Mycologia

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Two novel Fusarium species that cause canker disease of prickly ash (Zanthoxylum bungeanum) in northern form a novel clade with Fusarium torreyae

Xue Zhou, Kerry O’Donnell, Takayuki Aoki, Jason A. Smith, Matthew T. Kasson & Zhi-Min Cao

To cite this article: Xue Zhou, Kerry O’Donnell, Takayuki Aoki, Jason A. Smith, Matthew T. Kasson & Zhi-Min Cao (2016) Two novel Fusarium species that cause canker disease of prickly ash (Zanthoxylum bungeanum) in northern China form a novel clade with Fusarium torreyae, Mycologia, 108:4, 668-681 To link to this article: http://dx.doi.org/10.3852/15-189

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Download by: [University of Florida] Date: 03 March 2017, At: 09:57 Mycologia, 108(4), 2016, pp. 668–681. DOI: 10.3852/15-189 # 2016 by The Mycological Society of America, Lawrence, KS 66044-8897

Two novel Fusarium species that cause canker disease of prickly ash (Zanthoxylum bungeanum) in northern China form a novel clade with Fusarium torreyae

Xue Zhou of three fusaria on Z. bungeanum resulted in consistent College of Forestry, Northwest A&F University, Taicheng canker symptoms from which these three fusaria were Road, Yangling, , China 712100 recovered. The two novel fusaria, however, induced Kerry O’Donnell significantly larger lesions than FSSC 6. Herein, the Mycotoxin Prevention and Applied Microbiology Research two novel prickly ash pathogens are formally described Unit, National Center for Agricultural Utilization Research, as F. zanthoxyli and F. continuum. Agricultural Research Service, US Department of Agriculture, Key words: Fusarium continuum, F. zanthoxyli, Peoria, Illinois 60604-3999 genealogical concordance, molecular phylogenetics, Takayuki Aoki morphology, RPB2, TEF1 Genetic Resources Center (MAFF), National Agriculture and Food Research Organization, 2-1-2 Kannondai, Tsukuba, INTRODUCTION Ibaraki 305-8602, Japan Commonly known as prickly ash, Zanthoxylum bungea- Jason A. Smith num (Rutaceae) is an economically important tree School of Forest Resources and Conservation, University of species in dry, mountainous areas in several provinces Florida, Gainesville, Florida 32611-0680 in northern China. In the past two and one-half Matthew T. Kasson decades a canker disease has resulted in diminished Division of Plant and Soil Sciences, West Virginia University, production of prickly ash, a common peppery spice − Morgantown, West Virginia 26506 6108 used in Asian cuisine derived from at least two species Zhi-Min Cao1 of Zanthoxylum including Z. bungeanum. The pericarp College of Forestry, Northwest A&F University, of this plant also is used in traditional Chinese medi-

Taicheng Road, Yangling, Shaanxi, China 712100 cine (Tang et al. 2014). Symptoms of the prickly ash disease include branch and stem cankers, dieback and occasional tree mortality. In initial studies con- Abstract: Canker disease of prickly ash (Zanthoxylum ducted on canker disease of Z. bungeanum (hereafter bungeanum) has caused a decline in the production abbreviated CDZB) in Shaanxi and provinces of this economically important spice in northern the putative pathogen isolated from cankered stems China in the past 25 y. To identify the etiological agent, was identified as Fusarium sambucium Fuckel based 38 fungal isolates were recovered from symptomatic on morphological data (Cao et al. 1992, 2010). tissues from trees in five provinces in China. These iso- Subsequently the CDZB isolates were re-identified as lates were identified by conducting BLASTN queries F. lateritium Nees (Xie 2012). These contrasting identi- of NCBI GenBank and phylogenetic analyses of DNA fications highlight the need for the CDZB isolates to be sequence data from the nuclear ribosomal internal characterized further with molecular systematic data. transcribed spacer region (ITS rDNA), a portion of Phylogenetic species recognition based on genealogi- the translation elongation factor 1-a (TEF1) gene, and cal concordance (Taylor et al. 2000) has made a signif- genes encoding RNA polymerase II largest (RPB1) icant impact on Fusarium systematics over the past two and second largest (RPB2) subunits. Results of these decades (Aoki et al. 2014). Portions of the translation analyses suggested that 30/38 isolates belonged to two elongation factor 1-a gene (TEF1, Geiser et al. 2004) novel fusaria most closely related to the Florida torreya and the largest (RPB1) and second largest (RPB2)sub- (Torreya taxifolia Arn.) pathogen, Fusarium torreyae units of RNA polymerase II genes have proven utility in Florida and Georgia. These three canker-inducing for inferring species limits and elucidating phyloge- tree pathogens form a novel clade within Fusarium here netic relationships within Fusarium (O’Donnell et al. designated the F. torreyae species complex (FTOSC). 2013). Accordingly the present study was conducted BLASTN queries of GenBank also revealed that 5/38 to (i) collect and analyze multilocus DNA sequence isolates recovered from cankers represented an unde- data phylogenetically together with morphological scribed phylogenetic species within the F. solani species data to identify the casual agents of Fusarium CDZB complex (FSSC) designated FSSC 6. Stem inoculations in the main prickly ash production areas of northern China, (ii) assess virulence of the fusaria on Z. bungea- Submitted 9 Sep 2015; accepted for publication 16 Feb 2016. num and (iii) formally describe two novel CDZB patho- 1 Corresponding author. E-mail: [email protected] gens morphologically as F. zanthoxyli and F. continuum.

668 ZHOU ET AL.: NOVEL PATHOGENS OF PRICKLY ASH 669

FIG. 1. Collection sites in five Chinese provinces where Fusarium zanthoxyli (N) and F. continuum (▴) were isolated from Zanthoxylum bungeanum. Note that the range of these two pathogens overlaps in Fuping County, which is in central Shaanxi province.

MATERIALS AND METHODS et al. 1998a). The nuclear ribosomal internal transcribed spacer region (ITS rDNA) and portions of TEF1, RPB1 and — Isolation of pathogenic fusaria. Cankers on Z. bungeanum RPB2 genes were PCR-amplified and sequenced with primers – were collected 2010 2013 mainly in Shaanxi, Gansu and published in the following: ITS rDNA (White et al. 1990), provinces but also in Shanxi and Hebei in TEF1 (O’Donnell et al. 1998b, 2008), RPB1 (O’Donnell China (FIG.1).Thirty-eightFusarium isolates (SUPPLEMENTARY et al. 2010) and RPB2 (Liu et al. 1999, Reeb et al. 2004). TABLE I) were recovered from surface-sterilized stem tissues PCR reactions were conducted in a total volume of 50 μL excised from the margins of cankers following the protocol of that contained 2 μL of each primer (10 μM), 25 μL26 Taq Smith et al. (2011). For long-term preservation all CDZB isolates PCR MasterMix (Biosci Biotech Co., , China), were stored in 40% glycerol at −80 C in the laboratory of 2 μL diluted (1 : 50) template DNA and 19 μL double- Zhi-Min Cao. In addition, two isolates of F. zanthoxyli and distilled water. After PCR amplification amplicons were sized F. continuum, including the ex-type isolates, were deposited in by gel electrophoresis on 1.5% agarose gels that were run in the ARS Culture Collection (NRRL) and the CBS-KNAW Biodi- 16 TAE buffer. Amplicons were purified and sequenced versityCentre(seeSUPPLEMENTARY TABLE I). by Sangon Biotech Ltd, , China. Herbarium specimens were deposited in the Mycological Herbarium of Forestry College, Northwest A&F University, Molecular phylogenetics.—Chromas 2 and Chromas Pro 1.7.5 Yangling, Shaanxi province, China (HMNWAFU). (Technelysium Pty Ltd, Brisbane, Australia), respectively, were used to manually edit chromatograms and assemble DNA extraction, PCR amplification and sequencing.— the contigs. Contigs were aligned with MUSCLE (Edgar Mycelium was cultured in potato dextrose broth ( et al. 2004) and the following two datasets were analyzed phylo- 2014) on a rotary shaker at 120 rpm for 7 d at 24 C. Mycelium genetically: (i) a 39-taxon RPB1-RPB2 dataset to place the was harvested over sterilized gauze, freeze-dried, and then CDZB isolates within Fusarium (FIG. 2), and (ii) a 31-taxon total genomic DNA was extracted with a CTAB (hexadecyltri- three-gene dataset to assess the genealogical exclusivity of methylammonium bromide) miniprep protocol (O’Donnell the two putatively novel CDZB pathogens, F. zanthoxyli and 670 MYCOLOGIA

FIG. 2. One of two most-parsimonious phylograms inferred from a combined RPB1-RPB2 dataset that strongly supported the monophyly of seven species complexes within Fusarium, including a novel clade of canker pathogens here designated the F. torreyae species complex. Fusarium torreyae is strongly supported as sister to the two novel Zanthoxylum bungeanum pathogens, F. zanthoxyli and F. continuum. Canker disease of Z. bungeanum (CDZB) also can be induced by isolates of phylogenetic species FSSC 6 in the FSSC. Sequences of the FSSC were used to root the phylogram. Boldface identifies the 11 isolates from Z. bungeanum cankers included in the analysis. The number above internodes represents ML-BS value based on 1000 pseudoreplicates of the data. The MP-BS value is indicated only when it differed by $ 5% the ML-BS value (ML-BS\MP-BS). PIC 5 parsimony informative character, MPTs 5 most-parsimonious trees, CI 5 consistency index, RI 5 retention index. ZHOU ET AL.: NOVEL PATHOGENS OF PRICKLY ASH 671

F. continuum (FIG. 3). The individual and combined parti- onto PDA. The cultures were incubated in the dark at eight tions were analyzed phylogenetically and clade support was temperatures 5–40 C at 5 C intervals. Cultures were exam- assessed by maximum parsimony bootstrapping (MP-BS) in ined after 1 and 5 d under a dissecting microscope, and col- PAUP* 4.0b10 (Swofford 2003), and maximum likelihood ony margins were marked on the reverse side of the Petri (ML) in GARLI 2.01 on XSEDE (Zwickl 2006) on the CIPRES dishes. Mean values of radial mycelial growth rate per day Science Gateway (https://www.phylo.org/portal2/login! were calculated by measuring the distance from 16 points input.action). The MP-BS analysis was conducted with on the colony margin to the center. Measurements were equally weighted characters, a heuristic search employing repeated twice and averaged. All microscopic studies and 1000 random sequence additions, TBR branch-swapping, measurements were made on isolates cultured on SNA. Mea- MAXTREES set at 5000 employing 1000 bootstrap replicates surements of 50 randomly selected conidia were taken based (Felsenstein 1985). ML bootstrapping was conducted on the number of septa and cultural condition, and minimal employing the GTR + Γ + I model of molecular evolution, and maximal sizes, arithmetic means and standard deviations which was selected with JModelTest (Posada 2008). DNA (SD) were obtained. Conidia, conidiophores and chlamydo‐ sequence data generated in the present study were deposited spores produced on SNA were viewed and photographed in GenBank (SUPPLEMENTARY TABLE I), and alignments and by light microscopy (Olympus, CX31RTSF, Japan) with or trees were submitted to TreeBASE (accession number without mounting them in water. Descriptive terms for conid‐ S17885, tree number Tr89189–Tr89193). ia and conidiophore morphology followed Nirenberg and O’Donnell (1998). Pathogenicity of fusaria to Zanthoxylum bungeanum.— To assess pathogenicity 38 isolates were inoculated onto RESULTS eight 7 y old Z. bungeanum cultivar Doujiao trees in the nurs‐ ery of the College of Forestry, Northwest A&F University. DNA sequence-based identification of the isolates.—ITS After a wound approximately 40 mm2 was made with a rDNA, TEF1 and RPB2 nucleotide sequences of the pointed tweezer on 1 or 2 y old branches of prickly ash, it 38 isolates recovered from Z. bungeanum cankers were was inoculated with a 5 mm diam potato dextrose agar used to query GenBank; TEF1 also was used to query (PDA) plug containing mycelium from a 14 d old culture FUSARIUM-ID (Geiser et al. 2004). Nucleotide of one of the 38 isolates (SUPPLEMENTARY TABLE I), after BLASTN queries revealed that Fusarium torreyae was which the inoculation site was wrapped with absorbent cot- ton dipped in sterile water and sealed tightly with a plastic the best match for 30/38 isolates (SUPPLEMENTARY

5 5 – film to keep humid. Every isolate was inoculated in two TABLE I) as follows: ITS rDNA 97%, TEF1 87 5 – wounds on two different branches. Wounds inoculated with 91% and RPB2 95 96%. Sequences of 5/38 isolates a sterile PDA plug served as the negative control. During that were nested within the FSSC (F201131–201135) the pathogenicity experiment the average temperature in revealed 99–100% identity to an unnamed phyloge- the field was 20 C during daytime and 12 C at night. The netic species designated FSSC 6 (O’Donnell et al. experiment was terminated after 22 d at which time inocu- 2008). The remaining three isolates were identified lated branches were removed from the trees and were taken via BLASTN queries of GenBank, using the partial to the lab where canker size was measured (FIG. 4). The aver- RPB2,asF. acuminatum (F201136, 100% identity to age lesion dimension was measured and used in the ANOVA HM068334.1 F. acuminatum NRRL 54216), Fusarium analysis with the LSD multiple comparison test employing sp. (F201237, 96% identity to JX171571.1 F. lateritium IBM SPSS Statistics 19 (https://www14.software.ibm.com/). To complete Koch’s postulates, the inoculated isolates were NRRL 13622) and an unnamed species within the re-isolated as described above and confirmed morphologi- F. incarnatum-equiseti species complex designated cally and molecularly by ITS sequence analysis. FIESC 1 (F201338, 100% identity to GQ505814.1 Fusar- ium sp. FIESC 1, O’Donnell et al. 2009). Morphological characterization.—Fourteen isolates of F. zanthoxyli and five of F. continuum were used in the mor- Molecular phylogenetics.—To place the two putatively phological study. Methods for determining phenotypic char- novel isolates within Fusarium,weconductedaMPanal‐ acters and mycelial growth rates followed Aoki et al. ysis of a 39-taxon RPB1-RPB2 dataset spanning the phy- (2003, 2005). Isolates were grown at 20 C in 9 cm plastic Petri logenetic breadth of the genus that included sequences dishes on PDA and synthetic nutrient-poor agar (SNA) with of five isolates of F. zanthoxyli and F. continuum (FIG.2). 6 or without placing a sterile 1 1 cm piece of filter paper MP analysis of the two-gene dataset outgroup-rooted on the SNA surface (Nirenberg and O’Donnell 1998) in on sequences of six members of the FSSC (FIG.2)based the dark, under continuous fluorescent light (Panasonic on more inclusive analyses (O’Donnell et al. 2013), in YZ36RR6500K) or under daylight. Colony morphology, which 1145/3368 characters were parsimony informa- color, odor and growth rate were based on cultures grown on PDA (Fang 1998). All colors are given according to the tive, found two most-parsimonious trees of 4486 steps 5 5 Methuen Handbook of Color (Kornerup and Wanscher (CI 0.42, RI 0.73) that differed only in the branch- 1978). Mycelial growth rates were calculated as described in ing order of F. illudens C. Booth NRRL 22090 within the Aoki et al. (2003). Agar blocks 5 mm diam were cut from FSSC. The ML analysis produced a topologically similar the margins of 2 wk old cultures on SNA and inoculated tree (data not shown). ML and MP bootstrapping 672 MYCOLOGIA

FIG. 3. Maximum parsimony (MP) phylograms inferred from 31 aligned DNA sequences of the three fusaria comprising the FTOSC, which included data from ITS rDNA, TEF1, RPB2 and the combined three gene dataset. The phylograms were rooted on sequences of F. torreyae. Note that F. zanthoxyli and F. continuum are strongly supported as genealogical exclusive sister taxa by ML and MP (ML-BS\MP-BS) bootstrapping. PIC 5 parsimony informative character, MPT(s) 5 most-parsimonious tree(s), CI 5 consistency index, RI 5 retention index. ZHOU ET AL.: NOVEL PATHOGENS OF PRICKLY ASH 673

FIGS.5–8. Canker symptoms exhibited by Zanthoxylum bungeanum cultivar Doujiao inoculated with (5) Fusarium zanthoxyli F201119, (6) F. continuum F201127 and (7) FIG. 4. Virulence of Fusarium zanthoxyli, F. continuum and Fusarium sp. FSSC 6 F201131. 8. Negative control lacking a FSSC 6 to Zanthoxylum bungeanum cultivar Doujiao. Bars discernible canker. represent means and ¡ standard errors (SEM) of isolate- lesion size of each pathogen and the uninoculated negative multilocus haplotypes. In addition, F. zanthoxyli isolates control. *P , 0.05 and **P , 0.01 compare size of lesions F201112 from Shaanxi and F201125 from Shanxi pos- induced by the three pathogens. sessed highly divergent TEF1 and RPB2 alleles, respec- tively, (FIG.3).Fusarium continuum isolates were supported 27 and 28 nodes, respectively, at $70% collected from Shandong (n 5 3) and Hebei (n 5 1) (FIG. 2) and provided strong support for a novel clade and isolates of F. zanthoxyli were from Gansu (n 5 10), (i.e. FTOSC) comprising F. torreyae and the two novel Shaanxi (n 5 14) and Shanxi (n 5 1). However, CDZB pathogens F. zanthoxyli and F. continuum whose F. continuum (F201030) and F. zanthoxyli (F201307) 5 monophyly (ML-BS/MP-BS 100%) and sister group were sympatric in Fuping County, Shaanxi (FIG.1). relationship were strongly supported (ML-BS/MP-BS 5 92%). Although the three partitions we sequenced Pathogenicity of isolates to Zanthoxylum bungeanum.— possessed different levels of phylogenetically informa- Thirty-five of the 38 Fusarium isolates tested induced tive characters (PIC) (TABLE I; ITS rDNA 5 2.9%, cankers (FIGS.5–7) and were recovered from the can- TEF1 5 17.1%, and RPB2 5 8.9%), analyses of the indi- ker margins when the pathogenicity experiment was vidual ITS rDNA, TEF1 and RPB2 partitions and the terminated after 22 d. Compared with isolates of F. combined three-gene dataset, using sequences of zanthoxyli and F. continuum, the single isolate of F. acu- F. torreyae to root the phylogenies, strongly supported minatum F201136, Fusarium sp. F201237 (F. lateritium the genealogical exclusivity F. zanthoxyli and F. con‐ species complex) and Fusarium sp. FIESC 1 F201338 tinuum (FIG. 3). Considerable allelic diversity was (F. incarnatum-equiseti species complex) induced lesions detected within both species, in that 20/24 F. zanthoxyli on Z. bungeanum cultivar Doujiao that were not signifi- and all five F. continuum isolates possessed unique cantly larger than the negative control (FIG. 8).

TABLE I. Tree statistics for individual partitions and combined dataset (see FIG.3)

MPTsa Tree length CIb RIc bpd AUTe PICf PIC/bpg ITS rDNA 1 21 1 1 548 2 16 0.029 TEF1 1 101 0.95 0.99 479 6 82 0.171 RPB2 3 181 0.96 0.99 1762 14 157 0.089 Combined 56 321 0.90 0.97 2789 22 255 0.091

a MPTs, most-parsimonious trees. b CI, consistency index. c RI, retention index. d bp, base pairs. e AUT, autapomorphy or a derived character unique to a particular taxon (i.e. not parsimony-informative). f PIC, parsimony-informative or shared derived character. g PIC/bp, parsimony-informative characters/bp. 674 MYCOLOGIA

FIGS.9–31. Morphology of Fusarium zanthoxyli cultured on SNA (9, 22, 30 cultured in the dark; 10–21, 23–29, 31 cultured under day light; 9–11 aerial view; 12–31 mounted in water). 9–11. Aerial conidia formed on conidiophores arising from hyphae on the agar surface. 12, 13. Branched or unbranched aerial conidiophores forming monophialides. 14–16. Falcate and short naviculate aerial conidia. 17–20. Branched or unbranched sporodochial conidiophores forming conidia. 21–24. Falcate ZHOU ET AL.: NOVEL PATHOGENS OF PRICKLY ASH 675

Therefore we focused on results of the pathogenicity directly on aerial mycelium, on substrate mycelium experiment obtained for isolates of F. zanthoxyli or in sporodochia. Sporodochia sometimes formed (n 5 25), F. continuum (n 5 5) and FSSC 6 (n 5 5). around the inoculum under fluorescent or daylight. Comparisons using Fisher’s LSD multiple comparison Aerial conidiophores and conidia mostly absent or test revealed that F. zanthoxyli and F. continuum pro- rarely formed on SNA. Aerial and sporodochial duced significantly larger lesions (mean 5 1.24 ¡ conidiophores and conidia not well differentiated. 0.03 [SEM] and mean 5 1.36 ¡ 0.05 [SEM], respec- Aerial and sporodochial conidiophores, if present, tively) than the negative control (mean 5 0.67 ¡ generally densely to sparsely branched but sometimes 0.01 [SEM]) (p , 0.01). Moreover, the five isolates unbranched, forming apical monophialides or some- of FSSC 6 induced significantly larger lesions (mean times intercalary phialides, up to 62.0 μm long and 5 0.94 ¡ 0.03 [SEM]) than the negative control 2.5–6.5 μm wide. Phialides subcylindrical to ampulli- (p , 0.01). While lesions induced by F. zanthoxyli form, often with a conspicuous collarette at the tip, and F. continuum did not differ in size (p . 0.05), up to 40.5 μm long and 2.5–5.0 μm wide. Aerial and both were significantly larger than those produced by sporodochial conidia morphologically indistinguish- , the five FSSC 6 isolates (p 0.01), indicating that able, typically falcate, slightly curved or straight, dorsi- F. zanthoxyli and F. continuum were more virulent to ventral, or sometimes fusiform, often widest in the Z. bungeanum cultivar Doujiao. upper half or less frequently at the midregion, taper- ing toward both ends, with an apical cell often rostrate, TAXONOMY sometimes acute, and a basal foot cell indistinct to dis‐ tinct with a conspicuous protrusion, (1–)3–5(–7)-septate; Fusarium zanthoxyli X. Zhou, T. Aoki, K. O’Donnell in darkness or under daylight, three-septate: 19.5–55.0 & Z.M. Cao, sp. nov. FIGS.9–31 6 3.0–6.0 μm in total range, 30.7–42.3 6 3.7–4.5 μm MycoBank MB809716 av. (ex-type: 25.3–48.0 6 3.0–5.0 μm, 39.1 ¡ 7.3 6 Typification: CHINA. SHAANXI PROVINCE: Tong- 4.3 ¡ 0.5 μm av. and SD); five-septate: 41.0–76.0 6 chuan city, Yaozhou , Sunyuan town, isolated 3.7–6.5 μm, 54.3–68.5 6 4.7–5.8 μm av. (ex-type: from stem tissue of diseased Zanthoxylum bungeanum, – 6 – μ ¡ 6 ¡ μ 4 Sep 2013, Xue Zhou Fyzs133-2013 (holotype 47.2 74.0 4.3 5.8 m, 62.3 5.9 5.0 0.3 m HMNWAFU XZ-Fyzs133-20130408, a dried culture of av. and SD). Occasionally shorter, naviculate, ellipsoi- F201311). Ex-type culture: F201311 5 NRRL 66285 5 dal to clavate conidia with acuate to pointed, rarely CBS 140838. rounded apex and pointed to rounded base less – – – Etymology: From Latin zanthoxyli, referring to the host, frequently formed on SNA, (0 )1 2( 3)-septate, – 6 – μ ‐ Zanthoxylum bungeanum. 7.5 28.0 2.0 4.0 m if present. Chlamydospores pres Colonies on PDA with mycelial growth rates of ent or absent, oblong to subglobose, smooth to rough, 0.6–2.4 mm/d at 20 C in the dark. Colony margins thick-walled, intercalary or terminal, solitary, in pairs mostly undulate, sometimes entire. Aerial mycelia or catenate, 3.0–16.5 6 3.0–8.5 μmwhenpresent. on PDA generally sparsely to moderately formed, Other isolates examined: All from stem tissue of diseased Z. some developed abundantly, then loose to densely bungeanum; CHINA. SHAANXI PROVINCE: city, floccose, sometimes felted, white (1A1), yellowish , Jiaoliao village, Aug 2011, Ning Xie Fbjj- white (2–4A2), pinkish white to pastel red (7A2–4) in 2011, F201105; CHINA. SHAANXI PROVINCE: Hancheng the dark, pastel red to reddish orange (7A4–6), pale city, Xuefeng town, Wang village, Aug 2011, Ning Xie Fhcw- red (7–9A3) under fluorescent or daylight, upon spor- 2011, F201108; CHINA. GANSU PROVINCE: Qinan ulation pale orange to deep orange (6A3–8). Pigmen- County, Yebao Township, Yangjiasi, Sep 2011, Ning Xie tation in the reverse pale yellow (2–4A3), pale orange Fqayy-2011, F201115; CHINA. GANSU PROVINCE: Gangu to light orange (6A3–4) or pale red to pastel red County, Ershilipu, Sep 2011, Ning Xie Fgge-2011, F201116; (7A3–4) in the dark, light orange to orange (6A4–7), CHINA. GANSU PROVINCE: , Daxiangshan pastel red to reddish orange (7A5–7) under fluores- town, Zhangjiajing village, Sep 2011, Ning Xie Fggz-2011, cent or daylight. Dark blue sclerotial bodies sometimes F201117; CHINA. GANSU PROVINCE: city, present under daylight. Odor sweet, sometimes absent. Qinzhou district, Guanzi town, Sep 2011, Ning Xie Ftqg- Sporulation on SNA generally relatively scarce either 2011, F201119; CHINA. GANSU PROVINCE: Wen County, r sporodochial conidia with rostrate apical cell and protruding basal foot cell and shorter conidia. 25, 26, 29. Catenate chlamydospores in hyphae and conidia. 27, 28, 30, 31. Short 1–3-septate, naviculate to clavate conidia with acute to rounded apex and pointed to rounded base. 9, 22 from F201119; 10, 11–16, 23, 30 from F201124; 17, 28 from F201311 (ex holotype); 18 from F201116; 19, 20, 26 from F201313; 21, 29 from F201108; 24 from F201218; 25 from F201307; 27 from F201322; 31 from F201117. Bars: 9–11, 18 5 50 μm, 12–17, 19–31 5 20 μm. 676 MYCOLOGIA

FIGS.32–53. Morphology of Fusarium continuum cultured on SNA (32, 33, 35–37, 39–41, 43, 44, 50, 53 cultured in the dark; 34, 38, 42, 45–49, 51, 52 cultured under day light; 32–35, 43, 44 aerial view; 36–42, 45–53 mounted in water). 32–35. Aerial conidia formed on conidiophores arising from hyphae on the agar surface. 36. Unbranched aerial conidiophores forming apical monophialides. 37–39. Falcate and short ellipsoidal to clavate aerial conidia. 40–45. Branched or unbranched sporodochial ZHOU ET AL.: NOVEL PATHOGENS OF PRICKLY ASH 677

Baoziba Township, Guanyinba, Sep 2011, Ning Xie Fwbg- rates were 1.4–2.6 mm/d at 30 C in the dark. Colony 2011, F201124; CHINA. GANSU PROVINCE: Qinan margins undulate. Aerial mycelia on PDA generally County, Yebao Township, Chenjiahe, Aug 2012, Jie-Fei moderately formed, loose to densely floccose, white Wang Fqayc1-2012, F201218; CHINA. GANSU PROVINCE: (1A1), light yellow to greenish yellow (1A5–7), pastel Qinan, Yebao Township, Anjiachuan, Sep 2012, Jie-Fei yellow (2–3A4), light orange (5A4), grayish yellow Wang Fqaya2-2012, F201220; CHINA. SHAANXI PROV‐ (3–4B4, 5B4–5), grayish violet (19D3–4) or dull INCE: Baoji city, Chencang district, Tianwang town, blue (22D4) in the dark, pale yellow to light yellow Miaoju village, Apr 2013, Jie-Fei Wang Fbjm1-2013, (3–4A4), pale red to pastel red (7A3–4), grayish F201301; CHINA. SHAANXI PROVINCE: Fuping County, orange to grayish red (6–7B3), deep violet to dark vio- Lei village, Sep 2013, Xue Zhou Ffpl-13-2013, F201307; let (16E–F8) or bluish gray to dull blue (23E2–4) CHINA. SHAANXI PROVINCE: Hancheng city, Zhiyang under fluorescent or daylight, upon sporulation light town, village, Jun 2013, Ning Xie Fhcd-2013, orange to orange (6A4–7), or grayish orange (6B3–4). F201309 5 NRRL 66217 5 CBS 140839; CHINA. Light yellow or light orange exudate sometimes pres‐ SHAANXI PROVINCE: city, Yaozhou district, ent. Pigmentation in the reverse grayish yellow – – – Sunyuan town, Sep 2013, Xue Zhou Fyzs132-2013, F201313; (2 3B6, 4B5 6), light orange (5A4 5), grayish orange – – CHINA. GANSU PROVINCE: Wen County, Baoziba (4B7 8), or light brown to dark brown (6D F5) in the – Township, Sidouping village, Oct 2013, Xue Zhou Fwbs1- dark, light orange to brownish orange (5A C5), light – 2013, F201322. brown (6D5), or gray to grayish brown (7D1 3) under Distribution: Gansu, Shanxi and Shaanxi provinces, fluorescent or daylight. Dark blue sclerotial bodies China. sometimes present under daylight. Odor sweet. Sporu- Notes: The key morphological character that distin- lation on SNA generally relatively rapid and abundant guishes F. zanthoxyli from other species within the directly on aerial mycelium, on substrate mycelium FTOSC is the production of falcate conidia that typi- or in sporodochia. Aerial conidia formed earlier than cally possess a rostrate apex, together with aerial and sporodochial conidia on SNA. Aerial and sporodochial sporodochial conidiophores that are not well differen- conidiophores and conidia not well differentiated tiated. Fusarium zanthoxyli is similar to F. torreyae in that and form a morphological continuum. Aerial and sporodochial conidiophores formed sparsely to abun- both species form long, slender sporodochial conidia. However, conidia in F. zanthoxyli typically are widest dantly, unbranched, sparsely or densely branched, forming intercalary or apical monophialides, up to in the upper half whereas those of F. torreyae are usually 60.5 μm long and 2.5–6.0 μm wide. Phialides subcylin‐ widest at the mid-region. In addition, F. zanthoxyli drical or ampuliform, often with a conspicuous collar- produces fewer 7–9-septate conida than F. torreyae. ette at the tip, up to 38.5 μm long and 1.5–4.5 μm The rostrate apical cell and protruding basal cell of wide. Aerial and sporodochial conidia morphologically F. zanthoxyli represent additional morphological fea- indistinguishable, typically falcate and gradually curved, tures that differentiate it from F. torreyae. dorsiventral, or sometimes fusiform, mostly widest in Fusarium continuum X. Zhou, T. Aoki, K. O’Donnell & the upper half, sometimes at the midregion, tapering Z.M. Cao, sp. nov. FIGS.32–53 and gradually curving toward both ends, with an MycoBank MB809718 apical cell mostly acuminate, sometimes pointed, and Typification: CHINA. SHAANXI PROVINCE: Fuping a basal foot cell indistinct to distinct, sometimes with County, Lei village, isolated from stem tissue of a discernible protrusion; (1–)3–5(–6)-septate; in dark- diseased Z. bungeanum, Aug 2010, Ning Xie Ffpl-10- ness three-septate: 16.0–48.0 6 3.0–5.5 μm, 34.5–38.9 6 2010 (holotype HMNWAFU NX-Ffpl-10-20100851,a 4.3–4.7 μm av. (ex-type: 22.3–44.0 6 4.0–5.6 μm, dried culture of F201030). Ex-type culture: F201030 34.5 ¡ 5.1 6 4.7 ¡ 0.4 μm av. and SD); five-septate: 5 NRRL 66286 5 CBS 140841. 33.0–67.0 6 4.0–5.8 μm, 45.3–57.4 6 4.9–5.0 μm av. Etymology: from Latin continuus, based on the morphologi- (ex-type: 33.0–53.1 6 4.1–5.5 μm, 45.3 ¡ 3.3 6 5.0 ¡ cal continuum of aerial and sporodochial conidiophores 0.2 μm av. and SD); On SNA sometimes shorter, and conidia. ellipsoidal, naviculate to clavate conidia also formed, Colonies on PDA with mycelial growth rates of straight or curved, with a rounded, rarely pointed 1.0–1.5 mm/d at 20 C in the dark, but maximal growth apex and a truncate or rounded base, 0–2(–3)-septate, r conidiophores forming conidia. 46, 47. Falcate sporodochial conidia with acuminate apical cell and shorter conidia. 48, 49. Solitary, paired or catenate chlamydospores in conidia. 50–53. Short 0–3-septate, ellipsoidal, naviculate to clavate conidia with rounded to acute apex and rounded or truncate base. 32, 34, 38, 40, 41, 43, 44, 48, 49, 53 from F201127; 33, 35, 36, 47 from F201128; 37, 39 from F201129; 42, 45, 46, 51, 52 from F201030 (ex holotype); 50 from F201126. Bars: 32–35, 43, 44 5 50 μm; 36–42, 45–53 5 20 μm. 678 MYCOLOGIA

FIGS. 54, 55. Radial growth rates of (54) Fusarium zanthoxyli and (55) Fusarium continuum per d on PDA at eight temperatures 5–40 C. Thick horizontal and vertical bars indicate means and total ranges, respectively, of each species (number of isolates examined in parentheses).

4.0–26.5 6 2.0–5.0 μm when present. Chlamydospores rounded to truncated base on SNA. These two species formed on hyphae or in conidia, subglobose to oblong, also can be distinguished by differences in their radial smooth to rough, thick-walled, intercalary or terminal, mycelial growth rates. Average radial mycelial growth solitary, in pairs or catenate, 3.0–12.5 6 2.5–8.0 μm. rates on PDA in the dark at eight temperatures Other isolates examined: All isolated from stem tissue 5–40 C were calculated for 14 isolates of F. zanthoxyli of diseased Z. bungeanum; CHINA. SHANDONG PROV‐ and five isolates of F. continuum and are summarized INCE: city, Shanting district, Sep 2011, Ning (FIGS. 54, 55). Optimal temperature for mycelial Xie Fst-2011, F201126 5 NRRL 66218 5 CBS 140840; growth was 25 C for all isolates of F. zanthoxyli and CHINA. SHANDONG PROVINCE: city, Boshan dis- one isolate (F201126) of F. continuum and 30 C for trict, Yijialou, Sep 2011, Ning Xie Fbsy-2011, F201127; 4/5 isolates of F. continuum.Mycelialgrowthat25C – – CHINA. SHANDONG PROVINCE: , was 0.8 2.6 mm/d for F. zanthoxyli and 1.2 1.8 mm/ – Shiqiao village, Sep 2011, Ning Xie Fyys-2011, F201128; dforF. continuum; growth rate at 30 C was 0.2 1.3 – CHINA. HEBEI PROVINCE: She County, Wangjinzhuang mm/d for F. zanthoxyli and 1.4 2.6 mm/d for F. village, Sep 2011, Ning Xie Fhsw-2011, F201129. continuum. Distribution: Shandong, Hebei, and Shaanxi prov‐ inces, China. DISCUSSION Notes: Fusarium continuum can be differentiated from The primary findings of the present study include the other members of the FTOSC by the production discovery of a novel clade of canker-inducing fusaria, of shorter aerial and sporodochial conidia and rare here designated as FTOSC, comprising F. torreyae,a production of 7–9-septate conidia, together with the pathogen of the critically endangered Florida torreya undifferentiated aerial and sporodochial conidiophores. (Torreya taxifolia) in northern Florida and southwest‐ Of the three species within the FTOSC, conidia of ern Georgia (Smith et al. 2011, Aoki et al. 2013) and F. torreyae are the longest, followed by F. zanthoxyli two novel pathogens of prickly ash trees (Zanthoxylum and F. continuum, when conidia with the same number bungeanum) in northern China that are described of septa were compared. Fusarium continuum and formally herein as F. zanthoxyli and F. continuum. F. zanthoxyli produce morphologically similar multisep- Molecular clock estimates place the divergence of the tate, falcate conidia that are usually widest in the upper FTOSC in the mid-Eocene , 40 Mya (O’Donnell et al. half and only rarely at the midregion. By contrast those 2013), but it remains an open question whether this produced by F. torreyae are long, slender, gradually clade first evolved in the Old or New World. Further- curved, and most frequently widest at the mid-region. more, it remains to be determined whether F. torreyae Conidia produced by F. continuum can be distinguished is native to North America and restricted to T. taxifolia. from those of F. zanthoxyli in that the latter possess Surveys for F. torreyae on Torreya endemic to China are a rostrate apical cell and a conspicuous ventrally pro- warranted because it is the modern area of diversity truding basal cell. In addition, F. continuum also forms of this genus (Li et al. 2001) and because the putative ellipsoidal, clavate to naviculate, straight or slightly Asian origin of the CDZB pathogens could indicate curved conidia with a mostly rounded apex and that the most recent common ancestor of the FTOSC ZHOU ET AL.: NOVEL PATHOGENS OF PRICKLY ASH 679 evolved in Asia. Additional surveys also are needed Several species of Fusarium are capable of causing to characterize the host range and geographic distribu- cankers on woody plants, and mixed infections fre- tion of the two novel CDZB pathogens. The available quently occur. For example, a particular FSSC species data suggests their ranges are distinct, with F. zanthoxyli occasionally was found co-occurring with F. torreyae in distributed across three provinces from Gansu and cankers on Florida torreya (Torreya taxifolia) (Smith Shaanxi in the northwest to Shanxi in the north of et al. 2011). Although both species could induce China, whereas F. continuum is mainly distributed in cankers, F. torreyae is considered to be the primary Shandong in the east of China. The two prickly ash pathogen due to increased virulence and consistent pathogens, however, are sympatric in Fuping County isolation from a large number of cankers. By contrast of Shaanxi province (FIG.1,SUPPLEMENTARY TABLE I). the available data indicates the FSSC taxon should be Although a sexual cycle has not been found in the regarded as an opportunistic ( J. Smith pers comm). three FTOSC species in the laboratory, the high multi- In addition, F. solani f. sp. xanthoxyli, a well-known locus haplotype diversity we detected within F. zanthoxy‐ fungus in Japan within the FSSC has been reported li and F. continuum suggests that these pathogens may to cause trunk-blight of a closely related tree species, possess a heterothallic sexual reproductive mode. Zanthoxylum piperatum (L.) DC. under the name of Nec- A similar finding in the soybean sudden death syn- tria elegans Yamamoto & Maeda (Yamamoto et al. 1957, drome pathogen, F. tucumaniae T. Aoki et al., led to Matuo and Snyder 1961, Sakurai and Matuo 1961). the discovery of a sexual cycle by conducting laboratory However, this fungus was shown to represent an unde- crosses under light and temperature conditions optimal scribed phylogenetic species of Fusarium designated for perithecial production (Covert et al. 2007). Knowl- FSSC 22 (O’Donnell et al. 2008), which is morphologi- edge of a sexual cycle in the CDZB pathogens has cally different from the CDZB-associated Fusarium spe- important implications for disease management and cies from China. Although multiple species frequently control because sexually reproducing pathogens are are isolated from a host in disease etiology studies, much more likely to overcome host resistance. In addi- pathogenicity and virulence testing are essential to tion to the high SNP diversity that we detected within complete Koch’s postulates, as is identification of the F. zanthoxyli it is worth mentioning that highly divergent fungal species most frequently isolated from symptom‐

TEF1, RPB2 and ITS rDNA alleles were detected in iso- atic tissues. lates of F. zanthoxyli from Shaanxi, Shanxi and Gansu, In addition to Fusarium species other microbes have respectively. Of interest, the ITS rDNA of F201125 been reported to induce cankers on Z. bungeanum, for from Shanxi differed at only two nucleotide positions example, Phytophthora spp. (Xie et al. 2013). Because from that of F. torreyae, suggesting possible hybridization these cankers are typical of those caused by other between F. zanthoxyli and a F. torreyae-like species. Fusarium spp. (i.e. FSSC 6 and F. torreyae) it is possible In the present study we characterized a novel plant that two different stem diseases have been confused disease designated Canker disease of Zanthoxylum on this host. Also a jewel beetle (Agrilus sp.) frequently bungeanum (CDZB) and completed Koch’s postulates. colonizes wounds resulting from CDZB (Li et al. 1988), Lesion sizes caused by the three most frequently iso- causing further damage to this host. Whether the lated Fusarium species recovered from prickly ash trees wounds caused by this wood-boring beetle serve as showing symptoms of CDZB were used as a proxy for infection courts for these pathogens warrants further their virulence on this host. Results of the pathogenic‐ study. ity experiment revealed that isolates of the two novel The 38 fusaria isolated from Zanthoxylum bungeanum CDZB-associated Fusarium species exhibited higher cankers in the present study were identified and virulence than that of FSSC 6 and single isolates of placed in Fusarium by a two-step process that first three other Fusarium species. This finding establishes involved BLASTN queries of NCBI GenBank and that F. zanthoxyli and F. continuum are the major FUSARIUM-ID (Geiser et al. 2004), using a partial pathogenetic Fusarium species on Z. bungeanum. Iso- TEF1 sequence as the query, followed by phylogenetic lates of F. zanthoxyli and F. continuum also were con- analyses of a RPB1-RPB2 dataset that spanned the phy- firmed to be pathogenic to Z. bungeanum cultivars logenetic breadth of Fusarium (O’Donnell et al. 2013). Dahongpao (including Fengjiao and Hancheng- Results of both analyses indicated that the two primary Dahongpao), Youjiao and Mijiao (Xie 2012). Longer- CDZB pathogens were most closely related to F. torreyae term inoculation studies are needed to help elucidate (Smith et al. 2011, Aoki et al. 2013). Morphological whether the causal agents can ultimately kill trees species recognition is challenging for nonspecialists under more field based conditions. It is also of inter- in Fusarium (Gerlach and Nirenberg 1982, Nelson et al. est to test these isolates against other members of 1983, Leslie and Summerell 2006), as evidenced by the the Rutaceae, especially those that are economically phenotypic identification of F. torreyae as F. lateritium important (i.e. citrus). (El Gholl 1985) and the CDZB pathogens as F. 680 MYCOLOGIA sambucinum (Cao et al. 1992, 2010) and F. lateritium rostrum sp. nov., F. tucumaniae and F. virguliforme. Mycolo- (Xie et al. 2012), although Fusarium isolates from gia 46:162–183, doi:10.1007/s10267-005-0235-y wooden substrates often have been identified as ———, Smith JA, Mount LL, Geiser DM, O’Donnell K. 2013. F. lateritium without detailed morphological analyses. Fusarium torreyae sp. nov., a pathogen causing canker Fusarium torreyae can be distinguished from F. zanthoxyli disease of Florida torreya (Torreya taxifolia), a critically and F. continuum by its host, Florida torreya, and phe- endangered conifer restricted to northern Florida and southwestern Georgia. Mycologia 105:312–319, notypically by the frequent production of seven-septate doi:10.3852/12-262 sporodochial conidia and the absence of aerial coni- Cao Z-M, Liang Y-M, Ma X. 1992. A study on stem rot of dia. In contrast F. continuum produces abundant coni- prickly ash. J Northwest Forestry College 7:58–63. dia that are shorter than F. torreyae, rarely produces ———, Ming YL, Cheng D, Zhang H. 2010. Resistance of seven-septate sporodochial conidia and occasionally prickly ash to stem rot and pathogenicity differentiation produces dark blue sclerotia. Fusarium zanthoxyli of Fusarium sambucinum. 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