also been re-examined. Although we could not find any trace of an asexual morph in this specimen, the sexual morph was well consistent with the specimens on M. coromandelianum from Argentina (Table 1). We did not try to sequencing of this specimen due to its too old origin and minute amount of fungal material. 3.3. Molecular phylogeny
Fig. 2. SEM micrograms of Brasiliomyces malachrae on Malvastrum coromandelianum. A: Conidiophores with catenescent conidia. B: Chasmothecia, conidia, and hyphal appressoria (shown by triangle). C: Chasmothecium. D: Broken chasmothecium showing asci and ascospores. Bars: 50 mm.
DNA sequences were determined for two Brasiliomyces samples, TSU-MUMH 3093 (asexual morph) and TSU-MUMH 3119 (sexual morph), collected in Corrientes (Northeastern Argentina). DNA sequences of 18S rRNA gene þ ITS region, and ITS region þ50 end of 28S rRNA gene including D1/D2 domains were determined for TSUMUMH 3093 and 3119, respectively. Both sequences were identical with each other in ITS region, indicating that the examined asexual morph is conspecific with the analysed sexual morph. We thus combined the two sequences into one sequence of 3104 nucleotide
Please cite this article in press as: Cabrera, M. G., et al., Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.04.003
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M.G. Cabrera et al. / Mycoscience xxx (2018) 1e6
Table 1 Morphological characteristics of Brasiliomyces malachrae on M. coromandelianum and its holotype. Structure
This study
gas (1944) Vie
B. malachrae (holotype: NY 6488)
Hyphae Conidiophore Foot cell Conidium Chasmothecium Appendage number Ascus number Ascus dimension Ascospore number Ascospore dimension
30e57 2.5e7.5 mm 45e100 8.7e12.5 mm 25e65 7.5e12.5 mm 25e37.5 15e20 mm 45e98 mm 3e4 3 30e80 26e60 mm 4e6 16e30 10e18 mm
3e4 mm 30e50 3e5 mm NAa 24e35 10e13 mm 50e60 mm NA 3e4 36e40 28e30 mm 4 22e24 15e17 mm
NA NA NA NA 50e75 mm 4e6 3 30e40 mm 5 29 14 mm
a
NA: not available.
length. BLAST search using the ITS sequence as a query showed that powdery mildew species were listed as the highest score to this sequence, but the cover range of the alignments were less than 50%, and the highest similarity was only 88%. For example, powdery mildew taxa with highest score were Erysiphe adunca (LC028970, 88%), E. fernandoae (AB693962, 88%), E. glycines (LC028953, 88%), etc. This result indicates that B. malachrae belongs to the Erysiphaceae, but is distantly related to all other powdery mildew taxa. Because we were not able to make a reliable alignment in ITS sequences, we used a combined sequence of 18S, 5.8S and partial sequence of 28S rRNA gene region for the phylogenetic analyses. This DNA sequence was aligned with 35 sequences retrieved from DNA databases (Mori, Sato, & Takamatsu, 2000b, 2000a). Byssoascus striatosporus (AF062817) was used as outgroup according to Mori et al. (2000a). The alignment data matrix consisted of 36 sequences and 2742 characters, of which 424 (15.5%) characters were variable and 257 (9.4%) characters were informative for parsimony analysis. A total of nine equally parsimonious trees with 1122 steps were constructed by the MP analysis. Likelihood values were calculated for these trees by PAUP* and a tree with the highest likelihood value was given in Fig. 3. Brasiliomyces malachrae was sister to the Erysiphe clade. Although BS supports of this branch were not strong (MP ¼ 50%, ML ¼ 74%), this branch also appeared in a strict consensus tree. This Erysiphe þ Brasiliomyces clade was then sister to the combined clade of Microidium and Neo€ erysiphe. But this was not supported by BS. 4. Discussion
the asexual morph of B. malachrae. The tribes Erysipheae, Golovinomyceteae and Phyllactinieae form a large clade, but the branching order of these three tribes is still unknown (Mori et al., 2000a). Both Erysipheae and Phyllactinieae have conidia produced singly. Similarly, Golovinomyceteae and Phyllactinieae commonly have 2(e4)-spored asci, and Erysipheae and Golovinomyceteae are ecto-parasitic. Thus, the phylogenetic relationships among these three tribes are obscure just based on morphology. In the present phylogenetic analyses, the tribe Golovinomyceteae was divided into two groups, i.e., the Golovinomyces þ Arthrocladiella group and the Neo€ erysiphe group. The Neo€ erysiphe group further clustered with Microidium, and this combined clade formed a large clade together with the Erysiphe þ Brasiliomyces clade. Neo€ erysiphe and Microidium are distinguished from Golovinomyces and Arthrocladiella in having lobed hyphal appressoria and 2e8-spored asci (vs nipple-shape hyphal appressoria and 2(e4)-spored asci in the latter group). These characteristics of Neo€ erysiphe and Microidium are shared by the genus Erysiphe and B. malachrae. The result of the present phylogenetic analyses suggests evolutionary link among Neo€ erysiphe, Microidium, B. malachrae, and Erysiphe. The tribes Erysipheae and Phyllactinieae share conidia produced singly. Since these tribes do not form a common clade, the noncatenescent conidial formation may be evolved independently in the respective tribes. However, because only weak BS supports were obtained in the branching order among these three tribes, further phylogenetic analyses using other DNA regions are required to clarify the branching order among these three tribes.
4.1. Asexual morph and phylogeny
4.2. Brasiliomyces malachrae and B. malvastri
The catenescent conidial formation without distinct fibrosin bodies and the conidial germ tubes similar to Reticuloidium-type of B. malachrae are shared with the genus Golovinomyces. On the other hand, lobed hyphal appressoria are a character shared with the genus Erysiphe. Thus, it is difficult to discuss phylogenetic relationships of B. malachrae with other known powdery mildew genera just based on morphology (Braun & Cook, 2012). The phylogenetic analyses using 18S þ 5.8S þ 28S rRNA gene sequences showed that B. malachrae is situated at the boundary between the tribes Golovinomyceteae and Erysipheae, and sister to the Erysiphe clade. Brasiliomyces malachrae can be clearly distinguished from the genus Erysiphe in forming catenescent conidia (vs conidia formed singly in Erysiphe). The combined clade of Erysiphe þ Brasiliomyces was sister to the Microidium and Neo€ erysiphe clade although this was not supported by BS. The conidial formation in true chains and lobed hyphal appressoria of B. malachrae are shared by Microidium and Neo€ erysiphe. Thus, the result of the present phylogenetic analyses seems to be supported by the morphological characteristics of
Brasiliomyces malachrae was first described as Erysiphe malachrae based on a specimen on Malachra capitata (Seaver & Chardon, 1926). This species was then transferred to the newly introduced genus Salmonia as S. malachrae (Blumer & Müller, 1964). The genus Salmonia was then reduced to synonymy with the genus Brasiliomyces based on comparison of type specimens (Blumer, 1967), and E. malachrae was reallocated to Brasiliomyces (Boesewinkel, 1980). Zheng (1984) reduced B. malvastri to synonymy with B. malachrae also based on type specimens. However, Boesewinkel (1980) distinguished B. malvastri from B. malachrae on the basis of the size of conidia. Brasiliomyces malvastri and B. malachrae commonly infect host plants of the family Malvaceae, but the former species infects hosts of genus Malvastrum, and the latter occurs on Malachra species. Recent phylogenetic analyses often revealed that morphology-based species are actually species complex consisting of cryptic species confined to hosts of plant genera or species (Braun et al., 2006; Meeboon, Siahaan, Fujioka, & Takamatsu, 2017; Siahaan, Sakamoto, Shinoda, & Takamatsu, 2018; Takamatsu et al.,
Please cite this article in press as: Cabrera, M. G., et al., Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.04.003
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Fig. 3. Phylogenetic analysis of Brasiliomyces malachrae on Malvastrum coromandelianum based on the 18S, 5.8S, and 28S rRNA gene sequences. This tree is a phylogram with the highest likelihood value among the nine equally parsimonious trees with 1122 steps, 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 (70%) values by the maximum parsimony (MP) and maximum likelihood (ML) methods were shown on/under the respective branches.
2007). Thus, the synonymy of these two species should be reinvestigated by molecular analyses. 4.3. Phylogeny of Brasiliomyces DNA sequence data of Brasiliomyces spp. have not yet been deposited in DNA databases except for B. trinus, a species that was later transferred to the genus Erysiphe. Thus, the phylogenetic affinity of the genus Brasiliomyces has remained unknown for a long time. DNA sequences from other Brasiliomyces species are urgently required to clarify the phylogeny of this genus. Species of Erysiphe sect. Californiomyces, previously usually assigned to the genus Brasiliomyces, share chasmothecia with thin, single-layered peridia with true Brasiliomyces spp. Molecular phylogenetic analyses clearly showed that this character evolved at least twice independently along with the lineage leading to Brasiliomyces and Erysiphe
sect. Californiomyces. The four Brasiliomyces species described in Braun and Cook (2012) infect hosts of distantly related plant families, such as Malvaceae, Fabaceae, and Sapindaceae, and are confined to small subtropical or tropical distribution areas, such as Central & South America, Southeast Asia, South Africa, and Hawaii (Braun & Cook, 2012). Under these circumstances, it cannot be ruled out that the chasmothecia with single-layered peridia of Brasiliomyces constitute a polyphyletic character that evolved several times independently to adapt tropical or subtropical environments. Comprehensive phylogenetic analyses of this genus are urgently required. Disclosure The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the
Please cite this article in press as: Cabrera, M. G., et al., Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.04.003
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countries where they were performed. Voucher The specimens examined were deposited in CTES and a part of them also in Phytopathology Laboratory UNNE, Corrientes city and additional duplicates also in HAL and TSU. Acknowledgements The authors wish to acknowledge the kind assistance by Drs. G. Vobis and M. Havrylenko, CRUB Comahue University, especially for constructive comments, and Herbarium NY for providing holotype specimen of B. malachrae. We also thank Uwe Braun for critical reading the previous version of the manuscript. This work was financially supported in part by a Grant-in-Aid for Scientific Research (No. 16K07613 and 16F16097) from the Japan Society for the Promotion of Science to ST. References Amano, K. (1986). Host range and geographical distribution of the powdery mildew fungi. Tokyo: Japan Scientific Societies Press. Blumer, S. (1967). Echte Mehltaupilze (Erysiphaceae). Jena: G. Fischer Verlag. Blumer, S., & Müller, E. (1964). Über zwei Mehltauarten auf Baumwollein Peru (in German). Phytopathologische Zeitschrift, 50, 379e385. Boesewinkel, H. J. (1980). The morphology of imperfect states of powdery mildews (Erysiphaceae). The Botanical Review, 46, 167e224. Braun, U. (1981). Taxonomic studies in the genus Erysiphe I. Generic delimitation and position in the system of the Erysiphaceae. Nova Hedwigia, 34, 679e719. Braun, U. (1987). A monograph of the Erysiphales (powdery mildews). Beihefte zur Nova Hedwigia, 89, 1e700. Braun, U., & Cook, R. T. A. (2012). Taxonomic manual of the Erysiphales (powdery mildews). CBS Biodiversity Series No. 11. Utrecht: CBS-KNAW Fungal Biodiversity Centre. Braun, U., Takamatsu, S., Heluta, V., Limkaisang, S., Divarangkoon, R., Cook, R. T. A., et al. (2006). Phylogeny and taxonomy of powdery mildew fungi of Erysiphe sect. Uncinula on Carpinus species. Mycological Progress, 5, 139e153. https:// doi.org/10.1007/s11557-006-0509-6. ficas Argentinas. Enciclopedia Argentina de Cabrera, A. L. (1976). Regiones Fitogeogra Agricultura y Jardinería (2nd ed.). Buenos Aires: ACME. Tomo 2(1) (in Spanish). Carmichael, J. W. (1955). Lacto-fuchsine: A new medium for mounting fungi. Mycologia, 47, 611. D'Ambrogio de Argüeso, A. (1986). Manual de T ecnicas en Histología Vegetal. Buenos Aires: Hemisferio Sur S.A (in Spanish). Dimitri, M. J. (1980). Enciclopedia Argentina de Agricultura y Jardinería. (3rd. Ed.). Tomo 1 (Vol. 2). Buenos Aires: ACME SACI (in Spanish). Edgar, R. C. (2004). MUSCLE: Multiple sequence alignment with high accuracy and
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Please cite this article in press as: Cabrera, M. G., et al., Morphology and molecular phylogeny of Brasiliomyces malachrae, a unique powdery mildew distributed in Central and South America, Mycoscience (2018), https://doi.org/10.1016/j.myc.2018.04.003