m y c o s c i e n c e x x x ( 2 0 1 3 ) 1 e9 Available online at www.sciencedirect.com journal homepage: www.elsevier.com/locate/myc Full paper Puccinia galiiuniversa, a new caricicolous rust fungus systemically inhabiting Galium aparine in its spermogonialeaecial stage5 Izumi Okane a,*, Yuichi Yamaoka a, Makoto Kakishima a, Junichi Peter Abe a, Kazuo Obata b a b Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan Ibaraki Nature Museum, 700 Osaki, Bando, Ibaraki 306-0622, Japan article info abstract Article history: Field observations and inoculation experiments revealed that spermogonia and aecia Received 16 December 2012 produced on systemically infected plants of Galium aparine and uredinia and telia on Carex Received in revised form maackii are different stages of the life cycle of a Puccinia fungus. By comparative 27 May 2013 morphology with allied species, the fungus was concluded to be a new species and named Accepted 30 May 2013 as Puccinia galiiuniversa. Available online xxx ª 2013 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. Keywords: Cyperaceae Life cycle Rubiaceae Rust fungus Taxonomy 1. Introduction Systemically infected plants of Galium aparine L. [¼ G. spurium L. var. echinospermon (Wallr.) Hayek] were found in the riparian vegetation along the Tone River and along Sugao Marsh, Ibaraki, Central Honshu, Japan. All leaves of the infected plants had spermogonia (type 4 of Hiratsuka and Cummins 1963) and aecia (aecidium type of Sato and Sato 1985). Based on morphological observations, this rust seemed to be a spermogonialeaecial stage of a heteroecious species of Puccinia or Uromyces. An undescribed Puccinia species with hyaline teliospores germinable without dormancy was collected on Carex maackii Maxim, in the same riparian vegetation. Seventy-two caricicolous rusts have been reported in Japan (Kakishima and Sato 1980; Kakishima and Sato 1982; Harada 1986; Hiratsuka et al. 1992; Okane and Kakishima 1992; Kakishima et al. 1999; Ono 2001; Ono et al. 2001). Among them, 70 species producing uredinial and/or telial stages on Carex species belong to the genus Puccinia Pers., and the other two are Uromyces caricis-brunneae Y. Morimoto and Uredo caricisincisae S. Ito ex S. Ito et Murayama. No caricicolouseheteroecious rust fungi producing spermogonialeaecial stages on Galium and other rubiaceous species have been reported previously, not only in Japan, but also in other regions (Saccardo 1888, 1899, 1905; Cunningham 1924; Arthur 1934; McKenzie 1998; Wang and Zhuang 1998; Kuo and Chen 1999; Kim et al. 5 Contribution No. 252. * Corresponding author. Tel./fax: þ81 29 853 6687. E-mail address: okane.izumi.fw@u.tsukuba.ac.jp (I. Okane). 1340-3540/$ e see front matter ª 2013 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.myc.2013.05.008 Please cite this article in press as: Okane I, et al., Puccinia galiiuniversa, a new caricicolous rust fungus systemically inhabiting Galium aparine in its spermogonialeaecial stage, Mycoscience (2013), http://dx.doi.org/10.1016/j.myc.2013.05.008 2 m y c o s c i e n c e x x x ( 2 0 1 3 ) 1 e9 Table 1 e Results of the field observation. Locality Tone River, Togashira, Toride, Ibaraki Pref. Sugao Marsh, Osaki, Bando, Ibaraki Pref. Date of survey Presence of host plant/rust sorusa G. aparine C. maackii þ/I þ/ þ/ þ/I þ/I þ/ þ/ þ/I þ/I þ/ þ/I þ/ þ/II, III þ/II, III þ/ þ/II þ/II, III þ/II, III þ/ þ/ þ/II, III þ/ 29 Apr 2010 14 Nov 2010 10 Jan 2011 3 May 2011 5 Jun 2011 16 Nov 2011 4 Dec 2011 22 Apr 2012 11 Apr 2009 19 Dec 2010 7 May 2011 a þ: Host present, I: aecial stage, II: uredinial stage, III: telial stage, : no sorus. 2004; Azbukina 2005; Kazeroni et al. 2010; Singh and Palni 2011; Bahcecioglu and Kabaktepe 2012). In the present study, we carried out a field survey and inoculation experiments to confirm the life cycle connection between the rust fungus on C. maackii and G. aparine. We performed morphological analysis of the rust fungus in comparison with related Puccinia and/or Aecidium species reported from Carex, Galium, and other rubiaceous plants to determine its taxonomy. This rust is described as a new heteroecious species belonging to the genus Puccinia. 2. Materials and methods 2.1. Field survey We visited the riparian vegetation along the Tone River, Togashira, Toride, Ibaraki, and that along Sugao Marsh, Osaki, Bando, Ibaraki and Otsukado-machi, Jyoso, Ibaraki, Central Honshu, Japan between Apr 2009 and Apr 2012 (Table 1). Spore production of the rust on C. maackii and G. aparine was observed. 2.2. Inoculation experiments with aeciospores collected in the field Aeciospores on systemically infected G. aparine were collected from the riparian vegetation along the Tone River, Togashira, and that along Sugao Marsh, Osaki, and Otsukado-machi (Table 2). Carex maackii was collected from the riparian vegetation along the Tone River, Togashira and that along Sugao Marsh, Osaki. They were planted in plastic pots and grown in a growth cabinet at a controlled temperature of ca. 20  C under 16 h light (50 mmol m 2 s 1): 8 h dark photoperiod. Young fresh leaves of C. maackii in the pots were inoculated with aeciospores using a small (ca. 3  3 mm) filter paper dusted with spores using a scalpel and then placed on the lower surface of the leaves. After keeping in a moist chamber overnight, the plants were incubated in a growth cabinet under the above conditions. They were observed continuously for 3 week after inoculation. 2.3. Inoculation experiments with basidiospores Teliospores produced on C. maackii were collected from the riparian vegetation along the Tone River, Togashira on 16 Oct 2011 and 4 Dec 2011 (Table 3). They were also collected from the riparian vegetation along Sugao Marsh, Osaki and Otsukado-machi. Small seedlings of G. aparine were collected from a field in Azuma, Tsukuba, Ibaraki, Japan. This field is located in an urban area far from large rivers and ponds. It was used to survey the rust flora between 2006 and 2008 (Yamaoka et al. 2009), and also occasionally since then. No rust infection on G. aparine had been detected previously. The collected seedlings were planted in plastic pots and incubated in a growth cabinet under the same conditions as described for C. maackii. When inoculated on 28 Oct 2011, the seedlings had two to three verticillate leaves. Leaves of C. maackii with telia were cut into segments w5 mm in length, which were placed on top of the shoots of G. aparine in pots and incubated at 20  C in the dark for 2 days. The inoculated plants were incubated in a growth cabinet under the same conditions as described the aeciospore Table 2 e Aeciospores on Galium aparine used in inoculation experiments and results of inoculation experiments with the aeciospores. No. As-1 As-2 As-3 As-4 Aeciopsores as inoculum Inoculation experiments with aeciospores to Carex maackii Locality of collection Date of collection Voucher specimen no. Date of inoculation Date of the first appearance of uredinia (days after inoculation) Voucher specimen no. Tone River, Togashira, Toride, Ibaraki Pref. Tone River, Togashira, Toride Ibaraki Pref. Sugao Marsh, Osaki, Bando, Ibaraki Pref. Sugao Marsh, Otsukado-machi, Jyoso, Ibaraki Pref. 3 May 2011 TSH-R 11875 3 May 2011 16 May 2011 (13 days) TSH-R 11898 3 May 2011 TSH-R 11876 3 May 2011 16 May 2011 (13 days) TSH-R 11899 7 May 2011 TSH-R 11891 7 May 2011 25 May 2011 (18 days) TSH-R 11904 7 May 2011 TSH-R 11892 7 May 2011 25 May 2011 (18 days) TSH-R 11905 Please cite this article in press as: Okane I, et al., Puccinia galiiuniversa, a new caricicolous rust fungus systemically inhabiting Galium aparine in its spermogonialeaecial stage, Mycoscience (2013), http://dx.doi.org/10.1016/j.myc.2013.05.008 3 m y c o s c i e n c e x x x ( 2 0 1 3 ) 1 e9 Table 3 e Teliospores on C. maackii used in inoculation experiments and results of the inoculation experiments with basidiospores to G. aparine. No. Ts-1 Ts-2 Ts-3 Teliospores tested as inoculum Inoculation experiments with basidiospores produced on the teliospores Locality of collection Date of collection Voucher specimen no. Date of inoculation Date of the first appearance of aecia (days after inoculation)a Numbers of infected plants/numbers of inoculated plants Voucher specimen no. Togashira, Toride, Ibaraki Pref. 16 Oct 2011 TSH-R 11881 4 Dec 2011 TSH-R 11883 21 Nov 2011 (24 days) e 21 Nov 2011 (18 days) e 15 Jan 2012 (29 days) 13/14 0/12 8/18 0/13 18/19 TSH-R 11896 Togashira, Toride, Ibaraki Pref. Togashira, Toride, Ibaraki Pref. 28 Oct 2011 Control 3 Nov 2011 Control 17 Dec 2011 4 Dec 2011 TSH-R 11884 17 Dec 2011 Control 15 Jan 2012 (29 days) e 16/17 0/15 TSH-R 11897 TSH-R 11900 TSH-R 11901 a e: no sign. experiment. They were observed continuously for 2 months after inoculation. 2.4. Inoculation experiments with aeciospores Aeciospores produced in the aecia obtained from the inoculation experiment using basidiospores were used as inocula (Table 4). Young fresh leaves of C. maackii in the pots were inoculated with aeciospores. After storage in a moist chamber overnight, the plants were incubated in a growth cabinet and observed continuously for 3 week after inoculation. 2.5. Morphological observation The telia and uredinia on C. maackii and the spermogonia and aecia on G. aparine collected in the field and obtained from inoculation experiments were used for morphological observations. Specimens examined are listed in the species descriptions. The spores or thin sections of sori were mounted in a drop of lactophenol solution on glass slides for observations by light microscopy (LM). To examine the morphology and for size measurement, 20e50 spores from each specimen were examined. Morphological observations were made by LM and scanning electron microscopy (SEM). For SEM, sori or spores obtained from dry specimens were attached to specimen holders by double-sided adhesive tape and coated with platinumepalladium in a high vacuum with a Hitachi E-1030 Ion Sputter (Hitachi, Tokyo, Japan). They were examined with a Hitachi S-4200 SEM operating at 15 kV. To compare the morphological features of the present rust fungus with other related rust fungi, dried specimens, the original descriptions and/or other reports were examined (Table 5). The statistical significance of differences in dimension data of specimens of the present rust compared to those of specimens of other related rusts was calculated by one-way ANOVA followed by Dunnett’s post-hoc test using SPSS ver. 20.0 for Windows (SPSS, Chicago, IL). 2.6. Sequence analysis of 28S rDNA D1/D2 and internal transcribed spacer (ITS) regions of the present rust DNA was extracted from aeciospores from an aecia obtained in the inoculation experiment with basidiospores. DNA extraction was performed according to the method described by Virtudazo et al. (2001). Polymerase chain reaction (PCR) was conducted using a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA) under the conditions used by Virtudazo et al. (2001). The D1/D2 region was amplified using the primer pair, NL1 (50 -GCATATCAATAAGCGGAGGAAAAG-30 ) Table 4 e Results of inoculation experiments with aeciospores, which were obtained from inoculation experiments using basidiospores, to C. maackii. Inoculum Ts-1e1 (¼ TSH-R 11896) Ts-1e2 (¼ TSH-R 11897) Ts-2e3 (¼ TSH-R 11900) Ts-3e3 (¼ TSH-R 11901) Date of inoculation 4 Jan 2012 19 Dec 2011 4 Jan 2012 27 Jan 2012 27 Jan 2012 Date of the first appearance of uredinia (days after inoculation) 24 12 24 17 17 Jan 2012 (20 days) Jan 2012 (24 days) Jan 2012 (20 days) Feb 2012 (21 days) Feb 2012 (21 days) Voucher specimen no. TSH-R 11902 TSH-R 11903 Please cite this article in press as: Okane I, et al., Puccinia galiiuniversa, a new caricicolous rust fungus systemically inhabiting Galium aparine in its spermogonialeaecial stage, Mycoscience (2013), http://dx.doi.org/10.1016/j.myc.2013.05.008 4 Species and host plants The present fungus 0 I: Galium aparine II III: Carex maackii Puccinia caricisfediae 0 I: Lysimachia japonica II III: Carex fedia var. miyabei Puccinia hyalina 0 I: unknown II III: Carex baccans, C. siderosticta Puccinia mandshurica 0 I: unknown II III: Carex siderosticta Type of spermogonia Aeciospore Size (mm) Urediniospore Wall Surface thickness (mm) Size (mm) Wall thickness (mm) 0.8e1.5 Teliospore Surface Size (mm) Echinulate 27e43  9e14 Wall Apical thickness wall (mm) thickness (mm) Type 4 13e16  10e14 0.4e1.3 Verrucose 15e22  10e14 Type 4 17e20  16e20 (14e18**  12e15**)a 0.7e0.8 (0.7e1.0)a Verrucose 20e27  15e19 1 (0.8e1.7)b Echinulate 38e60  11e15 (0.7e1.3**)b (2.1e5.7)b b b (17e26**  15e20**) (34e53**  11e16**) 18e25  16e21 (21.1e26.5**  17.5e20.3**)c 1e2 (0.8e1.4)c Echinulate 31e60  13e20 (30.3e44.6*  10.9e15.2**)c 28e36  18e25 (23.8e30.7**  17.9e22.5**)d 1e1.5 Echinulate 36e50  10e15 (1.5e2.1**)d (29.2e44.2**  9.7e13.5*)d **p < 0.01, *p < 0.05 in statistic test between the data of the present rust and those of other rusts. Blank: no data. a Data of No. 10790 on Lysimachia japonica, JAPAN: Aomori, Nishigaoka, Hirosaki, 25 Jun. 1980, Y. Harada. b Data of No. 11690 (holotype) on Carex fedia var. miyabei, JAPAN: Aomori, Hirosaki (obtained aeciospore inoculation), 14 Aug. 1981, Y. Harada. c Data of TSH-R1266 (¼ HMAS 41753) on Carex sp., CHINA: Fujian, Pucheng, 26 Oct. 1980, J.-Y. Zhuang. d Data of TNS-F-188496 on Carex siderosticta, JAPAN: Tosa, Matsuo-saka, Hadamura, 11 Nov. 1929, T. Yoshinaga. e Data derived from Dietel (1905b); Arthur (1934); Harada (1986); Hiratsuka et al. (1992); Wang and Zhuang (1998); Kuo and Chen (1999). 0.4e1.5 1e5 Pedicel (mm) 19e59 Pale yellow to hyaline 30e60 (e80) (25e50)b Pale yellow (0.9e1.6**)c 8e10 Up to 45 (3.2e8.2**)c (13.3e40.4)c (0.6e1.5**)d (3.3e5.1)d Wall color Pale yellow to nearly colorless 14 Pale (4.6e16.5**)d yellow m y c o s c i e n c e x x x ( 2 0 1 3 ) 1 e9 Please cite this article in press as: Okane I, et al., Puccinia galiiuniversa, a new caricicolous rust fungus systemically inhabiting Galium aparine in its spermogonialeaecial stage, Mycoscience (2013), http://dx.doi.org/10.1016/j.myc.2013.05.008 Table 5 e Morphological characters and host plants of four rust species producing pale-yellow to hyaline teliospores on Carexe. m y c o s c i e n c e x x x ( 2 0 1 3 ) 1 e9 and NL4 (50 -GGTCCGTGTTTCAAGACGG-30 ) (O’Donnell 1993). The ITS region was amplified using primers ITS5 (50 -GGAAGTAAAAGTCGTAACAAGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ) (White et al. 1990). Purified PCR products were then directly sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) with the same primers as used for PCR amplification. Sequencing was performed using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). 3. Results 3.1. Field survey The results of the field survey are shown in Table 1. In November, small seedlings of G. aparine were confirmed in the riparian vegetation. There was no sign of infection. Uredinia and telia were produced on leaves of C. maackii. More telia were produced on leaves of C. maackii collected in December and January. The first signs of systemic infection by the rust on G. aparine were observed in April. Systemically infected shoots and plants were paler in color and had shorter leaves than healthy plants. Aecia were produced on the abaxial surface of all the verticillated leaves on the shoots (Fig. 1A). 3.2. Inoculation experiments Four samples of aecial stage on G. aparine collected in the riparian vegetation along the Tone River and Sugao Marsh were used as inocula (Table 2). Thirteen or eighteen days after inoculation, whitish uredinia were produced on the abaxial surface of the leaves of C. maackii (Table 2). Three samples of telia on C. maackii collected in the riparian vegetation along the Tone River were used as inocula (Table 3). Eighteen to twenty-nine days after inoculation, spermogonia and aecia were produced on verticillate leaves of G. aparine (Table 3). When they appeared, aecia were produced all of the verticillate leaves. Once they appeared, all the verticillated leaves expanded later produced aecia; i.e., the shoots were systemically infected. However, the older parts did not produce aecia. Aeciospores resulting from the inoculation experiments with basidiospores were used as inocula. Twenty to twentyfour days after inoculation, whitish uredinia were produced on the abaxial surface of the leaves of C. maackii (Table 4). 3.3. Morphology The morphological characteristics of the present rust and other rusts are shown in Table 5. There were no significant morphological differences in the spermogonialeaecial stage of the present rust between infections obtained naturally or by basidiospore inoculation on G. aparine, and in the uredinialetelial stages between infections formed either naturally or by aeciospore inoculation on C. maackii. Germ pores of urediniospore of the present rust were obscure even after staining with lactophenol cotton-blue solution. 3.4. 5 Taxonomy Puccinia galiiuniversa Okane & Yamaoka, sp. nov. Figs. 1 and 2 MycoBank no.: MB 802495. Differs from other caricicolous rusts in having smaller urediniospores and teliospores, apical shape of teliospores, systemic infection on G. aparine in its spermogonialeaecial stage. This rust turns the spermogonialeaecial host sterile. Holotype. II, III on Carex maackii, JAPAN: Ibaraki, Togashira, Toride, 4 Dec. 2011, Y. Yamaoka, TNS-F-38438. Etymology: Latin Galium and universus ¼ whole, referring to its systemic infection of the spermogonialeaecial host, Galium aparine. Spermogonia hypophyllous, scattered, yellow to brown, subepidermal, and flask-shaped (type 4 of Hiratsuka and Cummins 1963, Fig. 1B), 99e143 mm high  104e152 mm wide (mean  SE ¼ 124  2.5  130  2.8). Aecia hypophyllous, scattered (Fig. 1A), cupulate with peridia (Fig. 1C and D), and pale yellow. Peridial cells irregular polygonal with a concave inner wall, verrucose (Fig. 1C), 18e27  13e19 mm (mean  SE ¼ 22.4  0.54  16.3  0.30). Aeciospores globose to subglobose, often angular (Fig. 1E), 13e16  10e14 mm (mean  SE ¼ 14.4  0.11  11.7  0.12), 1.0e1.5 in length/width (mean ¼ 1.2). Walls 0.4e1.3 mm thick (mean  SE ¼ 0.8  0.02), colorless, and verrucose with granules (type 5 of Savile 1973, Fig. 1F). Uredinia hypophyllous, scattered, erumpent, pale yellow without paraphyses. Urediniospores obovoid to ellipsoid (Fig. 2A), and 15e19(e22)  10e14 mm (mean  SE ¼ 17.2  0.18  11.8  0.12), 1.1e1.8 in length/width (mean ¼ 1.4). Walls 0.8e1.5 mm thick (mean  SE ¼ 1.0  0.02), pale yellowish to colorless and echinulate (Fig. 2B). Germ pores obscure. Telia hypophyllous, scattered, erumpent, pale yellow to hyaline without paraphyses. Teliospores fusiform to ellipsoid, constricted at the septa (Fig. 2C), and 27e43  9e14 mm (mean  SE ¼ 34.0  0.50  10.6  0.11). Walls 0.4e1.5 mm thick (mean  SE ¼ 0.7  0.02) with apical thickenings of 1e5 mm (mean  SE ¼ 3.5  0.11), pale yellow to colorless, and smooth. Pedicels 19e59 mm long (mean  SE ¼ 35.3  1.23) and persistent. Teliospores germinable without dormancy. Other specimens examined: 0, I on G. aparine, JAPAN: Ibaraki, Tone river, Togashira, Toride, 3 May 2011, Y. Yamaoka (YY), TSH-R 11875; TSH-R 11876; Ibaraki, Sugao Marsh, Osaki, Bando, 7 May 2011, YY, TSH-R 11891; Sugao Marsh, Otsukadomachi, Jyoso, 7 May 2011, YY, TSH-R 11892; Tsukuba (obtained from basidiospore inoculation), 31 Jan 2012, YY, TSH-R 11896; TSH-R 11897; 17 Feb 2012, YY, TSH-R 11900; TSH-R 11901. II, III on C. maackii, JAPAN: Ibaraki, Togashira, Toride, 16 Oct 2011, YY, TSH-R 11881; 4 Dec 2011, YY, TSH-R 11884; Tsukuba (obtained from aeciospore inoculation), 4 Jun 2011, YY, TSH-R 11898; TSH-R 11899; TSH-R 11904; TSH-R 11905; 13 Mar 2012, YY, TSH-R 11902; 5 Mar 2012, YY, TSH-R 11903. 3.5. Sequences of 28S rDNA D1/D2 and ITS regions of the present rust The successfully read ITS region sequences and partial sequence of the 28S rDNA D1/D2 region of the present rust fungus (Accession No. AB781702 and AB781703, respectively) were Please cite this article in press as: Okane I, et al., Puccinia galiiuniversa, a new caricicolous rust fungus systemically inhabiting Galium aparine in its spermogonialeaecial stage, Mycoscience (2013), http://dx.doi.org/10.1016/j.myc.2013.05.008 6 m y c o s c i e n c e x x x ( 2 0 1 3 ) 1 e9 Fig. 1 e Spermogonialeaecial stage of Puccinia galiiuniversa. A: Aecia on G. aparine. B: A vertical section of spermogonia and aecium on G. aparine. C: Aecium observed by SEM. D: A vertical section of aecium. E: Aeciospores. F: Aeciospore having granules on the surface observed by SEM. Bars A 10 mm; B & D 100 mm; C 200 mm; E 20 mm; F 5 mm. Please cite this article in press as: Okane I, et al., Puccinia galiiuniversa, a new caricicolous rust fungus systemically inhabiting Galium aparine in its spermogonialeaecial stage, Mycoscience (2013), http://dx.doi.org/10.1016/j.myc.2013.05.008 m y c o s c i e n c e x x x ( 2 0 1 3 ) 1 e9 7 Fig. 2 e Uredinialetelial stage of Puccinia galiiuniversa. A: Urediniospores. B: Urediniospores observed by SEM. C: Teliospores. Bars A 10 mm; B 5 mm; C 20 mm. analyzed by Basic Local Alignment Search Tool (BLAST) search. However, no identical sequences were retrieved from the DDBJ/ EMBL/GenBank nucleotide sequence databases. Although specimens of P. caricis-fediae Y. Harada were also examined to analyze the DNA sequences, these experiments were unsuccessful. 4. Discussion Inoculation experiments revealed that the present rust fungus is a heteroecious species of Puccinia with the spermogonialeaecial stage on G. aparine and the uredinial-telial stage on C. maackii. No rust fungi have been reported from C. maackii not only in Japan, but also in other regions in which the plant has been recorded (Wang and Zhuang 1998; Kuo and Chen 1999; Kim et al. 2004; Azbukina 2005). According to field survey and inoculation experiments, the almost colorless teliospores of the present rust were revealed to germinate without dormancy and infect G. aparine systemically. While the first signs of systemic infection of G. aparine were observed in early spring, infection by basidiospores arising from teliospores without dormancy may begin in early winter when the seedlings of G. aparine were confirmed in the field. In early spring when pollinators would be active, cross-fertilization between compatible spermogonia formed on the leaves may occur, and then the aecial stage may be formed. Further studies are required to confirm the timing of rustinfection and the latent mode of the rust after infection. The present rust forms colorless teliospores germinable without dormancy. While pale-colored teliospores have been described in several caricicolous species, i.e., Puccinia caricisfediae, P. hyalina Dietel, P. mandshurica Miura, P. microsora Körn. (Saccardo 1888; Dietel 1905b; Arthur 1934; Harada 1986; Hiratsuka et al. 1992; Wang and Zhuang 1998; Kuo and Chen 1999), the present rust can be distinguished from these rust fungi in being germinable without dormancy and/or having smaller spores (teliospores, urediniospores and aeciospores). Among these caricicolous Puccinia, P. caricis-fediae is most Please cite this article in press as: Okane I, et al., Puccinia galiiuniversa, a new caricicolous rust fungus systemically inhabiting Galium aparine in its spermogonialeaecial stage, Mycoscience (2013), http://dx.doi.org/10.1016/j.myc.2013.05.008 8 m y c o s c i e n c e x x x ( 2 0 1 3 ) 1 e9 similar to the present fungus in producing pale-yellow teliospores that are germinable without dormancy (Harada 1986). In addition, the spermogonialeaecial stage of this rust, which was named Aecidium lysimachiae-japonicae Dietel (1905a), systemically infect Lysimachia japonica Thunb. (Primulaceae). However, according to the description by Harada (1986) and observation of the holotype specimen (No. 11690, Hirosaki University), this species clearly possesses larger aeciospores, teliospores, and urediniospores (Table 5). In terms of morphology of apical shape of teliospores, P. caricis-fediae has a papilla on the apex, while the present rust forms teliospores with a thickened apical wall. In morphological observation of a specimen of the spermogonialeaecial stage of P. caricis-fediae (No. 10790, Hirosaki Univ.), the dimensions of aeciospores measured were 14e18  12e15 mm. These data correspond to those in the description by Dietel (1905a), as Aecidium lysimachiae-japonica Dietel. In addition to the host differences in the spermogonialeaecial stages of those rusts, namely, L. japonica (Primulaceae) for P. caricis-fediae and G. aparine (Rubiaceae) for the present rust, these rusts were clearly distinguished from each other in the morphological features of the uredinial-telial stage. Puccinia hyalina and P. mandshurica, whose spermogonialeaecial hosts are unknown, form significantly lager urediniospores and teliospores according to the descriptions (Dietel 1905b; Hiratsuka et al. 1992; Wang and Zhuang 1998; Kuo and Chen 1999). We examined specimens of the two rusts. While the teliospore sizes of these specimens were slightly smaller than described in the literature, they were significantly larger than those of the present rust. The same was true for urediniospore sizes (Table 5). In addition, the apical wall thickness of teliospores distinguishes P. hyalina from the present rust. Puccinia mandshurica is clearly distinguished from the present rust by the digitate apex and shorter pedicel of teliospores. Puccinia microsora, whose spermogonialeaecial host is also unknown, has significantly larger urediniospores (26e30  20e26 mm) and teliospores (35e64  13e19 mm) than those of the present rust (Saccardo 1888; Arthur 1934; Hiratsuka et al. 1992; Wang and Zhuang 1998). In addition, this rust is morphologically distinguished from the present rust in having uredinial paraphyses. While the specimens of this rust were not examined, its reported morphological characteristics are clearly distinguished from those of the present rust. Although several species of Puccinia, i.e., P. punctata Link, P. asperulina (Juel) Lagerh., P. difformis Kunze and P. valantiae Pers. have been found on Galium and other rubiaceous plants, all of these are autoecious species and clearly distinguished from the present fungus in morphology (Saccardo 1899, 1905; Cunningham 1924; McKenzie 1998; Kazeroni et al. 2010; Singh and Palni 2011; Bahcecioglu and Kabaktepe 2012). Consequently, the rust fungus was concluded to be a new species based on morphology and the host-parasite relationship. The ITS and partial 28S sequences of the present rust fungus were unique in a BLAST search. Maximum similarities are reported for P. silvatica (98% DQ917708) and P. caricina var. ribis-ferrugineae (98% DQ917694) in 28S and Uromyces pisi (93% AF180192) in ITS. In a result, no identical rusts were found: max identities in ITS ¼ 93% U. pisi AF180192; in D1D2 ¼ 98% P. silvatica DQ917708, 98% P. caricina var. ribis-ferrugineae DQ917694. Galium aparine (¼ G. spurium var. echinospermon) is commonly distributed through Japan, Asia to Europe and Africa (Satake et al. 1999b). This annual herb occurs widely in woodland margins, thickets, barrens or roadsides, whereas C. maackii is distributed at riparian vegetation or wet grassland in Japan, Korea, China and the Far East of Russia (Satake et al. 1999a; Katsuyama 2005). According to the Search System of Japanese Red Data (http://www.jpnrdb.com/index.html), C. maackii is categorized as endangered or near threatened by the drainage of swamps. Carex maackii has been reported to be a rare plant and to be distributed locally around the riparian vegetation along the Tone and Kokai Rivers in Ibaraki Prefecture (Suzuki et al. 1981). Accordingly, the distribution of the present rust fungus is likely to be limited to such riparian vegetation so that both host plants grow in close association. The field survey revealed that the plants of G. aparine systemically infected by the present rust were sterile or seedless. The rust infection seems to inhibit flowering of the infected plants. Further studies are required to clarify the mechanism by which this novel rust fungus inhibits flowering and fructification as well as to determine its ecological strategy. Acknowledgments We are grateful to Mr. A. Narushima, Ibaraki Nature Museum, for help in identification of plant specimens. We sincerely thank Dr. K. Tanaka, Hirosaki University, and National Museum of Nature and Science, Tsukuba, for the loan of specimens. This study was conducted under the Comprehensive Survey of Plants, Animals and Geology in Ibaraki Prefecture by Ibaraki Nature Museum 2009e2011. references Arthur JC, 1934. Manual of the rusts in the United States and Canada, reprinted with a supplement by Cummins GB (1962). Hafner, New York. Azbukina ZM, 2005. Lower plants, fungi, and bryophytes of the Russian Far East (in Russian). In: Rust fungi, vol 5. Dalnauka, Vladivostok. Bahcecioglu Z, Kabaktepe S, 2012. Checklist of rust fungi in Turkey. Mycotaxon 119: 494. Cunningham GH, 1924. The Uredinales, or rust-fungi, of New Zealand: supplement to Part 1; and Part 2. Transactions and Proceedings of the New Zealand Institute 55: 1e58. Dietel P, 1905a. Uredineae japonicae V. Botanische Jahrbücher fur Systematik, Pflanzengeschichte und Pflanzengeographie 34: 590. Dietel P, 1905b. Uredineae japonicae VI. Botanische Jahrbücher fur Systematik, Pflanzengeschichte und Pflanzengeographie 37: 99. Harada Y, 1986. Four new species of heteroecious Puccinia (Uredinales) on Carex from Japan. Transactions of the Mycological Society of Japan 27: 357e370. Hiratsuka N, Sato S, Katsuya K, Kakishima M, Hiratsuka Y, Kaneko S, Ono Y, Sato T, Harada Y, Hiratsuka T, Nakayama K, 1992. The rust flora of Japan. Tsukuba-shuppankai, Tsukuba. Hiratsuka Y, Cummins GB, 1963. Morphology of the spermogonia of the rust fungi. Mycologia 55: 487e507. Kakishima M, Sato S, 1980. Three Puccinia species parasitic on Carex. Transactions of the Mycological Society of Japan 21: 35e45. Please cite this article in press as: Okane I, et al., Puccinia galiiuniversa, a new caricicolous rust fungus systemically inhabiting Galium aparine in its spermogonialeaecial stage, Mycoscience (2013), http://dx.doi.org/10.1016/j.myc.2013.05.008 m y c o s c i e n c e x x x ( 2 0 1 3 ) 1 e9 Kakishima M, Sato S, 1982. Puccinia velutina: a new caricicolous rust and a perfect state of Aecidium elaeagni. Mycologia 74: 427e432. Kakishima M, Yokoi M, Harada Y, 1999. Puccinia carici-adenocauli, a new rust fungus on Carex, and its anamorph, Aecidium adenocauli. Mycoscience 40: 503e507. Katsuyama T, 2005. Carex of Japan (in Japanese). Bun-ichi Co. Ltd, Tokyo. Kazeroni EG, Abbasi M, Rezaee S, 2010. Additions to the rust fungi (Pucciniales) of Fars Province, southern Iran. Iran Journal of Plant Pathology 45: 31e35. Kim SH, Lee MW, Lee TS, 2004. Pucciniaceae (Rust fungi) in Korea. Journal of Huazhong Agricultural University 23: 106e112. Kuo HC, Chen ZC, 1999. Notes on Puccinia species on Cyperaceae in Taiwan. Taiwania 44: 503e513. McKenzie EHC, 1998. Rust fungi of New Zealand e an introduction, and list of recorded species. New Zealand Journal of Botany 36: 233e271. O’Donnel K, 1993. Fusarium and its near relatives. In: Reynolds DR, Taylor JW (eds), The fungal holomorph: mitotic, meiotic and pleomorphic speciation in fungal systematics. CAB International, Wallingford, pp 225e233. Okane I, Kakishima M, 1992. Puccinia nigrolinearis, a new caricicolous rust, and its anamorph, Aecidium elaeagniumbellatae. Transactions of the Mycological Society of Japan 33: 497e503. Ono Y, Kakishima M, Ishimiya K, 2001. Aecidium dispori is the aecial anamorph of Puccinia albispora, sp. nov. (Uredinales). Mycoscience 42: 149e153. Ono Y, 2001. Puccinia speciosa, sp. nov., host-alternates between Tricyrtis and Carex in Japan. Mycologia 93: 796e801. Saccardo PA, 1888. Sylloge fungorum omnium hucusque cognitorum VII 600e686. Saccardo PA, 1899. Sylloge fungorum omnium hucusque cognitorum XIV 381. 9 Saccardo PA, 1905. Sylloge fungorum omnium hucusque cognitorum XVII 319. Satake Y, Ohwi J, Kitamura S, Watari S, Tominari T, 1999a (in Japanese). Wild Flowers of Japan; herbaceous plants (incl. dwarf subshrubs), vol I. Heibonsha, Tokyo. Satake Y, Ohwi J, Kitamura S, Watari S, Tominari T, 1999b (in Japanese). Wild Flowers of Japan; herbaceous plants (incl. dwarf subshrubs), vol III. Heibonsha, Tokyo. Sato T, Sato S, 1985. Morphology of aecia of the rust fungi. Transactions of the British Mycological Society 85: 223e238. Savile DBO, 1973. Aeciospore type in Puccinia and Uromyces attacking Cyperaceae, Juncaceae and Poaceae. Reports of the Tottori Mycological Institute 10: 225e241. Singh AS, Palni UT, 2011. Diversity and distribution of rust fungi in central Hymalayan region. Journal of Phytology 3: 49e59. Suzuki M, Shimizu O, Ami T, Yasu M, Fujita H, Nakazaki Y, Wada N, Noguchi T, 1981. Flora of Ibaraki Prefecture (in Japanese). Ibaraki-ken Shokubutsusi Kankoukai, Mito. Virtudazo EV, Nakamura H, Kakishima M, 2001. Phylogenetic analysis of sugarcane rusts based on sequences of ITS, 5.8S rDNA and D1/D2 regions of LSU rDNA. Journal of General Plant Pathology 67: 28e36. Wang Y, Zhuang J, 1998 (in Chinese). Uredinales (1), Flora fungorum Sinicorum, vol 10. Science Press, Beijing. White TJ, Bruns T, Lee S, Taylor J, 1990. Amplification and direct sequencing of fungal ribosomal DNA for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JI, White TJ (eds), PCR protocols: a guide to the methods and applications. Academic Press, San Diego, pp 315e322. Yamaoka Y, Abe J, Kakishima M, 2009. Rust fungi from the west district and Mt. Tsukuba, Ibaraki Prefecture. In: Kurisu N (ed) Report of comprehensive surveys of plants, animals and geology in Ibaraki Prefecture by the Ibaraki Nature Museum, Fungal flora from the west district and Mt. Tsukuba, Ibaraki Prefecture (2006e2008). Yatabe Printing, Tsukuba, pp 5e28. Please cite this article in press as: Okane I, et al., Puccinia galiiuniversa, a new caricicolous rust fungus systemically inhabiting Galium aparine in its spermogonialeaecial stage, Mycoscience (2013), http://dx.doi.org/10.1016/j.myc.2013.05.008