ORIGINAL ARTICLE

A new species of Devriesia causing sooty blotchand 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# German Mycological Society and Springer-Verlag Berlin Heidelberg 2013

Abstract Sooty blotch and flyspeck is a fungal diseasecomplex that colonizes a wide range of plants. In a surveyof host plants for sooty blotch and flyspeck fungi, weobtained an isolate associated with discrete flyspeck symp-tom on stems of the Rubber Trees, Ficus elastica, fromHaikou, Hainan, China. A pure culture was obtained, andthe nuclear ITS region of rDNA was amplified by PCR.Molecular phylogeny inferred from ITS sequence data sug-gested that it was close to Devriesia strelitziae, reportedfrom South Africa. Morphologically, it was characterizedby longer conidia, more conidial septa, and having chlamy-dospores. Therefore, we describe our isolate as a novelspecies of Devriesia.

Keywords Microfungi . Taxonomy . Teratosphaeriaceae .

Hyphomycete . Phylogeny

Introduction

Fungi in the sooty blotch and flyspeck complex colonize thesurfaces of stems, twigs, leaves, and fruits of a wide range ofplants, resulting in a black or sooty appearance. Sootyblotch and flyspeck fungi are epiphytes and cause no pre-harvest losses or fruit decay (Colby 1920). However, thesmudges and stipples of sooty blotch fungi often result in

downgrading of fruit from fresh-market grade to processinguse (Gleason et al. 2011).

Progress in understanding the disease has been slowed bydifficulty in isolating, culturing, and identifying sootyblotch and flyspeck fungi, as well as a paucity of fungalstructures on fruit, although sooty blotch and flyspeck hasbeen studied for nearly 180 years (Hickey 1960). The num-ber of fungal species causing sooty blotch and flyspeck waspreviously underestimated (Johnson et al. 1997). Now, it hasbecome clear that the sooty blotch and flyspeck complexcomprises as many as 80 putative species, mostly within theCapnodiales, according to surveys in the USA, Europe, andChina (Batzer et al. 2005, 2008; Díaz Arias et al. 2010;Frank et al. 2010; Gleason et al. 2011; Li et al. 2012; Sun etal. 2008; Yang et al. 2010).

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 inChina. One new species from China is formally describedand illustrated here. Preliminary phylogenetic analysis basedon ITS sequences was carried out to confirm the new species.

Materials and methods

Isolates

Rubber Trees, Ficus elastica, stems with signs displaying thediscrete speck mycelial type (Gleason et al. 2011) were col-lected in Jinniuling Park in Haikou City, Hainan Province,China, in October 2011. Thalli on the stem were transferreddirectly from colonies to potato dextrose agar (PDA) slants in asterile environment and cultured at 25 °C for 1 month indarkness (Sun et al. 2003). Hyphal tips were then transferredto oatmeal agar (OA), malt extract agar (MEA), and syntheticnutrient-poor agar (SNA) plates. In order to measure and

W. Li :Y. Xiao :C. Wang : J. Dang :C. Chen : L. Gao :G. Sun (*)State Key Laboratory of Crop Stress Biology in Arid Areas andCollege of Plant Protection, Northwest A&F University,Yangling, Shaanxi Province 712100, Chinae-mail: sgy@nwsuaf.edu.cn

J. C. Batzer :M. L. GleasonDepartment of Plant Pathology and Microbiology, Iowa StateUniversity, Ames, IA 50011, USA

Mycol ProgressDOI 10.1007/s11557-012-0885-z

observe fungal structures, the isolates were allowed to growonto an adjacent, sterile cover slip that had been partiallyinserted into the agar surface at a 60° angle (Li et al. 2011).Microscopic examination was made after 7 days of incubation

at 25 °C. Thirty measurements per relevant microscopic struc-ture were gathered where possible. Sterile water was used asmounting media for microscopy. Colony descriptions (surfaceand reverse) weremade after 2 weeks of growth onmalt extract

Table 1 Taxa includedin 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

Fig. 1 One of 4 equallyparsimonious trees determinedfrom ITS sequences. Bootstrapsupport values (>50 %) basedon 1,000 replicates are shown atthe nodes (consistency index=0.7352, retention index=0.7253, rescaled consistencyindex=0.5332). The tree isrooted to Schizothyrium pomiand our isolate is presented inbold

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agar (MEA), OA, and SNA plates at 25±1 °C in the dark.Subsequently, pure cultures were stored in glycerol at −80 °C.

DNA extraction, PCR, and sequencing

Genomic DNA for polymerase chain reaction (PCR) wasobtained according to the protocol of Li et al. (2011). Primerpairs ITS1-F and ITS4 (White et al. 1990) were used toamplify the internal transcribed spacer (ITS) region of nu-clear ribosomal DNA. The PCR reactions were carried outwith Taq polymerase, 1×PCR buffer, 2 mMMgCl2, 0.2 mMof each dNTP, 0.4 μM of each primer, and 2 μl of templateDNA, and were made up to a total volume of 25 μl withsterile water. Reactions were performed on a Bio-Rad PCRSystem S1000™ Thermal Cycler. The amplification condi-tions were: initial denaturation at 94 °C for 90 min followed

by 35 cycles of denaturation at 94 °C for 35 s, annealing at52 °C for 60 s, extension at 72 °C for 1 min, and a finalextension step at 72 °C for 10 min. The PCR products weresequenced by Sangon Biotech, Shanghai, China.

Sequence alignment

The ITS nucleotide sequences generated in this study wereadded to other sequences with high homology that weredownloaded from GenBank (Table 1) as the result of aBLAST search. Sequences were imported into BioEdit5.0.9.1 (Hall 1999). Preliminary alignments were performedusing CLUSTAL-X (Thompson et al. 1997), then manuallyadjusted. Phylogenetic analysis of aligned DNA sequenceswas carried out with PAUP v.4.0b 10 for 32-bit MicrosoftWindows (Swofford 2003). Heuristic searches were

Fig. 2 Devriesia ficus. a Signson stem of Ficus elastica withclose-up view. b Colony onOA. c Colony on SNA. d–fConidiophores. g–i Conidia inchains. j Chlamydospores.Scale bars (d, e, j) 5 μm (f–i)10 μm

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performed with 1,000 random sequence additions. Cladestability was assessed by 1,000 bootstrap replications.Other measures calculated for parsimony analysis includedtree length, consistency index (CI), retention index (RI),rescaled consistency index (RC), and homoplasy index (HI).

The sequences of ITS described in this study were de-posited in GenBank; accession number JX294932 for theisolate LWHHK-7. Schizothyrium pomi was used as theoutgroup taxon. Alignment and the representative tree weresaved in TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S13677).

Results

Phylogenetic analysis

The ITS alignments contained 17 taxa including the out-group, and 560 characters were used for the analysis. Ofthese characters, 126 were parsimony-informative, 115 wereparsimony-uninformative, and 319 were constant. From themost parsimonious tree with 472 steps (CI=0.7352, RI=0.7253, RC=0.5332), two major clades were resolved(Fig. 1). One clade, with 100 % bootstrap support, includedfive species in Devriesia. The other major clade had abootstrap value of 99 %. Our strain clustered together as asub-clade with Devriesia strelitziae (100 % bootstrap valuesupport). However, there were 15 nucleotide differencesbetween D. strelitziae and the isolate LWHHK-7, indicatingthat our strain might represent a new species.

Taxonomy

Devriesia ficus G.Y. Sun & Wenhuan Li, sp. nov. (Figs. 2, 3)MycoBank no.: MB802336Colonies sporulating on OA. Mycelium consisting of

smooth, branched, septate, pale brown hyphae, 2–3 μmwide; chlamydospores intercalary, globose, 4–6 μm diam,pale brown, smooth. Conidiophores lateral on creeping hy-phae, highly variable in length, at times macronematous, butalso micronematous, reduced to conidiogenous cells; un-branched, erect, solitary, subcylindrical, straight to slightlycurved, pale brown, smooth, thick-walled, (2–)7–31(−37)×2–3(−3.5) μm, 0–4(–5)-septate. Conidiogenous cells termi-nal on conidiophores, (4.5–)5.5–11(−17)×2–3 μm, palebrown, proliferating sympodially with terminal polyblasticloci; loci minutely thickened, somewhat refractive, 0.5–1 μm wide. Ramoconidia pale brown, smooth, subcylindri-cal, (8–)10–17×1.5–2.5 μm, 0–2(−3)-septate. Conidia palebrown, finely verruculose, fusoid-ellipsoidal to subcylindri-cal, obclavate, apex obtuse, base truncate, (0–)1–2(–3)-sep-tate, (6–)8–20(−23)×1.5–3 μm, occurring in unbranched orloosely branched chains; hila somewhat thickened and dark-ened, 0.5–1 μm diam. Sexual state not observed.

Cultural characteristics Colonies after 2 weeks at 25 °Cin the dark on OA spreading, planar to slightly convex, withvelvety aerial mycelium and smooth margin; surface mouse-gray, reverse pale mouse-gray; colonies reaching up to10 mm diam after 2 weeks. On SNA, spreading, withmoderate aerial mycelium; aerial mycelium dark olive, outerregion pale olive; with transparent droplets formed on the

Fig. 3 Microscopic structure ofDevriesia ficus (drawn from theholotype). a Conidiophoreswith conidia. b Conidia. cRamoconidia. dChlamydospores. Scale bar5 μm

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surface; colonies reaching up to 9 mm diam after 2 weeks.On MEA, erumpent, spreading, with folded surface,olivaceous-grey, colonies reaching up to 9 mm diam after2 weeks. Cultures of D. ficus did not grow at 37 °C.

Appearance on stem surface: Shiny, black, sclerotium-like bodies scattered on the surface without mycelial mat;round to oval, 70–110(−130) μm diam; densely arranged 4–5/mm2.

Holotype China, Hainan Province, Haikou City, 20°0′26″N, 110°20′31″E, on stems of Ficus elastica Roxb. exHornem, 16 Oct. 2011, W.H. Li, Holotype HMAS244345(= LWHHK-7) (dried culture), ex-type CGMCC 3.15004 (=LWHHK-7).

Etymology: Named after the host from which it wascollected, Ficus.

Notes: Devriesia spp. share a similar micromorphology,namely the production of pigmented conidiophores, withbranched, acropetal chains of non- or multi-septate, usuallysomewhat pigmented conidia, and somewhat darkened co-nidial hila and scars, as well as the formation of chlamydo-spores in culture. Phylogenetically, our strain clusters closeto Devriesia strelitziae (GenBank EU436763; Identities=474/497(95 %); Gaps=9/497(2 %), but the latter species iseasily distinguishable by its smaller conidia [(6–)9–10.5(−17) μm] and fewer conidial septae [0(−2)-septate](Arzanlou and Crous 2008). Moreover, Devriesia strelitziaediffers from our strain by lacking chlamydospores. Wetherefore describe our isolate as a novel species ofDevriesia.

Discussion

The genus Devriesia was introduced by Seifert et al. (2004).A total of 16 species have been recorded: Devriesia aca-diensis, D. agapanthi, D. americana, D. chlamydospora, D.fraseriae, D. hilliana, D. lagerstroemiae, D. pseudoamer-icana, D. queenslandica, D. shelburniensis, D. staurophora,D. strelitziae, D. strelitziicola, D. thermodurans, D. tardi-crescens and D. xanthorrhoeae (Arzanlou and Crous 2008;Crous et al. 2007, 2010a, b, 2011, 2012; Crous andGroenewald 2011; Frank et al. 2010; Seifert et al. 2004;Schoch et al. 2009). Although the genus is paraphyleticbased on phylogenetic analysis of LSU sequences, Franket al. (2010) refrained from describing new genera becausethey thought that more taxa and strains should be added firstin order to resolve possible synapomorphies supporting thephylogenetics by morphology. In concordance with thisviewpoint, we set up the novel taxon under the concept ofDevriesia s.l.

Members of Devriesia s.str. are thermotolerant and usu-ally occur in soil, whereas those of Devriesia s.l. are usuallyassociated with leaf spots, or occur on dead plant debris as

saprobes, and are not thermotolerant (Crous et al. 2007,2009; Koukol 2010). Previously, only one species has beenreported as a causal agent of sooty blotch and flyspeck onapples (Frank et al. 2010). This is the first report of sootyblotch and flyspeck species on Ficus. Our current researchadds a species as a new member of this genus.

Acknowledgements This work was supported by the National Nat-ural Science Foundation of China (31170015, 31171797), the 111Project from Education Ministry of China (B07049), Top Talent Proj-ect of Northwest A&F University and the earmarked fund for ModernAgro-industry Technology Research System (nycytx-08-04-04).

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