Mycol Progress (2013) 12:733–738 DOI 10.1007/s11557-012-0885-z

ORIGINAL ARTICLE

A new species of Devriesia causing sooty blotch and flyspeck on Rubber Trees in China

Wenhuan Li & Yunxue Xiao & Chunsheng Wang & Jiling Dang & Chen Chen & Liu Gao & Jean C. Batzer & Guangyu Sun & Mark L. Gleason

Received: 27 September 2012 /Revised: 18 December 2012 /Accepted: 21 December 2012 /Published online: 16 January 2013 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2013

Abstract Sooty blotch and flyspeck is a fungal disease downgrading of fruit from fresh-market grade to processing complex that colonizes a wide range of plants. In a survey use (Gleason et al. 2011). of host plants for sooty blotch and flyspeck fungi, we Progress in understanding the disease has been slowed by obtained an isolate associated with discrete flyspeck symp- difficulty in isolating, culturing, and identifying sooty tom on stems of the Rubber Trees, Ficus elastica, from blotch and flyspeck fungi, as well as a paucity of fungal Haikou, Hainan, China. A pure culture was obtained, and structures on fruit, although sooty blotch and flyspeck has the nuclear ITS region of rDNA was amplified by PCR. been studied for nearly 180 years (Hickey 1960). The num- Molecular phylogeny inferred from ITS sequence data sug- ber of fungal species causing sooty blotch and flyspeck was gested that it was close to Devriesia strelitziae, reported previously underestimated (Johnson et al. 1997). Now, it has from South Africa. Morphologically, it was characterized become clear that the sooty blotch and flyspeck complex by longer conidia, more conidial septa, and having chlamy- comprises as many as 80 putative species, mostly within the dospores. Therefore, we describe our isolate as a novel Capnodiales, according to surveys in the USA, Europe, and species of Devriesia. China (Batzer et al. 2005, 2008; Díaz Arias et al. 2010; Frank et al. 2010; Gleason et al. 2011; Li et al. 2012;Sunet Keywords Microfungi . Taxonomy . Teratosphaeriaceae . al. 2008; Yang et al. 2010). Hyphomycete . Phylogeny Taxonomic study of Devriesia began in 2004 (Seifert et al. 2004), and since then 16 species have been documented. However, Devriesia has not previously been reported in Introduction China. One new species from China is formally described and illustrated here. Preliminary phylogenetic analysis based Fungi in the sooty blotch and flyspeck complex colonize the on ITS sequences was carried out to confirm the new species. surfaces of stems, twigs, leaves, and fruits of a wide range of plants, resulting in a black or sooty appearance. Sooty blotch and flyspeck fungi are epiphytes and cause no pre- Materials and methods harvest losses or fruit decay (Colby 1920). However, the smudges and stipples of sooty blotch fungi often result in Isolates

: : : : : : Rubber Trees, Ficus elastica, stems with signs displaying the W. Li Y. Xiao C. Wang J. Dang C. Chen L. Gao discrete speck mycelial type (Gleason et al. 2011)werecol- * G. Sun ( ) lected in Jinniuling Park in Haikou City, Hainan Province, State Key Laboratory of Crop Stress Biology in Arid Areas and College of Plant Protection, Northwest A&F University, China, in October 2011. Thalli on the stem were transferred Yangling, Shaanxi Province 712100, China directly from colonies to potato dextrose agar (PDA) slants in a e-mail: [email protected] sterile environment and cultured at 25 °C for 1 month in darkness (Sun et al. 2003). Hyphal tips were then transferred J. C. Batzer : M. L. Gleason Department of Plant Pathology and Microbiology, Iowa State to oatmeal agar (OA), malt extract agar (MEA), and synthetic University, Ames, IA 50011, USA nutrient-poor agar (SNA) plates. In order to measure and 734 Mycol Progress (2013) 12:733–738

Table 1 Taxa included in DNA analyses Species Strain GenBank Reference

Devriesia acadiensis DAOM 232211 AY692095 Seifert et al. 2004 D. agapanthi CPC 19833 JX069875 Crous et al. 2012 D. americana STE-U 5121 AY251068 Crous et al. 2007 D. ficus LWHHK-7 JX294932 This paper D. fraseriae CBS 128217 HQ599602 Crous et al. 2010b D. hilliana CPC 15382 GU214633 Schoch et al. 2009 D. lagerstroemiae CPC 14403 GU214634 Schoch et al. 2009 D. pseudoamericana CPC 16174 GU570527 Frank et al. 2010 D. queenslandica CPC 17306 JF951148 Crous et al. 2011 D. shelburniensis DAOM 232218 AY692094 Seifert et al. 2004 D. staurophora DAOM 230744 AY692086 Seifert et al. 2004 D. strelitziae CBS 122379 EU436763 Arzanlou and Crous 2008 D. strelitziicola X1045 GU214635 Schoch et al. 2009 D. tardicrescens CBS 128770 JF499840 Crous and Groenewald 2011 D. thermodurans DAOM 226677 AY692088 Seifert et al. 2004 D. xanthorrhoeae CBS 128219 HQ599605 Crous et al. 2010a Schizothyrium pomi CBS 228.57 EF134947 Batzer et al. 2008 observe fungal structures, the isolates were allowed to grow at 25 °C. Thirty measurements per relevant microscopic struc- onto an adjacent, sterile cover slip that had been partially ture were gathered where possible. Sterile water was used as inserted into the agar surface at a 60° angle (Li et al. 2011). mounting media for microscopy. Colony descriptions (surface Microscopic examination was made after 7 days of incubation and reverse) were made after 2 weeks of growth on malt extract

Fig. 1 One of 4 equally parsimonious trees determined from ITS sequences. Bootstrap support values (>50 %) based on 1,000 replicates are shown at the nodes (consistency index= 0.7352, retention index= 0.7253, rescaled consistency index=0.5332). The tree is rooted to Schizothyrium pomi and our isolate is presented in bold Mycol Progress (2013) 12:733–738 735

Fig. 2 Devriesia ficus. a Signs on stem of Ficus elastica with close-up view. b Colony on OA. c Colony on SNA. d–f Conidiophores. g–i Conidia in chains. j Chlamydospores. Scale bars (d, e, j)5μm(f–i) 10 μm

agar (MEA), OA, and SNA plates at 25±1 °C in the dark. by 35 cycles of denaturation at 94 °C for 35 s, annealing at Subsequently, pure cultures were stored in glycerol at −80 °C. 52 °C for 60 s, extension at 72 °C for 1 min, and a final extension step at 72 °C for 10 min. The PCR products were DNA extraction, PCR, and sequencing sequenced by Sangon Biotech, Shanghai, China.

Genomic DNA for polymerase chain reaction (PCR) was Sequence alignment obtained according to the protocol of Li et al. (2011). Primer pairs ITS1-F and ITS4 (White et al. 1990) were used to The ITS nucleotide sequences generated in this study were amplify the internal transcribed spacer (ITS) region of nu- added to other sequences with high homology that were clear ribosomal DNA. The PCR reactions were carried out downloaded from GenBank (Table 1)astheresultofa with Taq polymerase, 1×PCR buffer, 2 mM MgCl2, 0.2 mM BLAST search. Sequences were imported into BioEdit of each dNTP, 0.4 μM of each primer, and 2 μl of template 5.0.9.1 (Hall 1999). Preliminary alignments were performed DNA, and were made up to a total volume of 25 μl with using CLUSTAL-X (Thompson et al. 1997), then manually sterile water. Reactions were performed on a Bio-Rad PCR adjusted. Phylogenetic analysis of aligned DNA sequences System S1000™ Thermal Cycler. The amplification condi- was carried out with PAUP v.4.0b 10 for 32-bit Microsoft tions were: initial denaturation at 94 °C for 90 min followed Windows (Swofford 2003). Heuristic searches were 736 Mycol Progress (2013) 12:733–738

Fig. 3 Microscopic structure of Devriesia ficus (drawn from the holotype). a Conidiophores with conidia. b Conidia. c Ramoconidia. d Chlamydospores. Scale bar 5 μm

performed with 1,000 random sequence additions. Clade Taxonomy stability was assessed by 1,000 bootstrap replications. Other measures calculated for parsimony analysis included Devriesia ficus G.Y. Sun & Wenhuan Li, sp. nov. (Figs. 2, 3) tree length, consistency index (CI), retention index (RI), MycoBank no.: MB802336 rescaled consistency index (RC), and homoplasy index (HI). Colonies sporulating on OA. Mycelium consisting of The sequences of ITS described in this study were de- smooth, branched, septate, pale brown hyphae, 2–3 μm posited in GenBank; accession number JX294932 for the wide; chlamydospores intercalary, globose, 4–6 μm diam, isolate LWHHK-7. Schizothyrium pomi was used as the pale brown, smooth. Conidiophores lateral on creeping hy- outgroup taxon. Alignment and the representative tree were phae, highly variable in length, at times macronematous, but saved in TreeBase (http://purl.org/phylo/treebase/phylows/ also micronematous, reduced to conidiogenous cells; un- study/TB2:S13677). branched, erect, solitary, subcylindrical, straight to slightly curved, pale brown, smooth, thick-walled, (2–)7–31(−37)× 2–3(−3.5) μm, 0–4(–5)-septate. Conidiogenous cells termi- Results nal on conidiophores, (4.5–)5.5–11(−17)×2–3 μm, pale brown, proliferating sympodially with terminal polyblastic Phylogenetic analysis loci; loci minutely thickened, somewhat refractive, 0.5– 1 μm wide. Ramoconidia pale brown, smooth, subcylindri- The ITS alignments contained 17 taxa including the out- cal, (8–)10–17×1.5–2.5 μm, 0–2(−3)-septate. Conidia pale group, and 560 characters were used for the analysis. Of brown, finely verruculose, fusoid-ellipsoidal to subcylindri- these characters, 126 were parsimony-informative, 115 were cal, obclavate, apex obtuse, base truncate, (0–)1–2(–3)-sep- parsimony-uninformative, and 319 were constant. From the tate, (6–)8–20(−23)×1.5–3 μm, occurring in unbranched or most parsimonious tree with 472 steps (CI=0.7352, RI= loosely branched chains; hila somewhat thickened and dark- 0.7253, RC=0.5332), two major clades were resolved ened, 0.5–1 μm diam. Sexual state not observed. (Fig. 1). One clade, with 100 % bootstrap support, included Cultural characteristics Colonies after 2 weeks at 25 °C five species in Devriesia. The other major clade had a in the dark on OA spreading, planar to slightly convex, with bootstrap value of 99 %. Our strain clustered together as a velvety aerial mycelium and smooth margin; surface mouse- sub-clade with Devriesia strelitziae (100 % bootstrap value gray, reverse pale mouse-gray; colonies reaching up to support). However, there were 15 nucleotide differences 10 mm diam after 2 weeks. On SNA, spreading, with between D. strelitziae and the isolate LWHHK-7, indicating moderate aerial mycelium; aerial mycelium dark olive, outer that our strain might represent a new species. region pale olive; with transparent droplets formed on the Mycol Progress (2013) 12:733–738 737 surface; colonies reaching up to 9 mm diam after 2 weeks. saprobes, and are not thermotolerant (Crous et al. 2007, On MEA, erumpent, spreading, with folded surface, 2009; Koukol 2010). Previously, only one species has been olivaceous-grey, colonies reaching up to 9 mm diam after reported as a causal agent of sooty blotch and flyspeck on 2 weeks. Cultures of D. ficus did not grow at 37 °C. apples (Frank et al. 2010). This is the first report of sooty Appearance on stem surface: Shiny, black, sclerotium- blotch and flyspeck species on Ficus. Our current research like bodies scattered on the surface without mycelial mat; adds a species as a new member of this genus. round to oval, 70–110(−130) μm diam; densely arranged 4– 5/mm2. Acknowledgements This work was supported by the National Nat- Holotype China, Hainan Province, Haikou City, 20°0′26″ ural Science Foundation of China (31170015, 31171797), the 111 ′ ″ Project from Education Ministry of China (B07049), Top Talent Proj- N, 110°20 31 E, on stems of Ficus elastica Roxb. ex ect of Northwest A&F University and the earmarked fund for Modern Hornem, 16 Oct. 2011, W.H. Li, Holotype HMAS244345 Agro-industry Technology Research System (nycytx-08-04-04). (= LWHHK-7) (dried culture), ex-type CGMCC 3.15004 (= LWHHK-7). Etymology: Named after the host from which it was References collected, Ficus. Notes: Devriesia spp. share a similar micromorphology, namely the production of pigmented conidiophores, with Arzanlou M, Crous PW (2008) Devriesia strelitziae Arzanlou & Crous, branched, acropetal chains of non- or multi-septate, usually sp. nov. Fungal Planet, No. 22 somewhat pigmented conidia, and somewhat darkened co- Batzer JC, Gleason ML, Harrington TC, Tiffany LH (2005) Expansion of the sooty blotch and flyspeck complex on apples based on nidial hila and scars, as well as the formation of chlamydo- analysis of ribosomal DNA gene sequences and morphology. spores in culture. Phylogenetically, our strain clusters close Mycologia 97:1268–1286 to Devriesia strelitziae (GenBank EU436763; Identities= Batzer JC, Díaz Arias MM, Harrington TC, Gleason ML, Groenewald JZ, 474/497(95 %); Gaps=9/497(2 %), but the latter species is Crous PW (2008) Four species of Zygophiala (Schizothyriaceae, – – Capnodiales) are associated with the sooty blotch and flyspeck easily distinguishable by its smaller conidia [(6 )9 10.5 complex on apple. Mycologia 100:246–258 (−17) μm] and fewer conidial septae [0(−2)-septate] Colby AS (1920) Sooty blotch of pomaceous fruits. Trans Illinois State (Arzanlou and Crous 2008). Moreover, Devriesia strelitziae Acad Sci 13:139–179 ’ differs from our strain by lacking chlamydospores. We Crous PW, Groenewald JZ (2011) Why everlastings don tlast. Persoonia 26:70–84 therefore describe our isolate as a novel species of Crous PW, Braun U, Schubert K, Groenewald JZ (2007) Delimiting Devriesia. Cladosporium from morphologically similar genera. Stud Mycol 58:33–56 Crous PW, Schoch CL, Hyde KD, Wood AR, Gueidan C, de Hoog GS, Groenewald JZ (2009) Phylogenetic lineages in the Capnodiales. Discussion Stud Mycol 64:17–47 Crous PW, Groenewald JZ, Pascoe IG, Edwards J (2010a) Devriesia The genus Devriesia was introduced by Seifert et al. (2004). xanthorrhoeae. Fungal planet 67. Persoonia 25:154–155 A total of 16 species have been recorded: Devriesia aca- Crous PW, Groenewald JZ, Shivas RG (2010b) Devriesia fraseriae. Fungal planet 65. Persoonia 25:150–151 diensis, D. agapanthi, D. americana, D. chlamydospora, D. Crous PW, Groenewald JZ, Shivas RG, Edwards J, Seifert KA, Alfenas fraseriae, D. hilliana, D. lagerstroemiae, D. pseudoamer- AC, Alfenas RF, Burgess TI, Carnegie AJ, Hardy GE, StJ HN, icana, D. queenslandica, D. shelburniensis, D. staurophora, Hüberli D, Jung T, Louis-Seize G, Okada G, Pereira OL, Stukely D. strelitziae, D. strelitziicola, D. thermodurans, D. tardi- MJC, Wang W, White GP, Young AJ, McTaggart AR, Pascoe IG, Porter IJ, Quaedvlieg W (2011) Fungal planet description sheets: crescens and D. xanthorrhoeae (Arzanlou and Crous 2008; 69–91. Persoonia 26:108–156 Crous et al. 2007, 2010a, b, 2011, 2012; Crous and Crous PW, Summerell BA, Shivas RG, Burgess TI, Decock CA, Dreyer Groenewald 2011; Frank et al. 2010; Seifert et al. 2004; LL, Granke LL, Guest DI, Hardy GEStJ, Hausbeck MK, Hüberli D, Schoch et al. 2009). Although the genus is paraphyletic Jung T, Koukol O, Lennox CL, Liew ECY, Lombard L, McTaggart AR, Pryke JS, Roets F, Saude C, Shuttleworth LA, Stukely MJC, based on phylogenetic analysis of LSU sequences, Frank Vánky K, Webster BJ, Windstam ST, Groenewald JZ (2012) Fungal et al. (2010) refrained from describing new genera because Planet Description Sheets: 107–127. Persoonia 28:138–182 they thought that more taxa and strains should be added first Díaz Arias MM, Batzer JC, Harrington TC, Wang Wong A, Bost SC, in order to resolve possible synapomorphies supporting the Cooley DR, Ellis MA, Hartman JR, Rosenberger DA, Sundin GW, Sutton TB, Travis JW, Wheeler MJ, Yoder KS, Gleason phylogenetics by morphology. In concordance with this ML (2010) Diversity and biogeography of sooty blotch and fly- viewpoint, we set up the novel taxon under the concept of speck fungi on apple in the Eastern and Midwestern United States. Devriesia s.l. Phytopathology 100:345–355 Members of Devriesia s.str. are thermotolerant and usu- Frank J, Crous PW, Groenewald JZ, Oertel B, Hyde KD, Phengsintham P, Schroers HJ (2010) Microcyclospora and Microcyclosporella: ally occur in soil, whereas those of Devriesia s.l. are usually novel genera accommodating epiphytic fungi causing sooty associated with leaf spots, or occur on dead plant debris as blotch on apple. Persoonia 24:93–105 738 Mycol Progress (2013) 12:733–738

Gleason ML, Batzer JC, Sun GY, Zhang R, Díaz Arias MM, Sutton TB, CA, Phillips AJL, Phongpaichit S, Pointing SB, Pujade-Renaud Crous PW, Ivanović M, McManus PS, Cooley DR, Mayr U, Weber V, Raja HA, Rivas Plata E, Robbertse B, Ruibal C, Sakayaroj J, RWS, Yoder KS, Del Ponte EM, Biggs AR, Oertel B (2011) A new Sano T, Selbmann L, Shearer CA, Shirouzu T, Slippers B, view of sooty blotch and flyspeck. Plant Dis 95:368–383 Suetrong S, Tanaka K, Volkmann-Kohlmeyer B, Wingfield MJ, Hall TA (1999) BioEdit: a user-friendly biological sequence alignment Wood AR, Woudenberg JHC, Yonezawa H, Zhang Y, Spatafora editor and analysis program for Windows 95/98/NT. Nucleic JW (2009) A class-wide phylogenetic assessment of Acids Symp Ser 41:95–98 Dothideomycetes. Stud Mycol 64:1–15 Hickey KD (1960) The sooty blotch and flyspeck disease of apple with Seifert KA, Nickerson NL, Corlett M, Jackson ED, Louis-Seize G, Davies emphasis on variation within Gloeodes pomigena (SCW.) Colby. RJ (2004) Devriesia, a new hyphomycete genus to accommodate Ph.D diss. Pennsylvania State University, State College, PA heat-resistant, cladosporium-like fungi. Can J Bot 82:914–926 Johnson EM, Sutton TB, Hodges CS (1997) Etiology of apple sooty Sun GY, Zhang R, Zhang Z, Zhang M (2003) Isolation of sooty blotch blotch disease in North Carolina. Phytopathology 78:88–95 and flyspeck fungi from apple surface by picking up the thalli. Koukol O (2010) Revision of “Septonema ochraceum” revealed three new Acta Phytopathol Sin 33:479–480 species of Venturiaceae and Herpotrichiellaceae. Mycol Prog 9:369–378 Sun GY, Zhang R, Li HY, Gleason ML (2008) Diversity of fungi causing Li HY, Sun GY, Batzer JC, Crous PW, Groenewald JZ, Karakaya A, flyspeck-like signs on apple in China. Phytopathology 98:S153 Gleason ML (2011) Scleroramularia gen. nov. associated with Swofford DL (2003) PAUP* Phylogenetic analysis using parsimony sooty blotch and flyspeck of apple and pawpaw from the Northern (*and other methods) version 4. Sinauer, Sunderland Hemisphere. Fungal Divers 46:53–66 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG Li HY, Sun GY, Zhai XR, Batzer JC, Mayfield DA, Crous PW, (1997) The CLUSTAL_X windows interface: flexible strategies Groenewald JZ, Gleason ML (2012) Dissoconiaceae associated for multiple sequence alignment aided by quality analysis tools. with sooty blotch and flyspeck on fruits in China and the United Nucleic Acids Res 25:4876–4882 States. Persoonia 28:113–125 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct Schoch CL, Crous PW, Groenewald JZ, Boehm EWA, Burgess TI, de sequencing of fungal ribosomal RNA genes for phylogenetics. Gruyter J, de Hoog GS, Dixon LJ, Grube M, Gueidan C, Harada Academic, New York Y, Hatakeyama S, Hirayama K, Hosoya T, Huhndorf SM, Hyde Yang HL, Sun GY, Batzer JC, Crous PW, Groenewald JZ, Gleason ML KD, Jones EBG, Kohlmeyer J, Kruys Ǻ, Li YM, Lücking R, (2010) Novel fungal genera and species associated with the sooty Lumbsch HT, Marvanová L, Mbatchou JS, McVay AH, Miller blotch and flyspeck complex on apple in China and the USA. AN, Mugambi GK, Muggia L, Nelsen MP, Nelson P, Owensby Persoonia 24:29–37