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Short title: Novel pathogens of prickly ash
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
College of Forestry, Northwest A&F University, Taicheng Road, Yangling, Shaanxi China
712100
Kerry O’Donnell
Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for
Agricultural Utilization Research, Agricultural Research Service, U.S. Department of
Agriculture, Peoria, Illinois 60604-3999
Takayuki Aoki
Genetic Resources Center (MAFF), National Institute of Agrobiological Sciences, 2-1-2
Kannondai, Tsukuba, Ibaraki 305-8602, Japan
Jason A. Smith
School of Forest Resources and Conservation, University of Florida, Gainesville, Florida
32611-0680
Matthew T. Kasson
Division of Plant and Soil Sciences, West Virginia University, Morgantown, West Virginia
26506-6108
Zhi-Min Cao1
College of Forestry, Northwest A&F University, Taicheng Road, Yangling, Shaanxi China
712100
In Press at Mycologia, preliminary version published on April 7, 2016 as doi:10.3852/15-189
Copyright 2016 by The Mycological Society of America.
Abstract: Canker disease of prickly ash (Zanthoxylum bungeanum) has caused a decline in
the production of this economically important spice in northern China in the past 25 y. To
identify the etiological agent, 38 fungal isolates were recovered from symptomatic tissues
from trees in five provinces in China. These isolates were identified by conducting BLASTN
queries of NCBI GenBank and phylogenetic analyses of DNA sequence data from the nuclear
ribosomal internal transcribed spacer region (ITS rDNA), a portion of the translation
elongation factor 1-α (TEF1) gene, and genes encoding RNA polymerase II largest (RPB1)
and second largest (RPB2) subunits. Results of these analyses suggested that 30/38 isolates
belonged to two novel fusaria most closely related to the Florida torreya (Torreya taxifolia
Arn.) pathogen, Fusarium torreyae in Florida and Georgia. These three canker-inducing tree
pathogens form a novel clade within Fusarium here designated the F. tor reyae species
complex (FTOSC). BLASTN queries of GenBank also revealed that 5/38 isolates recovered
from cankers represented an undescribed phylogenetic species within the F. solani species
complex (FSSC) designated FSSC 6. Stem inoculations of three fusaria on Z. bungeanum
resulted in consistent canker symptoms from which these three fusaria were recovered. The
two novel fusaria, however, induced significantly larger lesions than FSSC 6. Herein, the two
novel prickly ash pathogens are formally described as F. zanthoxyli and F. continuum.
Key words: Fusarium continuum, F. zanthoxyli, genealogical concordance, molecular
phylogenetics, morphology, RPB2, TEF1
INTRODUCTION
Commonly known as prickly ash, Zanthoxylum bungeanum (Rutaceae) is an economically
important tree species in dry, mountainous areas in several provinces in northern China. In
the past two and one-half decades a canker disease has resulted in diminished production of
prickly ash, a common peppery spice used in Asian cuisine derived from at least two species
of Zanthoxylum including Z. bungeanum. The pericarp of this plant also is used in traditional
Chinese medicine (Tang et al. 2014). Symptoms of the prickly ash disease include branch and
stem cankers, dieback and occasional tree mortality. In initial studies conducted on canker
disease of Z. bungeanum (hereafter abbreviated CDZB) in Shaanxi and Gansu provinces the
putative pathogen isolated from cankered stems was identified as Fusarium sambucium
Fuckel based on morphological data (Cao et al. 1992, 2010). Subsequently the CDZB isolates
were re-identified as F. lateritium Nees (Xie 2012). These contrasting identifications
highlight the need for the CDZB isolates to be characterized further with molecular
systematic data. Phylogenetic species recognition based on genealogical concordance (Taylor
et al. 2000) has made a significant impact on Fusarium systematics over the past two decades
(Aoki et al. 2014). Portions of the translation elongation factor 1-α gene (TEF1, Geiser et al.
2004) and the largest (RPB1) and second largest (RPB2) subunits of RNA polymerase II
genes have proven utility for inferring species limits and elucidating phylogenetic
relationships within Fusarium (O’Donnell et al. 2013). Accordingly the present study was
conducted to (i) collect and analyze multilocus DNA sequence data phylogenetically together
with morphological data to identify the casual agents of Fusarium CDZB in the main prickly
ash production areas of northern China, (ii) assess virulence of the fusaria on Z. bungeanum
and (iii) formally describe two novel CDZB pathogens morphologically as F. zanthoxyli and
F. continuum.
MATERIALS AND METHODS
Isolation of pathogenic fusaria.—Cankers on Z. bungeanum were collected 2010–2013 mainly in Shaanxi,
Gansu and Shandong provinces but also in Shanxi and Hebei in China (FIG. 1). Thirty-eight Fusarium isolates
(SUPPLEMENTARY TABLE I) were recovered from surface-sterilized stem tissues excised from the margins of
cankers following the protocol of Smith et al. (2011). For long-term preservation all CDZB isolates were stored
in 40% glycerol at −80 C in the laboratory of Zhi-Min Cao. In addition two isolates of F. zanthoxyli and F.
continuum, including the ex-type isolates, were deposited in the ARS Culture Collection (NRRL) (see
SUPPLEMENTARY TABLE I).
Herbarium specimens were deposited in the Mycological Herbarium of Forestry College, Northwest
A&F University, Yangling, Shaanxi province, China (HMNWAFU).
DNA extraction, PCR amplification and sequencing.—Mycelium was cultured in potato dextrose broth
(Liang et al. 2014) on a rotary shaker at 120 rpm for 7 d at 24 C. Mycelium was harvested over sterilized gauze,
freeze-dried, and then total genomic DNA was extracted with a CTAB (hexadecyltrimethylammonium bromide)
miniprep protocol (O’Donnell et al. 1998a). The nuclear ribosomal internal transcribed spacer region (ITS
rDNA) and portions of TEF1, RPB1 and RPB2 genes were PCR-amplified and sequenced with primers
published in the following: ITS rDNA (White et al. 1990), TEF1 (O’Donnell et al. 1998b, 2008), RPB1
(O’Donnell et al. 2010) and RPB2 (Liu et al. 1999, Reeb et al. 2004, Hofstetter et al. 2007). PCR reactions were
conducted in a total volume of 50 μL that contained 2 μL of each primer (10 μM), 25 μL 2× Taq PCR
MasterMix (Biosci Biotech Co, Hangzhou, China), 2 μL diluted (1:50) template DNA and 19 μL
double-distilled water. After PCR amplification amplicons were sized by gel electrophoresis on 1.5% agarose
gels that were run in 1× TAE buffer. Amplicons were purified and sequenced by Sangon Biotech Ltd, Shanghai,
China.
Molecular phylogenetics.—Chromas 2 and Chromas Pro 1.7.5 (Technelysium Pty Ltd, Brisbane, Australia),
respectively, were used to manually edit chromatograms and assemble the contigs. Contigs were aligned with
MUSCLE (Edgar 2004) and the following two datasets were analyzed phylogenetically: (i) a 39-taxon
RPB1-RPB2 dataset to place the CDZB isolates within Fusarium (FIG. 2), and (ii) a 31-taxon three-gene dataset
to assess the genealogical exclusivity of the two putatively novel CDZB pathogens, F. zanthoxyli and F.
continuum (FIG. 3). The individual and combined partitions were analyzed phylogenetically and clade support
was assessed by maximum parsimony bootstrapping (MP-BS) in PAUP* 4.0b10 (Swofford 2003), and
maximum likelihood (ML) in GARLI 2.01 on XSEDE (Zwickl 2006) on the CIPRIS Science Gateway
(https://www.phylo.org/portal2/login!input.action). The MP-BS analysis was conducted with equally weighted
characters, a heuristic search employing 1000 random sequence additions, TBR branch-swapping, MAXTREES set
at 5000 employing 1000 bootstrap replicates (Felsenstein 1985). ML bootstrapping was conducted employing
the GRT + Γ + I model of molecular evolution, which was selected with JModelTest (Posada 2008). DNA
sequence data generated in the present study were deposited in GenBank (SUPPLEMENTARY TABLE I), and
alignments and trees were submitted to TreeBASE (accession number S17885, tree number Tr89189–Tr89193).
Pathogenicity of fusaria to Zanthoxylum bungeanum.—To assess pathogenicity 38 isolates were inoculated onto
eight 7 y old Z. bungeanum cultivar Doujiao trees in the nursery of the College of Forestry, Northwest A&F
University. After a wound approximately 40 mm2 was made with a pointed tweezer on 1 or 2 y old branches of
prickly ash, it was inoculated with a 5 mm diam potato dextrose agar (PDA) plug containing mycelium from a
14 d old culture of one of the 38 isolates (SUPPLEMENTARY TABLE I), after which the inoculation site was
wrapped with absorbent cotton dipped in sterile water and sealed tightly with a plastic film to keep humid.
Every isolate was inoculated in two wounds on two different branches. Wounds inoculated with a sterile PDA
plug served as the negative control. During the pathogenicity experiment the average temperature in the field
was 20 C during daytime and 12 C at night. The experiment was terminated after 22 d at which time inoculated
branches were removed from the trees and were taken to the lab where canker size was measured (FIG. 4). The
average lesion dimension was measured and used in the ANOVA analysis with the LSD multiple comparison
test employing IBM SPSS Statistics 19 (https://www14.software.ibm.com/). To complete Koch’s postulates, the
inoculated isolates were re-isolated as described above and confirmed morphologically and molecularly by ITS
sequence analysis.
Morphological characterization.—Fourteen isolates of F. zanthoxyli and five of F. continuum were used in the
morphological study. Methods for determining phenotypic characters and mycelial growth rates followed Aoki
et al. (2003, 2005). Isolates were grown at 20 C in 9 cm plastic Petri dishes on PDA and synthetic nutrient-poor
agar (SNA) with or without placing a sterile 1 × 1 cm piece of filter paper on the SNA surface (Nirenberg and
O’Donnell 1998) in the dark, under continuous fluorescent light (Panasonic YZ36RR6500K) or under daylight.
Colony morphology, color, odor and growth rate were based on cultures grown on PDA (Fang 1998). All colors
are given according to the Methuen Handbook of Color (Kornerup and Wanscher 1978). Mycelial growth rates
were calculated as described in Aoki et al. (2003). Agar blocks 5 mm diam were cut from the margins of 2 wk
old cultures on SNA and inoculated onto PDA. The cultures were incubated in the dark at eight temperatures
5–40 C at 5 C intervals. Cultures were examined after 1 and 5 d under a dissecting microscope, and colony
margins were marked on the reverse side of the Petri dishes. Mean values of radial mycelial growth rate per day
were calculated by measuring the distance from 16 points on the colony margin to the center. Measurements
were repeated twice and averaged. All microscopic studies and measurements were made on isolates cultured on
SNA. Measurements of 50 randomly selected conidia were taken based on the number of septa and cultural
condition, and minimal and maximal sizes, arithmetic means and standard deviations (SD) were obtained.
Conidia, conidiophores and chlamydospores produced on SNA were viewed and photographed by light
microscopy (Olympus, CX31RTSF, Japan) with or without mounting them in water. Descriptive terms for
conidia and conidiophore morphology followed Nirenberg and O’Donnell (1998).
RESULTS
DNA sequence-based identification of the isolates.—ITS rDNA, TEF1 and RPB2 nucleotide
sequences of the 38 isolates recovered from Z. bungeanum cankers were used to query
GenBank; TEF1 also was used to query FUSARIUM-ID (Geiser et al. 2004). Nucleotide
BLASTN queries revealed that Fusarium torreyae was the best match for 30/38 isolates
(SUPP LEME NTARY TA BLE I) as follows: ITS rDNA = 97%, TEF1 = 87–91% and RPB2 =
95–96%. Sequences of 5/38 isolates that were nested within the FSSC (F201131–201135)
revealed 99–100% identity to an unnamed phylogenetic species designated FSSC 6
(O’Donnell et al. 2008). The remaining three isolates were identified via BLASTN queries of
GenBank, using the partial RPB2, as F. acuminatum (F201136, 100% identity to
HM068334.1 F. acuminatum NRRL 54216), Fusarium sp. (F201237, 96% identity to
JX171571.1 F. lateritium NRRL 13622) and an unnamed species within the F.
incarnatum-equiseti species complex designated FIESC 1 (F201338, 100% identity to
GQ505814.1 Fusarium sp. FIESC 1, O’Donnell et al. 2009).
Molecular phylogenetics.—To place the two putatively novel isolates within Fusarium, we
conducted a MP analysis of a 39-taxon RPB1-RPB2 dataset spanning the phylogenetic
breadth of the genus that included sequences of five isolates of F. zanthoxyli and F. continuum
(FIG. 2). MP analysis of the two-gene dataset outgroup-rooted on sequences of six members
of the FSSC (FIG. 2) based on more inclusive analyses (O’Donnell et al. 2013), in which
1145/3368 characters were parsimony informative, found two most-parsimonious trees of
4486 steps (CI = 0.42, RI = 0.73) that differed only in the branching order of F. illudens C.
Booth NRRL 22090 within the FSSC. The ML analysis produced a topologically similar tree
(data not shown). ML and MP bootstrapping supported 27 and 28 nodes, respectively, at
≥70% (FIG. 2) and provided strong support for a novel clade (i.e. FTOSC) comprising F.
torreyae and the two novel CDZB pathogens F. zanthoxyli and F. continuum whose
monophyly (ML-BS/MP-BS = 100%) and sister group relationship were strongly supported
(ML-BS/MP-BS = 92%). Although the three partitions we sequenced possessed different
levels of phylogenetically informative characters (PIC) (TABLE I; ITS rDNA = 2.9%, TEF1 =
17.1%, and RPB2 = 8.9%), analyses of the individual ITS rDNA, TEF1 and RPB2 partitions
and the combined three-gene dataset, using sequences of F. torreyae to root the phylogenies,
strongly supported the genealogical exclusivity F. z anthoxyl i and F. continuum (FIG. 3).
Considerable allelic diversity was detected within both species, in that 20/24 F. zan tho xyl i
and all five F. continuum isolates possessed unique multilocus haplotypes. In addition F.
zanthoxyli isolates F201112 from Shaanxi and F201125 from Shanxi possessed highly
divergent TEF1 and RPB2 alleles, respectively, (FIG. 3). Fusarium continuum isolates were
collected from Shandong (n = 3) and Hebei (n = 1) and isolates of F. zanthoxyli were from
Gansu (n = 10), Shaanxi (n = 14) and Shanxi (n = 1). However, F. continuum (F201030) and
F. zanthoxyli (F201307) were sympatric in Fuping County, Shaanxi (FIG. 1).
Pathogenicity of isolates to Zanthoxylum bungeanum.—Thirty-five of the 38 Fusarium
isolates tested induced cankers and were recovered from the canker margins when the
pathogenicity experiment was terminated after 22 d. Compared with isolates of F. zanthoxyli
and F. continuum (FIGS. 5, 6), the single isolate of F. acuminatum F201136, Fusarium sp.
F201237 (F. lateritium species complex) and Fusarium sp. FIESC 1 F201338 (F.
incarnatum-equiseti species complex) induced lesions on Z. bungeanum cultivar Doujiao that
were not significantly larger than the negative control (FIG. 8). Therefore we focused on
results of the pathogenicity experiment obtained for isolates of F. zanthoxyli (n = 25), F.
continuum (n = 5) and FSSC 6 (n = 5). Comparisons using Fisher’s LSD multiple comparison
test revealed that F. zanthoxyli and F. continuum produced significantly larger lesions (mean
= 1.24 ± 0.03 [SEM] and mean = 1.36 ± 0.05 [SEM], respectively) than the negative control
(mean = 0.67 ± 0.01 [SEM]) (p < 0.01). Moreover, the five isolates of FSSC 6 induced
significantly larger lesions (mean = 0.94 ± 0.03 [SEM]) than the negative control (p < 0.01).
While lesions induced by F. zanthoxyli and F. continuum did not differ in size (p > 0.05), both
were significantly larger than those produced by the five FSSC 6 isolates (p < 0.01),
indicating that F. zanthoxyli and F. continuum were more virulent to Z. bungeanum cultivar
Doujiao.
TAXONOMY
Fusarium zanthoxyli X. Zhou, T. Aoki, K. O’Donnell & Z. M. Cao, sp. nov. FIGS.
9–31
MycoBank MB809716
Typification: CHINA. SHAANXI PROVINCE: Tongchuan city, Yaozhou district, Sunyuan
town, isolated from stem tissue of diseased Zanthoxylum bungeanum, 4 Sep 2013, Xue Zhou
Fyzs133-2013 (holotype HMNWAFU XZ-Fyzs133-20130408, a dried culture of F201311).
Ex-type culture: F201311 = NRRL 66285.
Etymology: From Latin zanthoxyli, referring to the host, Zanthoxylum bungeanum.
Colonies on PDA with mycelial growth rates of 0.6–2.4 mm/d at 20 C in the dark.
Colony margins mostly undulate, sometimes entire. Aerial mycelia on PDA generally
sparsely to moderately formed, some developed abundantly, then loose to densely floccose,
sometimes felted, white (1A1), yellowish white (2–4A2), pinkish white to pastel red (7A2–4)
in the dark, pastel red to reddish orange (7A4–6), pale red (7–9A3) under fluorescent or
daylight, upon sporulation pale orange to deep orange (6A3–8). Pigmentation in the reverse
pale yellow (2–4A3), pale orange to light orange (6A3–4) or pale red to pastel red (7A3–4) in
the dark, light orange to orange (6A4–7), pastel red to reddish orange (7A5–7) under
fluorescent or daylight. Dark blue sclerotial bodies sometimes present under daylight. Odor
sweet, sometimes absent. Sporulation on SNA generally relatively scarce either directly on
aerial mycelium, on substrate mycelium or in sporodochia. Sporodochia sometimes formed
around the inoculum under fluorescent or daylight. Aerial conidiophores and conidia mostly
absent or rarely formed on SNA. Aerial and sporodochial conidiophores and conidia not well
differentiated. Aerial and sporodochial conidiophores, if present, generally densely to
sparsely branched but sometimes unbranched, forming apical monophialides or sometimes
intercalary phialides, up to 62.0 μm long and 2.5–6.5 μm wide. Phialides subcylindrical to
ampulliform, often with a conspicuous collarette at the tip, up to 40.5 μm long and 2.5–5.0
μm wide. Aerial and sporodochial conidia morphologically indistinguishable, typically
falcate, slightly curved or straight, dorsiventral, or sometimes fusiform, often widest in the
upper half or less frequently at the midregion, tapering toward both ends, with an apical cell
often rostrate, sometimes acute, and a basal foot cell indistinct to distinct with a conspicuous
protrusion, (1–)3–5(–7)-septate; in darkness or under daylight, three-septate: 19.5–55.0 ×
3.0–6.0 μm in total range, 30.7–42.3 × 3.7–4.5 μm av. (ex-type: 25.3–48.0 × 3.0–5.0 μm, 39.1
± 7.3 × 4.3 ± 0.5 μm av. and SD); five-septate: 41.0–76.0 × 3.7–6.5 μm, 54.3–68.5 × 4.7–5.8
μm av. (ex-type: 47.2–74.0 × 4.3–5.8 μm, 62.3 ± 5.9 × 5.0 ± 0.3 μm av. and SD).
Occasionally shorter, naviculate, ellipsoidal to clavate conidia with acuate to pointed, rarely
rounded apex and pointed to rounded base less frequently formed on SNA,
(0–)1–2(–3)-septate, 7.5–28.0 ×2.0–4.0 μm if present. Chlamydospores present or absent,
oblong to subglobose, smooth to rough, thick-walled, intercalary or terminal, solitary, in pairs
or catenate, 3.0–16.5 × 3.0–8.5 μm when present.
Other isolates examined: All from stem tissue of diseased Z. bungeanum; CHINA. SHAANXI
PROVINCE: Baoji city, Chencang district, Jiaoliao village, Aug 2011, Ning Xie Fbjj-2011, F201105; CHINA.
SHAANXI PROVINCE: Hancheng city, Xuefeng town, Wang village, Aug 2011, Ning Xie Fhcw-2011, F201108;
CHINA. GANSU PROVINCE: Qinan County, Yebao Township, Yangjiasi, Sep 2011, Ning Xie Fqayy-2011,
F201115; CHINA. GANSU PROVINCE: Gangu County, Ershilipu, Sep 2011, Ning Xie Fgge-2011, F20 1116;
CHINA. GANSU PROVINCE: Gangu County, Daxiangshan town, Zhangjiajing village, Sep 2011, Ning Xie
Fggz-2011, F201117; CHINA. GANSU PROVINCE: Tianshui city, Qinzhou district, Guanzi town, Sep 2011,
Ning Xie Ftqg-2011, F201119; CHINA. GANSU PROVINCE: Wen County, Baoziba Township, Guanyinba, Sep
2011, Ning Xie Fwbg-2011, F201124; CHINA. GANSU PROVINCE: Qinan County, Yebao Township,
Chenjiahe, Aug 2012, Jie-Fei Wang Fqayc1-2012, F201218; CHINA. GANSU PROVINCE: Qinan, Yebao
Township, Anjiachuan, Sep 2012, Jie-Fei Wang Fqaya2-2012, F201220; CHINA. SHAANXI PROVINCE:
Baoji city, Chencang district, Tianwang town, Miaoju village, Apr 2013, Jie-Fei Wang Fbjm1-2013, F201301;
CHINA. SHAANXI PROVINCE: Fuping County, Lei village, Sep 2013, Xue Zhou Ffpl-13-2013, F201307;
CHINA. SHAANXI PROVINCE: Hancheng city, Zhiyang town, Dongying village, Jun 2013, Ning Xie
Fhcd-2013, F201309 = NRRL 66217; CHINA. SHAANXI PROVINCE: Tongchuan city, Yaozhou district,
Sunyuan town, Sep 2013, Xue Zhou Fyzs132-2013, F201313; CHINA. GANSU PROVINCE: Wen County,
Baoziba Township, Sidouping village, Oct 2013, Xue Zhou Fwbs1-2013, F201322.
Distribution: Gansu, Shanxi and Shaanxi provinces, China.
Notes: The key morphological character that distinguishes F. zanthoxyli from other
species within the FTOSC is the production of falcate conidia that typically possess a rostrate
apex, together with aerial and sporodochial conidiophores that are not well differentiated.
Fusarium zanthoxyli is similar to F. torreyae in that both species form long, slender
sporodochial conidia. However, conidia in F. zanthoxyli typically are widest in the upper half
whereas those of F. torreyae are usually widest at the mid-region. In addition F. zanthoxyli
produces fewer 7–9-septate conida than F. t or reyae. The rostrate apical cell and protruding
basal cell of F. zanthoxyli represent additional morphological features that differentiate it
from F. tor reyae.
Fusarium continuum X. Zhou, T. Aoki, K. O’Donnell & Z. M. Cao, sp. nov. FIGS. 32–53
MycoBank MB809718
Typification: CHINA. SHAANXI PROVINCE: Fuping County, Lei village, isolated from
stem tissue of diseased Z. bungeanum, Aug 2010, Ning Xie Ffpl-10-2010 (holotype
HMNWAFU NX-Ffpl-10-20100851, a dried culture of F201030). Ex-type culture: F201030 =
NRRL 66286.
Etymology: from Latin continuus, based on the morphological continuum of aerial and
sporodochial conidiophores and conidia.
Colonies on PDA with mycelial growth rates of 1.0–1.5 mm/d at 20 C in the dark, but
maximal growth rates were 1.4–2.6 mm/d at 30 C in the dark. Colony margins undulate.
Aerial mycelia on PDA generally moderately formed, loose to densely floccose, white (1A1),
light yellow to greenish yellow (1A5–7), pastel yellow (2–3A4), light orange (5A4), grayish
yellow (3–4B4, 5B4–5), grayish violet (19D3–4) or dull blue (22D4) in the dark, pale yellow
to light yellow (3–4A4), pale red to pastel red (7A3–4), grayish orange to grayish red
(6–7B3), deep violet to dark violet (16E–F8) or bluish gray to dull blue (23E2–4) under
fluorescent or daylight, upon sporulation light orange to orange (6A4–7), or grayish orange
(6B3–4). Light yellow or light orange exudate sometimes present. Pigmentation in the reverse
grayish yellow (2–3B6, 4B5–6), light orange (5A4–5), grayish orange (4B7–8), or light
brown to dark brown (6D–F5) in the dark, light orange to brownish orange (5A–C5), light
brown (6D5), or gray to grayish brown (7D1–3) under fluorescent or daylight. Dark blue
sclerotial bodies sometimes present under daylight. Odor sweet. Sporulation on SNA
generally relatively rapid and abundant directly on aerial mycelium, on substrate mycelium or
in sporodochia. Aerial conidia formed earlier than sporodochial conidia on SNA. Aerial and
sporodochial conidiophores and conidia not well differentiated and form a morphological
continuum. Aerial and sporodochial conidiophores formed sparsely to abundantly,
unbranched, sparsely or densely branched, forming intercalary or apical monophialides, up to
60.5 μm long and 2.5–6.0 μm wide. Phialides subcylindrical or ampuliform, often with a
conspicuous collarette at the tip, up to 38.5 μm long and 1.5–4.5 μm wide. Aerial and
sporodochial conidia morphologically indistinguishable, typically falcate and gradually
curved, dorsiventral, or sometimes fusiform, mostly widest in the upper half, sometimes at
the midregion, tapering and gradually curving toward both ends, with an apical cell mostly
acuminate, sometimes pointed, and a basal foot cell indistinct to distinct, sometimes with a
discernible protrusion; (1–)3–5(–6)-septate; in darkness three-septate: 16.0–48.0 × 3.0–5.5
μm, 34.5–38.9 × 4.3–4.7 μm av. (ex-type: 22.3–44.0 × 4.0–5.6 μm, 34.5 ± 5.1 × 4.7 ± 0.4 μm
av. and SD); five-septate: 33.0–67.0 × 4.0–5.8 μm, 45.3–57.4 × 4.9–5.0 μm av. (ex-type:
33.0–53.1 × 4.1–5.5 μm, 45.3 ± 3.3 × 5.0 ± 0.2 μm av. and SD); On SNA sometimes shorter,
ellipsoidal, naviculate to clavate conidia also formed, straight or curved, with a rounded,
rarely pointed apex and a truncate or rounded base, 0–2(–3)-septate, 4.0–26.5 × 2.0–5.0 μm
when present. Chlamydospores formed on hyphae or in conidia, subglobose to oblong,
smooth to rough, thick-walled, intercalary or terminal, solitary, in pairs or catenate, 3.0–12.5
× 2.5–8.0 μm.
Other isolates examined: All isolated from stem tissue of diseased Z. bungeanum; CHINA.
SHANDONG PROVINCE: Zaozhuang city, Shanting district, Sep 2011, Ning Xie Fst-2011, F201126 = NRRL
66218; CHINA. SHANDONG PROVINCE: Zibo city, Boshan district, Yijialou, Sep 2011, Ning Xie Fbsy-2011,
F201127; CHINA. SHANDONG PROVINCE: Yiyuan County, Shiqiao village, Sep 2011, Ning Xie Fyys-2011,
F201128; CHINA. HEBEI PROVINCE: She County, Wangjinzhuang village, Sep 2011, Ning Xie Fhsw-2011,
F201129.
Distribution: Shandong, Hebei, and Shaanxi provinces, China.
Notes: Fusarium continuum can be differentiated from other members of the FTOSC
by the production of shorter aerial and sporodochial conidia and rare production of
7–9-septate conidia, together with the undifferentiated aerial and sporodochial conidiophores.
Of the three species within the FTOSC, conidia of F. torreyae are the longest, followed by F.
zanthoxyli and F. continuum, when conidia with the same number of septa were compared.
Fusarium continuum and F. zanthoxyli produce morphologically similar multiseptate, falcate
conidia that are usually widest in the upper half and only rarely at the midregion. By contrast
those produced by F. torreyae are long, slender, gradually curved, and most frequently widest
at the mid-region. Conidia produced by F. continuum can be distinguished from those of F.
zanthoxyli in that the latter possess a rostrate apical cell and a conspicuous ventrally
protruding basal cell. In addition F. continuum also forms ellipsoidal, clavate to naviculate,
straight or slightly curved conidia with a mostly rounded apex and rounded to truncated base
on SNA. These two species also can be distinguished by differences in their radial mycelial
growth rates. Average radial mycelial growth rates on PDA in the dark at eight temperatures
5–40 C were calculated for 14 isolates of F. zanthoxyli and five isolates of F. continuum and
are summarized (FIG. 54, 55). Optimal temperature for mycelial growth was 25 C for all
isolates of F. zanthoxyli and one isolate (F201126) of F. continuum and 30 C for 4/5 isolates
of F. continuum. Mycelial growth at 25 C was 0.8–2.6 mm/d for F. zanthoxyli and 1.2–1.8
mm/d for F. continuum; growth rate at 30 C was 0.2–1.3 mm/d for F. zanthoxyli and 1.4–2.6
mm/d for F. continuum.
DISCUSSION
The primary findings of the present study include the discovery of a novel clade of
canker-inducing fusaria, here designated as FTOSC, comprising F. torreyae, a pathogen of
the critically endangered Florida torreya (Torreya taxifolia) in northern Florida and
southwestern Georgia (Smith et al. 2011, Aoki et al. 2013) and two novel pathogens of
prickly ash trees (Zanthoxylum bungeanum) in northern China that are described formally
herein as F. zanthoxyli and F. continuum. Molecular clock estimates place the divergence of
the FTOSC in the mid-Eocene ~ 40 Mya (O’Donnell et al. 2013), but it remains an open
question whether this clade first evolved in the Old or New World. Furthermore, it remains to
be determined whether F. t orreyae is native to North America and restricted to T. taxifolia.
Surveys for F. torreyae on Torreya endemic to China are warranted because it is the modern
area of diversity of this genus (Li et al. 2001) and because the putative Asian origin of the
CDZB pathogens could indicate that the most recent common ancestor of the FTOSC
evolved in Asia. Additional surveys also are needed to characterize the host range and
geographic distribution of the two novel CDZB pathogens. The available data suggests their
ranges are distinct, with F. zanthoxyli distributed across three provinces from Gansu and
Shaanxi in the northwest to Shanxi in the north of China, whereas F. continuum is mainly
distributed in Shandong in the east of China. The two prickly ash pathogens, however, are
sympatric in Fuping County of Shaanxi province (FIG. 1, SUPPLEMENTARY TABLE I).
Although a sexual cycle has not been found in the three FTOSC species in the
laboratory, the high multilocus haplotype diversity we detected within F. zanthoxyli and F.
continuum suggests that these pathogens may possess a heterothallic sexual reproductive mode.
A similar finding in the soybean sudden death syndrome pathogen, F. tucumaniae T. Aoki et
al., led to the discovery of a sexual cycle by conducting laboratory crosses under light and
temperature conditions optimal for perithecial production (Covert et al. 2007). Knowledge of a
sexual cycle in the CDZB pathogens has important implications for disease management and
control because sexually reproducing pathogens are much more likely to overcome host
resistance. In addition to the high SNP diversity that we detected within F. zanthoxyli it is
worth mentioning that highly divergent TEF1, RPB2 and ITS rDNA alleles were detected in
isolates of F. zanthoxyli from Shaanxi, Shanxi and Gansu, respectively. Of interest, the ITS
rDNA of F201125 from Shanxi differed at only two nucleotide positions from that of F.
torreyae, suggesting possible hybridization between F. zanthoxyli and a F. t or reyae-like
species.
In the present study we characterized a novel plant disease designated Canker disease
of Zanthoxylum bungeanum (CDZB) and completed Koch’s postulates. Lesion sizes caused
by the three most frequently isolated Fusarium species recovered from prickly ash trees
showing symptoms of CDZB were used as a proxy for their virulence on this host. Results of
the pathogenicity experiment revealed that isolates of the two novel CDZB-associated
Fusarium species exhibited higher virulence than that of FSSC 6 and single isolates of three
other Fusarium species. This finding establishes that F. zanthoxyli and F. continuum are the
major pathogenetic Fusarium species on Z. bungeanum. Isolates of F. zanthoxyli and F.
continuum also were confirmed to be pathogenic to Z. bungeanum cultivars Dahongpao
(including Fengjiao and Hancheng-Dahongpao), Youjiao and Mijiao (Xie 2012). Longer-term
inoculation studies are needed to help elucidate whether the causal agents can ultimately kill
trees under more field based conditions. It is also of interest to test these isolates against other
members of the Rutaceae, especially those that are economically important (i.e. citrus).
Several species of Fusarium are capable of causing cankers on woody plants, and
mixed infections frequently occur. For example, a particular FSSC species occasionally was
found co-occurring with F. t orre yae in cankers on Florida torreya (Torreya taxifolia) (Smith
et al. 2011). Although both species could induce cankers, F. torreyae is considered to be the
primary pathogen due to increased virulence and consistent isolation from a large number of
cankers. By contrast the available data indicates the FSSC taxon should be regarded as an
opportunistic (J. Smith pers comm). In addition F. solani f. sp. xanthoxyli, a well-known
fungus in Japan within the FSSC has been reported to cause trunk-blight of a closely related
tree species, Zanthoxylum piperatum (L.) DC. under the name of Nectria elegans Yam a mo t o
& Maeda (Yamamoto et al. 1957, Matuo and Snyder 1961, Sakurai and Matuo 1961).
However, this fungus was shown to represent an undefined phylogenetic species of Fusarium
designated FSSC 22 (O’Donnell et al. 2008), which is morphologically different from the
CDZB-associated Fusarium species from China. Although multiple species frequently are
isolated from a host in disease etiology studies, pathogenicity and virulence testing are
essential to complete Koch’s postulates, as is identification of the fungal species most
frequently isolated from symptomatic tissues.
In addition to Fusarium species other microbes have been reported to induce cankers
on Z. bungeanum, for example, Phytophthora spp. (Xie et al. 2013). Because these cankers
are typical of those caused by other Fusarium spp. (i.e. FSSC 6 and F. to rreyae) it is possible
that two different stem diseases have been confused on this host. Also a jewel beetle (Agrilus
sp.) frequently colonizes wounds resulting from CDZB (Li et al. 1988), causing further
damage to this host. Whether the wounds caused by this wood-boring beetle serve as
infection courts for these pathogens warrants further study.
The 38 fusaria isolated from Zanthoxylum bungeanum cankers in the present study
were identified and placed in Fusarium by a two-step process that first involved BLASTN
queries of NCBI GenBank and FUSARIUM-ID (Geiser et al. 2004), using a partial TEF1
sequence as the query, followed by phylogenetic analyses of a RPB1-RPB2 dataset that
spanned the phylogenetic breadth of Fusarium (O’Donnell et al. 2013). Results of both
analyses indicated that the two primary CDZB pathogens were most closely related to F.
torreyae (Smith et al. 2011, Aoki et al. 2013). Morphological species recognition is
challenging for nonspecialists in Fusarium (Gerlach and Nirenberg 1982, Nelson et al. 1983,
Leslie and Summerell 2006), as evidenced by the phenotypic identification of F. t orreyae as F.
lateritium (El Gholl 1985) and the CDZB pathogens as F. sambucinum (Cao et al. 1992, 2010)
and F. lateritium (Xie et al. 2012), although Fusarium isolates from wooden substrates often
have been identified as F. lateritium without detailed morphological analyses. Fusarium
torreyae can be distinguished from F. zanthoxyli and F. c ont inu um by its host, Florida torreya,
and phenotypically by the frequent production of seven-septate sporodochial conidia and the
absence of aerial conidia. In contrast F. continuum produces abundant conidia that are shorter
than F. t orreyae, rarely produces seven-septate sporodochial conidia and occasionally
produces dark blue sclerotia. Fusarium zanthoxyli occasionally produces aerial conidia and
falcate seven-septate sporodochial conidia with a distinct or indistinct ventrally protruding
basal foot cell and beak-like apical cell, but it only rarely produces shorter 0–3-septate
conidia. Because the tree space surrounding the F. torreyae, F. lat eri tiu m and F. buharicum
species complexes contains approximately 20 undescribed species lineages (O’Donnell
unpubl), which were resolved by phylogenetic species recognition based on genealogical
concordance (Taylor et al. 2000), we advocate employing multilocus DNA sequence data for
species identification and recognition within Fusarium coupled with detailed phenotypic
analyses including morphology. Through this approach we discovered a novel clade of tree
canker-inducing pathogens that includes the prickly ash pathogens F. zanthoxyli and F.
continuum in China and the Florida torreya pathogen, F. torreyae in southeastern United
States.
ACKNOWLEDGMENTS
This research was supported in part by the National Science-Technology Support Project (No. 2012BAD19B08).
The mention of company or trade products does not imply that they are endorsed or recommended by the US
Department of Agriculture over other companies or similar products not mentioned. The USDA is an equal
opportunity provider and employer. We thank Stacy Sink for assistance with the DNA sequence data and Ning
Xie and Jiefei Wang for help collecting specimens.
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LEGENDS
FIG. 1. Collection sites in five Chinese provinces where Fusarium zanthoxyli (●) 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.
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 = parsimony informative character, MPTs = most-parsimonious trees, CI = consistency index, RI =
retention index.
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 =
parsimony informative character, MPT(s) = most-parsimonious tree(s), CI = consistency index, RI = retention
index.
FIG. 4. Virulence of Fusarium zanthoxyli, F. continuum and FSSC 6 to Zanthoxylum bungeanum cultivar
Doujiao. Bars represent means and ± standard errors (SEM) of isolate-lesion size of each pathogen and the
uninoculated negative control. *p < 0.05 and **p < 0.01 compare size of lesions induced by the three pathogens.
FIGS. 5–8. Canker symptoms exhibited by Zanthoxylum bungeanum cultivar Doujiao inoculated with (5)
Fusarium zanthoxyli F201119, (6) F. continuum FZB201127 and (7) Fusarium sp. FSSC 6 F201131. 8. Negative
control lacking a discernible canker.
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 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 = 50 μm, 12–17, 19–31 = 20 μm.
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 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 = 50 μm; 36–42, 45–53 = 20 μm.
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).
FOOTNOTES
Submitted 9 Sep 2015; accepted for publication 16 Feb 2016.
1 Corresponding author: E-mail: zmcao@nwsuaf.edu.cn
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
