- Email: [email protected]
Setoidium castanopsidis, a new species of anamorphic Cystotheca (Ascomycota, Erysiphales) from Indonesia
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 2 7 4 e2 7 8
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/myc
Full paper
Setoidium castanopsidis, a new species of anamorphic Cystotheca (Ascomycota, Erysiphales) from Indonesia Jamjan Meeboon a, Iman Hidayat b, Susumu Takamatsu a,* a
Department of Bioresources, Graduate School, Mie University, 1577 Kurima-Machiya, Tsu 514-8507, Japan Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences - LIPI, Jl. Raya Jakarta-Bogor KM 46, Cibinong 16911, West Java, Indonesia b
article info
abstract
Article history:
A powdery mildew fungus belonging to the genus Setoidium (anamorph of Cystotheca) was
Received 31 August 2012
found on Castanopsis javanica in Cibodas Botanical Garden and mount Tangkuban Perahu,
Accepted 25 October 2012
West Java, Indonesia. The fungus is considered as a new species, namely, Setoidium cas-
Available online 7 January 2013
tanopsidis. Phylogenetic analyses of the 28S and ITS rDNA regions showed that S. castanopsidis formed a distinct lineage separated from Cy. tjibodensis, Cy. lanestris, and
Keywords:
Cy. wrightii. Setoidium castanopsidis also differs morphologically from Cy. tjibodensis in
Anamorph
having distinct appressoria (nipple-shaped), longer conidiophores with longer foot-cells,
Castanopsis
larger conidia, and being found on Ca. javanica. The teleomorphic state has not been
Erysiphaceae
found during the collection.
Molecular phylogeny
ª 2012 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Taxonomy
1.
Introduction
Setoidium (R.T.A. Cook, A.J. Inman & C. Billings) R.T.A. Cook & U. Braun is an anamorph genus in the family Erysiphaceae, linked to the teleomorph genus Cystotheca Berk. & M.A. Curtis. This genus is characterized by having mycelium bearing special pigmented aerial hyphae, falcate to filiform, a sinuate outline of the conidial chains, and germ tubes of the Fibroidium typeeorthotubus subtype (Cook et al. 1997; Braun and Cook 2012). Setoidium was recognized by Cook et al. (1997) as Oidium subgenus Setoidium (type species: Oidium japonicum Syd.). Currently only one species has been assigned to Setoidium, namely, S. murrayae (Hosag., U. Braun & Rabindran) U. Braun & R.T.A. Cook on Murraya paniculata (Rutaceae) from India (Braun and Cook 2012). There is no teleomorphic species linked to S. murrayae. All other Setoidium anamorphs, some of
them described as Oidium spp., are associated with known teleomorphs in Cystotheca so that a separate naming of these conidial states is not necessary and against the spirit of the current International Code of Nomenclature for algae, fungi, and plants. During a survey and collection of powdery mildews in Cibodas Botanical Garden and Mount Tangkuban Perahu (Indonesia) on Mar. 2011, two species of Castanopsis (Fagaceae) e Ca. argentea A. DC. and Ca. javanica Blume e were found being infected by powdery mildew. Specimens of Ca. argentea collected from Cibodas were infected by Cy. tjibodensis (Ga¨um.) Katumoto including its anamorphic state (Setoidium) (Meeboon et al. 2012). However, on specimens of Ca. javanica from Cibodas and mount Tangkuban Perahu only anamorphic states were found. Morphological examination showed that both specimens belong to a species different from the anamorph of Cy. tjibodensis. Therefore, we extracted the genomic
* Corresponding author. Tel.: þ81 59 231 9497; fax: þ81 59 231 9637. E-mail address: [email protected] (S. Takamatsu). 1340-3540/$ e see front matter ª 2012 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.myc.2012.10.004
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 2 7 4 e2 7 8
DNA from both specimens and conducted phylogenetic analyses based on the 28S and internal transcribed spacer (ITS) rDNA sequences.
2.
Materials and methods
2.1.
Morphological examination
Specimens were collected from Cibodas Botanical Garden and Mount Tangkuban Perahu, West Java province, in Java Island (Indonesia) on Mar. 2011. Details of host name, collection date, location, and collector were recorded. Herbarium samples were rehydrated before examination by boiling a small piece of infected leaf with the fungal mycelium downwards in a drop of lactic acid on a slide (Shin and La 1993). After boiling, the rehydrated mycelium was scraped off and mounted in lactic acid using a light microscope. Thirty conidia, conidiophores, foot- and mother-cells were measured per sample. Specimens were deposited at the National Museum of Nature and Science (TNS) and Mie University Mycological Herbarium (MUMH) in Japan, and Herbarium Bogoriense (BO) in Indonesia.
2.2.
Phylogenetic analysis
DNA extraction of the powdery mildew specimens was conducted according to the chelex method (Walsh et al. 1991) as described in Hirata and Takamatsu (1996). The 50 -end of the 28S (including the domains D1 and D2) and ITS rDNA regions were amplified by polymerase chain reaction (PCR) using the respective primer pairs: PM3 (Takamatsu and Kano 2001)/ TW14 (Mori et al. 2000) (28S), ITS5 (White et al. 1990)/PM6 (Takamatsu and Kano 2001) (ITS fragment 1), and PM7 (50 eRYYGACCCTCCACCCGTGYe30 þ 50 eRYYGACCCTCCCACC CGTGYe30 )/ITS4 (White et al. 1990) (ITS fragment 2). KOD FX Neo DNA polymerase (Toyobo, Japan) was used in the PCR reaction according to the manufacturer’s protocol. The amplicons of the 28S and ITS rDNA were sent to SolGent Co. Ltd. (Daejeon, South Korea) for sequencing using primer pairs NL1/NLP2 (28S) and ITS1/ITS4 (ITS), respectively. Representative sequences determined in this study were deposited in DNA Data Base of Japan (DDBJ) under the accession numbers AB743781eAB743784. Sequences generated from the respective 28S and ITS rDNA regions were aligned with sequences retrieved from DNA databases (DDBJ, EMBL, NCBI) using MUSCLE (Edgar 2004) implemented in MEGA 5 (Tamura et al. 2011). Sequences of Byssoascus striatisporus (G.L. Barron & C. Booth) Arx (U17912) and Sawadaea nankinensis (F.L. Tai) S. Takam. & U. Braun (AB353763) were used as outgroup for 28S and ITS rDNA data sets, respectively. The alignment was deposited in TreeBASE (http://www.treebase.org/) under the accession number S13331. Maximum parsimony (MP) analysis was done with the parsimony ratchet method (Nixon 1999) in PAUPRat ver. 1 (Sikes and Lewis 2001) and PAUP* 4.0b10 (Swofford 2002) for 28S rDNA data set. For ITS rDNA data set it was done by PAUP* with heuristic search. In both analyses, the ‘tree-bisection-reconstruction’ (TBR) algorithm was used as the heuristic search option. All sites were treated as unordered and unweighted, with gaps treated as missing
275
data. The strength of the internal branches of the resulting tree was tested with bootstrap (BS) analysis using 1000 replications (Felsenstein 1985). Tree scores, including tree length, CI, RI, and RC were also calculated.
3.
Results
3.1.
Phylogenetic analyses
A phylogenetic analysis of the 28S rDNA sequences was conducted in order to confirm the phylogenetic position of Setoidium on Ca. javanica among the Erysiphales. The alignment of the 28S rDNA contained 55 sequences and 818 total characters, of which 585 were constant, 59 were variable but parsimony-uninformative, and 174 were parsimonyinformative. From the 170 equally most parsimonious trees (MPTs) with 815 steps (CI ¼ 0.431, RI ¼ 0.749, RC ¼ 0.323) generated by the parsimony ratchet method, a tree with the highest likelihood value was obtained (Fig. 1). The 28S rDNA data set showed that sequences of the two Setoidium specimens on Ca. javanica have an identical sequence. This Setoidium clade was sister to the clade consisting of Cy. lanestris (Harkn.) Miyabe and Cy. wrightii Berk. & M.A. Curtis with 98% BS support. This result confirmed phylogenetically anamorph-teleomorph connection between Setoidiume Cystotheca as previously shown by Meeboon et al. (2012) on Cy. tjibodensis. Alignment of the ITS rDNA region contained 10 sequences and 479 characters, of which 382 were constant, 67 were variable but parsimony-uninformative, and 30 were parsimony-informative. The MP analysis resulted in two MPTs with a tree length of 122 steps (CI ¼ 0.885, RI ¼ 0.750, RC ¼ 0.664) (Fig. 2). Sequences of the two Setoidium specimens on Ca. javanica were identical. The phylogenetic tree also showed that the Setoidium sequences on Ca. javanica formed a distinct lineage separated from Cy. tjibodensis, Cy. lanestris and Cy. wrightii, which confirmed that the Setoidium on Ca. javanica is a species different from Cy. tjibodensis.
3.2.
Taxonomy
Setoidium castanopsidis Meeboon & S. Takam. sp. nov. Fig. 3aec. MycoBank no.: MB801230. Differs from the anamorphic state of Cy. tjibodensis in having amphigenous colonies, distinct nipple-shaped appressoria, longer conidiophores, longer foot-cells, larger conidia, and being found on Ca. javanica. Type: on Ca. javanica Blume (Fagaceae), Indonesia, West Java Province, Cianjur, Cibodas Botanical Garden, 14 Mar 2011, I. Hidayat, J. Meeboon, S. Takamatsu (Holotype, TNS-F-47858; Isotype, MUMH 5147). Ribosomal DNA sequence ex holotype: AB743783 (28S) and AB743781 (ITS). Etymology: Epithet derived from the host genus e Castanopsis. Mycelium on fruits and leaves, amphigenous, effuse, persistent to evanescent; hyphae almost straight to somewhat wavy, uniform or irregular in width; hyphal appressoria welldeveloped, nipple-shaped, single or opposite in pairs;
276
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 2 7 4 e2 7 8
Fig. 1 e Phylogenic tree of Setoidium castanopsidis inferred from the 28S rDNA sequences analysis using maximum parsimony method. The percentage bootstrap support (1000 replications; ‡70%) are shown on the branches.
conidiophores arising from superficial hyphal mother-cells, terminal to lateral, almost in the middle of mother cell, erect, straight, (94)112e198(245) (9)10e15(17) mm; foot-cells straight, cylindrical, (31)51e118(129) (9)10e16(17) mm, producing conidia in chains (catenescent) with sinuate
Fig. 2 e Phylogenic tree of Setoidium castanopsidis inferred from the rDNA ITS sequences analysis using maximum parsimony method. The percentage bootstrap support (1000 replications; ‡70%) are shown on the branches.
outline, conidia broadly ellipsoid-ovoid, (30) 34e39(42) (15.5)16.5e20(23) mm, with fibrosin bodies which are not very conspicuous. Germ tubes not observed. Additional collections examined (paratypes): on Ca. javanica (Fagaceae), Indonesia, West Java province, Bandung, Mount Tangkuban Perahu, 12 Mar. 2011, I. Hidayat, J. Meeboon, S. Takamatsu (MUMH 5123; BO-22655). Comments: Among 10 powdery mildew species infecting hosts of the plant genus Castanopsis (Fagaceae) worldwide (Amano 1986; Braun and Cook 2012), four species belong to Cystotheca and the remaining species to the genus Erysiphe R. Hedw. ex DC (Braun and Cook 2012). The genus Setoidium is linked to the teleomorph genus Cystotheca (type: Cy. wrightii) (Cook et al. 1997; Braun and Cook 2012). The distribution of Cystotheca on Castanopsis e Cy. esetacea Z.X. Chen & Y.J. Yao, Cy. lanestris, Cy. tjibodensis, Cy. wrightii e are mostly restricted to Asia except Cy. lanestris which is distributed in Asia and North America. During the collection of tropical powdery mildews in Indonesia (Mar 2011), we found Cy. tjibodensis and its anamorph on Ca. argentea at Cibodas Botanical Garden (Cianjur)
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 2 7 4 e2 7 8
277
Cy. tjibodensis]. Setoidium castanopsidis is also distinct from Cy. lanestris and Cy. wrightii by having amphigenous colonies, distinct nipple-shaped appressoria, and longer foot-cells. The conidiophores of S. castanopsidis are shorter than those of Cy. lanestris [(94)112e198(245) (9)10.5e15(17) mm vs (85) 130e280 10e17 mm in Cy. lanestris], but longer than in Cy. wrightii (70e110 10e14 mm) (Braun and Cook 2012). Cystotheca quercina N. Ahmad et al. (2006), another Cystotheca species on Fagaceae, differs from S. castanopsidis in having shorter conidiophores (80e110 mm long), smaller foot-cells (30) 35e45(50) 4e10 mm, and smaller conidia [(20) 25e30(35) 15e18 mm]. Among Cystotheca spp. and its anamorphs recognized by Braun and Cook (2012), S. murrayae has been reported having amphigenous colonies. However, S. murrayae is distinct from S. castanopsidis by having shorter conidiophores (140e165 12e16 mm), smaller foot-cells (30e50 6e9 mm), and smaller conidia (25e35 12e22 mm) (Braun and Cook 2012).
Disclosure The authors declare no conflict of interest. All the experiments undertaken in this study comply with the current laws of Japan.
Fig. 3 e Setoidium castanopsidis on Castanopsis javanica. a: Conidia; b: Conidiophore; c: Appressoria. Bar [ 20 mm.
(Meeboon et al. 2012) and S. castanopsidis sp. nov. on Ca. javanica at two locations e Cibodas Botanical Garden (Cianjur) and mount Tangkuban Perahu (Bandung). However, the teleomorphic state of the later species was not found in either location. The phylogenetic analyses showed that the two ITS sequences of S. castanopsidis on Ca. javanica were identical and formed a distinct lineage separated from Cy. tjibodensis, Cy. lanestris and Cy. wrightii (Figs. 1 and 2). Molecular results are supported by morphological data which showed that S. castanopsidis is distinct from the anamorphic state of Cystotheca species found on hosts of the plant genus Castanopsis, viz, Cy. esetacea, Cy. lanestris, Cy. tjibodensis, and Cy. wrightii. Cystotheca esetacea was reported to be found on Ca. fabri, Ca. tibetana, and Ca. fargesii in China, but no anamorphic state was described (Braun and Cook 2012). Anamorphic states of Cy. nanyuensis (J.L. Zhou) U. Braun on Quercus multinervis (Fagaceae) and Cy. indica M.S. Patil & Maham. on Calophyllum apetalum (Callophyllaceae) are also unknown (Braun and Cook 2012). Setoidium castanopsidis differs from Cy. tjibodensis in having amphigenous colonies, distinct appressoria (nipple-shaped), longer conidiophores [(94)112e198(245) (9)10e15(17) mm vs (94)101e147(158) (10)11e13(15) mm in Cy. tjibodensis], longer foot-cells [(31)51e118(129) (9) 10e16(17) mm vs (12)19e37(61) (9)11e14(19) mm in Cy. tjibodensis], and larger conidia [(30)34e39(42) (15) 16e20(23) mm vs (22)24e30(33) (16)18e22(23) mm in
Acknowledgments The authors wish to thank Dr. Uwe Braun for critical reading the manuscript. This work was financially supported in part by a Grant-in-Aid for Scientific Research (No. 23580061) from the Japan Society of the Promotion of Science to ST and MONBUKAGAKU SHO: MEXT (Ministry of Education, Culture, Science, and Technology) Scholarship of the Japanese Government awarded to JM.
references
Ahmad S, Sarbhoy AK, Kamal, Agarwal DK, 2006. Some new powdery mildews from Northern India. Indian Phytopathology 59: 219e222. Amano (Hirata) K, 1986. Host range and geographical distribution of the powdery mildew fungi. Japan Scientific Societies Press, Tokyo. Braun U, Cook RTA, 2012. Taxonomic manual of the Erysiphales (powdery mildews). CBS Biodiversity Series No.11. CBS, Utrecht. Cook RTA, Inman AJ, Billings C, 1997. Identification and classification of powdery mildew anamorphs using light and scanning electron microscopy and host range data. Mycological Research 101: 975e1002; http://dx.doi.org/10.1017/ S095375629700364X. Edgar RC, 2004. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5: 113; http://dx.doi.org/10.1186/1471-2105-5-113. Felsenstein J, 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783e791; http:// dx.doi.org/10.2307/2408678. Hirata T, Takamatsu S, 1996. Nucleotide diversity of rDNA internal transcribed spacers extracted from conidia and
278
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 2 7 4 e2 7 8
cleistothecia of several powdery mildew fungi. Mycoscience 37: 283e288; http://dx.doi.org/10.1007/BF02461299. Meeboon J, Hidayat I, Kramadibrata K, Nurcahyanto D, Siahaan SAS, Takamatsu S, 2012. Cystotheca tjibodensis (Erysiphaceae, Ascomycota): rediscovery in Java after 90 years and first finding of anamorph. Mycoscience 53: 1e5; http:// dx.doi.org/10.1007/s10267-011-0176-6. Mori Y, Sato Y, Takamatsu S, 2000. Evolutionary analysis of the powdery mildew fungi using nucleotide sequences of the nuclear ribosomal DNA. Mycologia 92: 74e93. Nixon KC, 1999. The Parsimony Ratchet, a new method for rapid parsimony analysis. Cladistics 15: 407e414. Shin HD, La YJ, 1993. Morphology of edge lines of chained immature conidia on conidiophores in powdery mildew fungi and their taxonomic significance. Mycotaxon 66: 445e451. Sikes DS, Lewis PO, 2001. Beta software, version 1. PAUPRat: PAUP* implementation of the parsimony ratchet. Distributed by the authors. Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, USA.
Swofford DL, 2002. PAUP*: phylogenetic analysis using parsimony (*and other methods), version 4.0b10. Sinauer, Sunderland, MA. Takamatsu S, Kano Y, 2001. PCR primers useful for nucleotide sequences of rDNA of the powdery mildew fungi. Mycoscience 42: 135e139. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731e2739; http://dx.doi.org/10.1093/molbev/ msr121. Walsh SP, Metzger DA, Higuchi R, 1991. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 10: 506e513. White TJ, Bruns TD, Lee SB, Taylor JW, 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp 315e322.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/myc
Full paper
Setoidium castanopsidis, a new species of anamorphic Cystotheca (Ascomycota, Erysiphales) from Indonesia Jamjan Meeboon a, Iman Hidayat b, Susumu Takamatsu a,* a
Department of Bioresources, Graduate School, Mie University, 1577 Kurima-Machiya, Tsu 514-8507, Japan Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences - LIPI, Jl. Raya Jakarta-Bogor KM 46, Cibinong 16911, West Java, Indonesia b
article info
abstract
Article history:
A powdery mildew fungus belonging to the genus Setoidium (anamorph of Cystotheca) was
Received 31 August 2012
found on Castanopsis javanica in Cibodas Botanical Garden and mount Tangkuban Perahu,
Accepted 25 October 2012
West Java, Indonesia. The fungus is considered as a new species, namely, Setoidium cas-
Available online 7 January 2013
tanopsidis. Phylogenetic analyses of the 28S and ITS rDNA regions showed that S. castanopsidis formed a distinct lineage separated from Cy. tjibodensis, Cy. lanestris, and
Keywords:
Cy. wrightii. Setoidium castanopsidis also differs morphologically from Cy. tjibodensis in
Anamorph
having distinct appressoria (nipple-shaped), longer conidiophores with longer foot-cells,
Castanopsis
larger conidia, and being found on Ca. javanica. The teleomorphic state has not been
Erysiphaceae
found during the collection.
Molecular phylogeny
ª 2012 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Taxonomy
1.
Introduction
Setoidium (R.T.A. Cook, A.J. Inman & C. Billings) R.T.A. Cook & U. Braun is an anamorph genus in the family Erysiphaceae, linked to the teleomorph genus Cystotheca Berk. & M.A. Curtis. This genus is characterized by having mycelium bearing special pigmented aerial hyphae, falcate to filiform, a sinuate outline of the conidial chains, and germ tubes of the Fibroidium typeeorthotubus subtype (Cook et al. 1997; Braun and Cook 2012). Setoidium was recognized by Cook et al. (1997) as Oidium subgenus Setoidium (type species: Oidium japonicum Syd.). Currently only one species has been assigned to Setoidium, namely, S. murrayae (Hosag., U. Braun & Rabindran) U. Braun & R.T.A. Cook on Murraya paniculata (Rutaceae) from India (Braun and Cook 2012). There is no teleomorphic species linked to S. murrayae. All other Setoidium anamorphs, some of
them described as Oidium spp., are associated with known teleomorphs in Cystotheca so that a separate naming of these conidial states is not necessary and against the spirit of the current International Code of Nomenclature for algae, fungi, and plants. During a survey and collection of powdery mildews in Cibodas Botanical Garden and Mount Tangkuban Perahu (Indonesia) on Mar. 2011, two species of Castanopsis (Fagaceae) e Ca. argentea A. DC. and Ca. javanica Blume e were found being infected by powdery mildew. Specimens of Ca. argentea collected from Cibodas were infected by Cy. tjibodensis (Ga¨um.) Katumoto including its anamorphic state (Setoidium) (Meeboon et al. 2012). However, on specimens of Ca. javanica from Cibodas and mount Tangkuban Perahu only anamorphic states were found. Morphological examination showed that both specimens belong to a species different from the anamorph of Cy. tjibodensis. Therefore, we extracted the genomic
* Corresponding author. Tel.: þ81 59 231 9497; fax: þ81 59 231 9637. E-mail address: [email protected] (S. Takamatsu). 1340-3540/$ e see front matter ª 2012 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.myc.2012.10.004
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 2 7 4 e2 7 8
DNA from both specimens and conducted phylogenetic analyses based on the 28S and internal transcribed spacer (ITS) rDNA sequences.
2.
Materials and methods
2.1.
Morphological examination
Specimens were collected from Cibodas Botanical Garden and Mount Tangkuban Perahu, West Java province, in Java Island (Indonesia) on Mar. 2011. Details of host name, collection date, location, and collector were recorded. Herbarium samples were rehydrated before examination by boiling a small piece of infected leaf with the fungal mycelium downwards in a drop of lactic acid on a slide (Shin and La 1993). After boiling, the rehydrated mycelium was scraped off and mounted in lactic acid using a light microscope. Thirty conidia, conidiophores, foot- and mother-cells were measured per sample. Specimens were deposited at the National Museum of Nature and Science (TNS) and Mie University Mycological Herbarium (MUMH) in Japan, and Herbarium Bogoriense (BO) in Indonesia.
2.2.
Phylogenetic analysis
DNA extraction of the powdery mildew specimens was conducted according to the chelex method (Walsh et al. 1991) as described in Hirata and Takamatsu (1996). The 50 -end of the 28S (including the domains D1 and D2) and ITS rDNA regions were amplified by polymerase chain reaction (PCR) using the respective primer pairs: PM3 (Takamatsu and Kano 2001)/ TW14 (Mori et al. 2000) (28S), ITS5 (White et al. 1990)/PM6 (Takamatsu and Kano 2001) (ITS fragment 1), and PM7 (50 eRYYGACCCTCCACCCGTGYe30 þ 50 eRYYGACCCTCCCACC CGTGYe30 )/ITS4 (White et al. 1990) (ITS fragment 2). KOD FX Neo DNA polymerase (Toyobo, Japan) was used in the PCR reaction according to the manufacturer’s protocol. The amplicons of the 28S and ITS rDNA were sent to SolGent Co. Ltd. (Daejeon, South Korea) for sequencing using primer pairs NL1/NLP2 (28S) and ITS1/ITS4 (ITS), respectively. Representative sequences determined in this study were deposited in DNA Data Base of Japan (DDBJ) under the accession numbers AB743781eAB743784. Sequences generated from the respective 28S and ITS rDNA regions were aligned with sequences retrieved from DNA databases (DDBJ, EMBL, NCBI) using MUSCLE (Edgar 2004) implemented in MEGA 5 (Tamura et al. 2011). Sequences of Byssoascus striatisporus (G.L. Barron & C. Booth) Arx (U17912) and Sawadaea nankinensis (F.L. Tai) S. Takam. & U. Braun (AB353763) were used as outgroup for 28S and ITS rDNA data sets, respectively. The alignment was deposited in TreeBASE (http://www.treebase.org/) under the accession number S13331. Maximum parsimony (MP) analysis was done with the parsimony ratchet method (Nixon 1999) in PAUPRat ver. 1 (Sikes and Lewis 2001) and PAUP* 4.0b10 (Swofford 2002) for 28S rDNA data set. For ITS rDNA data set it was done by PAUP* with heuristic search. In both analyses, the ‘tree-bisection-reconstruction’ (TBR) algorithm was used as the heuristic search option. All sites were treated as unordered and unweighted, with gaps treated as missing
275
data. The strength of the internal branches of the resulting tree was tested with bootstrap (BS) analysis using 1000 replications (Felsenstein 1985). Tree scores, including tree length, CI, RI, and RC were also calculated.
3.
Results
3.1.
Phylogenetic analyses
A phylogenetic analysis of the 28S rDNA sequences was conducted in order to confirm the phylogenetic position of Setoidium on Ca. javanica among the Erysiphales. The alignment of the 28S rDNA contained 55 sequences and 818 total characters, of which 585 were constant, 59 were variable but parsimony-uninformative, and 174 were parsimonyinformative. From the 170 equally most parsimonious trees (MPTs) with 815 steps (CI ¼ 0.431, RI ¼ 0.749, RC ¼ 0.323) generated by the parsimony ratchet method, a tree with the highest likelihood value was obtained (Fig. 1). The 28S rDNA data set showed that sequences of the two Setoidium specimens on Ca. javanica have an identical sequence. This Setoidium clade was sister to the clade consisting of Cy. lanestris (Harkn.) Miyabe and Cy. wrightii Berk. & M.A. Curtis with 98% BS support. This result confirmed phylogenetically anamorph-teleomorph connection between Setoidiume Cystotheca as previously shown by Meeboon et al. (2012) on Cy. tjibodensis. Alignment of the ITS rDNA region contained 10 sequences and 479 characters, of which 382 were constant, 67 were variable but parsimony-uninformative, and 30 were parsimony-informative. The MP analysis resulted in two MPTs with a tree length of 122 steps (CI ¼ 0.885, RI ¼ 0.750, RC ¼ 0.664) (Fig. 2). Sequences of the two Setoidium specimens on Ca. javanica were identical. The phylogenetic tree also showed that the Setoidium sequences on Ca. javanica formed a distinct lineage separated from Cy. tjibodensis, Cy. lanestris and Cy. wrightii, which confirmed that the Setoidium on Ca. javanica is a species different from Cy. tjibodensis.
3.2.
Taxonomy
Setoidium castanopsidis Meeboon & S. Takam. sp. nov. Fig. 3aec. MycoBank no.: MB801230. Differs from the anamorphic state of Cy. tjibodensis in having amphigenous colonies, distinct nipple-shaped appressoria, longer conidiophores, longer foot-cells, larger conidia, and being found on Ca. javanica. Type: on Ca. javanica Blume (Fagaceae), Indonesia, West Java Province, Cianjur, Cibodas Botanical Garden, 14 Mar 2011, I. Hidayat, J. Meeboon, S. Takamatsu (Holotype, TNS-F-47858; Isotype, MUMH 5147). Ribosomal DNA sequence ex holotype: AB743783 (28S) and AB743781 (ITS). Etymology: Epithet derived from the host genus e Castanopsis. Mycelium on fruits and leaves, amphigenous, effuse, persistent to evanescent; hyphae almost straight to somewhat wavy, uniform or irregular in width; hyphal appressoria welldeveloped, nipple-shaped, single or opposite in pairs;
276
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 2 7 4 e2 7 8
Fig. 1 e Phylogenic tree of Setoidium castanopsidis inferred from the 28S rDNA sequences analysis using maximum parsimony method. The percentage bootstrap support (1000 replications; ‡70%) are shown on the branches.
conidiophores arising from superficial hyphal mother-cells, terminal to lateral, almost in the middle of mother cell, erect, straight, (94)112e198(245) (9)10e15(17) mm; foot-cells straight, cylindrical, (31)51e118(129) (9)10e16(17) mm, producing conidia in chains (catenescent) with sinuate
Fig. 2 e Phylogenic tree of Setoidium castanopsidis inferred from the rDNA ITS sequences analysis using maximum parsimony method. The percentage bootstrap support (1000 replications; ‡70%) are shown on the branches.
outline, conidia broadly ellipsoid-ovoid, (30) 34e39(42) (15.5)16.5e20(23) mm, with fibrosin bodies which are not very conspicuous. Germ tubes not observed. Additional collections examined (paratypes): on Ca. javanica (Fagaceae), Indonesia, West Java province, Bandung, Mount Tangkuban Perahu, 12 Mar. 2011, I. Hidayat, J. Meeboon, S. Takamatsu (MUMH 5123; BO-22655). Comments: Among 10 powdery mildew species infecting hosts of the plant genus Castanopsis (Fagaceae) worldwide (Amano 1986; Braun and Cook 2012), four species belong to Cystotheca and the remaining species to the genus Erysiphe R. Hedw. ex DC (Braun and Cook 2012). The genus Setoidium is linked to the teleomorph genus Cystotheca (type: Cy. wrightii) (Cook et al. 1997; Braun and Cook 2012). The distribution of Cystotheca on Castanopsis e Cy. esetacea Z.X. Chen & Y.J. Yao, Cy. lanestris, Cy. tjibodensis, Cy. wrightii e are mostly restricted to Asia except Cy. lanestris which is distributed in Asia and North America. During the collection of tropical powdery mildews in Indonesia (Mar 2011), we found Cy. tjibodensis and its anamorph on Ca. argentea at Cibodas Botanical Garden (Cianjur)
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 2 7 4 e2 7 8
277
Cy. tjibodensis]. Setoidium castanopsidis is also distinct from Cy. lanestris and Cy. wrightii by having amphigenous colonies, distinct nipple-shaped appressoria, and longer foot-cells. The conidiophores of S. castanopsidis are shorter than those of Cy. lanestris [(94)112e198(245) (9)10.5e15(17) mm vs (85) 130e280 10e17 mm in Cy. lanestris], but longer than in Cy. wrightii (70e110 10e14 mm) (Braun and Cook 2012). Cystotheca quercina N. Ahmad et al. (2006), another Cystotheca species on Fagaceae, differs from S. castanopsidis in having shorter conidiophores (80e110 mm long), smaller foot-cells (30) 35e45(50) 4e10 mm, and smaller conidia [(20) 25e30(35) 15e18 mm]. Among Cystotheca spp. and its anamorphs recognized by Braun and Cook (2012), S. murrayae has been reported having amphigenous colonies. However, S. murrayae is distinct from S. castanopsidis by having shorter conidiophores (140e165 12e16 mm), smaller foot-cells (30e50 6e9 mm), and smaller conidia (25e35 12e22 mm) (Braun and Cook 2012).
Disclosure The authors declare no conflict of interest. All the experiments undertaken in this study comply with the current laws of Japan.
Fig. 3 e Setoidium castanopsidis on Castanopsis javanica. a: Conidia; b: Conidiophore; c: Appressoria. Bar [ 20 mm.
(Meeboon et al. 2012) and S. castanopsidis sp. nov. on Ca. javanica at two locations e Cibodas Botanical Garden (Cianjur) and mount Tangkuban Perahu (Bandung). However, the teleomorphic state of the later species was not found in either location. The phylogenetic analyses showed that the two ITS sequences of S. castanopsidis on Ca. javanica were identical and formed a distinct lineage separated from Cy. tjibodensis, Cy. lanestris and Cy. wrightii (Figs. 1 and 2). Molecular results are supported by morphological data which showed that S. castanopsidis is distinct from the anamorphic state of Cystotheca species found on hosts of the plant genus Castanopsis, viz, Cy. esetacea, Cy. lanestris, Cy. tjibodensis, and Cy. wrightii. Cystotheca esetacea was reported to be found on Ca. fabri, Ca. tibetana, and Ca. fargesii in China, but no anamorphic state was described (Braun and Cook 2012). Anamorphic states of Cy. nanyuensis (J.L. Zhou) U. Braun on Quercus multinervis (Fagaceae) and Cy. indica M.S. Patil & Maham. on Calophyllum apetalum (Callophyllaceae) are also unknown (Braun and Cook 2012). Setoidium castanopsidis differs from Cy. tjibodensis in having amphigenous colonies, distinct appressoria (nipple-shaped), longer conidiophores [(94)112e198(245) (9)10e15(17) mm vs (94)101e147(158) (10)11e13(15) mm in Cy. tjibodensis], longer foot-cells [(31)51e118(129) (9) 10e16(17) mm vs (12)19e37(61) (9)11e14(19) mm in Cy. tjibodensis], and larger conidia [(30)34e39(42) (15) 16e20(23) mm vs (22)24e30(33) (16)18e22(23) mm in
Acknowledgments The authors wish to thank Dr. Uwe Braun for critical reading the manuscript. This work was financially supported in part by a Grant-in-Aid for Scientific Research (No. 23580061) from the Japan Society of the Promotion of Science to ST and MONBUKAGAKU SHO: MEXT (Ministry of Education, Culture, Science, and Technology) Scholarship of the Japanese Government awarded to JM.
references
Ahmad S, Sarbhoy AK, Kamal, Agarwal DK, 2006. Some new powdery mildews from Northern India. Indian Phytopathology 59: 219e222. Amano (Hirata) K, 1986. Host range and geographical distribution of the powdery mildew fungi. Japan Scientific Societies Press, Tokyo. Braun U, Cook RTA, 2012. Taxonomic manual of the Erysiphales (powdery mildews). CBS Biodiversity Series No.11. CBS, Utrecht. Cook RTA, Inman AJ, Billings C, 1997. Identification and classification of powdery mildew anamorphs using light and scanning electron microscopy and host range data. Mycological Research 101: 975e1002; http://dx.doi.org/10.1017/ S095375629700364X. Edgar RC, 2004. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5: 113; http://dx.doi.org/10.1186/1471-2105-5-113. Felsenstein J, 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783e791; http:// dx.doi.org/10.2307/2408678. Hirata T, Takamatsu S, 1996. Nucleotide diversity of rDNA internal transcribed spacers extracted from conidia and
278
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 2 7 4 e2 7 8
cleistothecia of several powdery mildew fungi. Mycoscience 37: 283e288; http://dx.doi.org/10.1007/BF02461299. Meeboon J, Hidayat I, Kramadibrata K, Nurcahyanto D, Siahaan SAS, Takamatsu S, 2012. Cystotheca tjibodensis (Erysiphaceae, Ascomycota): rediscovery in Java after 90 years and first finding of anamorph. Mycoscience 53: 1e5; http:// dx.doi.org/10.1007/s10267-011-0176-6. Mori Y, Sato Y, Takamatsu S, 2000. Evolutionary analysis of the powdery mildew fungi using nucleotide sequences of the nuclear ribosomal DNA. Mycologia 92: 74e93. Nixon KC, 1999. The Parsimony Ratchet, a new method for rapid parsimony analysis. Cladistics 15: 407e414. Shin HD, La YJ, 1993. Morphology of edge lines of chained immature conidia on conidiophores in powdery mildew fungi and their taxonomic significance. Mycotaxon 66: 445e451. Sikes DS, Lewis PO, 2001. Beta software, version 1. PAUPRat: PAUP* implementation of the parsimony ratchet. Distributed by the authors. Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, USA.
Swofford DL, 2002. PAUP*: phylogenetic analysis using parsimony (*and other methods), version 4.0b10. Sinauer, Sunderland, MA. Takamatsu S, Kano Y, 2001. PCR primers useful for nucleotide sequences of rDNA of the powdery mildew fungi. Mycoscience 42: 135e139. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731e2739; http://dx.doi.org/10.1093/molbev/ msr121. Walsh SP, Metzger DA, Higuchi R, 1991. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 10: 506e513. White TJ, Bruns TD, Lee SB, Taylor JW, 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp 315e322.