Available via license: CC BY-NC 3.0
Content may be subject to copyright.
93
Mycobiology
Phylogenetic and Morphological Identification of
the Novel Pathogen of Rheum palmatum Leaf
Spot in Gansu, China
Yan Wang *, Amy O. Charkowski , Cuiyun Zeng , Tiantian Zhu , Huizhen Wang and Honggang Chen
Gansu University of Chinese Medicine, Lanzhou 730000, China
Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI 53706, USA
Abstract A new leaf spot disease was observed on leaves of Rheum palmatum (Chinese rhubarb) in Northwest China (Gansu
Province) starting in 2005. A Septoria-like fungus was isolated and completion of Koch’s postulates confirmed that the fungus was
the casual agent of the leaf spot disease. Morphology and molecular methods were combined to identify the pathogen. The
fungus produced conidiomata pycnidia and the conidia were 2~5 septate, 61.2~134.1 µm in length and 3.53~5.3 µm in width,
which is much larger than the known Spetoria species that infects Polygonaceae species. Phylogenic analysis of the internal
transcribed spacer region confirmed that this Septoria-like fungus is within the Septoria genus but distinct from known Septoria
species. Together, these morphological and phylogenetic data support that the R. palmatum infecting Septoria strain is a newly-
described plant pathogenic species.
Keywords Internal transcribed spacer rDNA, Novel pathogen, Rheum palmatum, Taxonomy
Rheum palmatum L., commonly called Chinese rhubarb, is
a medicinal plant. The air-dried root has been used as the
traditional Chinese herbal medicine (TCM) for over 2000
years. Although the pharmacological effects of R. palmatum
are that it is cathartic on the digestive movement of the
colon; it was found can also protects the damaged liver,
and has antitumor, antibacterial, anti-inflammation, and
anti-aging properties, the side effects are nausea, vomiting,
and dizziness, etc. [1-3]. R. palmatum widely grows either
in low altitude area (800 m to 2,000 m) with damp climate
or high altitude area (1,200 m to 3,500 m) with bleak climate.
In China, it distributed in the southwest, northwest, northeast
and north China. Gansu province, located in northwest
China, is the main production area of R. palmatum.
With the dozens of bioactive substances was found, the
use of the Chinese rhubarb become wider and wider. The
wild resources can not satisfy the pharmaceutical demands
any more leads to the expanding of the cultivation area.
Disease problem become the key factor on the yield and
quality of the plant. The production of R. palmatum was
affected by several pathogens, including ring spot (Ascochyta
rhei Ellis et Everhart), leaf spot (Septoria polygonorum Desm.)
[4], smut (Thecaphora schwarzmaniana Byzova) [5], rust
(Puccinia rhei-palmati B. Li) [6], gray spot (Phyllosticta rhei
Ellis et Everhart) [7], powdery mildew (Erysiphe polygoni
DC.) [8], grey mold (Botrytis sp.), root rot (Fusarium
oxysporium Schl.; Fusarium sp.; nematode) [9], and a virus
disease (cucumber mosaic cucumovirus) [10]. A new leaf
spot, caused by a Septoria-like fungus which is different from
the reported pathogen, S. polygonorum, was first found in
Weiyuan, Longxi, and Ming County in 2005 with 30~40%
disease incidence and 1~2 disease severity, because of
the increasing disease incidence and severity of necrotic
symptoms in the main Chinese rhubarb production areas
of Gansu, China. The pathogen of the disease need to be
confirmed and the control methods as well.
The genus Septoria Sacc. (with type S. cytisi Desm.) [11]
was introduced by Fries in 1819 [4]. The modern prescription
of Septoria is characterized by pycnidial conidiomata, filiform,
hyaline, smooth-walled multi-septate conidia [12-14]. It is
among the most common and widespread leaf-spotting
fungi worldwide [15, 16]. The identification of this genus
is largely based on the host and the biogeography location,
Research Article
Mycobiology 2016 June, 44(2): 93-98
http://dx.doi.org/10.5941/MYCO.2016.44.2.93
pISSN 1229-8093 • eISSN 2092-9323
© The Korean Society of Mycology
*Corresponding author
E-mail: gswangyan101@163.com
Received April 26, 2016
Revised May 30, 2016
Accepted June 7, 2016
This is an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted
non-commercial use, distribution, and reproduction in any medium,
provided the original work is properly cited.
94 Wang et al.
to date, the genus includes more than 3,000 published names
of species and infraspecific taxa [17], most of them are
synonyms. Teleomorphs are known for only a relatively small
number of Septoria species, and in all cases they are species
of Mycosphaerella Johanson (Dothideales, Ascomycota), a
genus with numerous plant pathogenic taxa and over 20
reported anamorph genera [18-20].
The objective of this research is to identify the pathogen,
the internal transcribed spacer (rDNA-ITS) region has
been amplified for phylogenetic identification purposes.
Furthermore, morphological criteria have been taken into
consideration to identify the pathogen.
MATERIALS AND METHODS
Diseased specimen collection and fungal isolate.
Specimens of Septoria-like leaf necrotic were collected in 5
different Rheum fields in Weiyuan, Gansu, China between
2008 and 2011. These tissues were dried between sheets of
newspaper and dispatched by courier for diagnosis and
isolation within 1 wk of collection. Leaf tissue with necrotic
lesions were surface sterilized in 1% sodium hypochlorite
for 3 min and then rinsed three times in sterile distilled
water. Small pieces (< 2 mm
2
) of tissue were excised from
the lesion margins, placed onto potato dextrose agar
(PDA) in darkness. To obtain single-spore colonies, single
pycnidia was picked from the surface-sterilized tissue and
then placed on a petri plate containing water agar (water
1,000 mL, agar 17 g), distributed across the plate surface
evenly using a sterile glass rod, and incubated at 20
o
C for
12 hr in the dark. Germinating conidia were subsequently
removed individually and placed onto PDA in darkness at
20
o
C. After 20 days, pick the mycelium from the margin of
the colony and transferred to PDA.
Morphology characterization. A single pycnidia on
leaf spot was gently picks up and placed in a small drop
of water on a slide. Press gently on the cover slip to allow
the dispersion of the conidia. Where possible, conidial
measurements were made using conidia from the leaf spots
with expelled spores. After the drop of water had evaporated,
1% cotton blue in lactic acid was added and a cover slip
applied. Conidia were measured randomly. For curved
conidia measurements were made on natural length. The
widths of both the conidia were measured at their widest
part [21]. Spore dimension and the presence of septa were
noted in each of 30 randomly selected conidia from a
single sporocarp, observations were made through bright
field microscopy at 400 magnification.
Cultural characteristics. Mycelium plugs, 7-mm in
diam., were removed from margins of colonies which had
been growing on PDA in the dark at 15
o
C for 60 days and
inoculated on PDA, malt extract agar (MEA), and oatmeal
agar (OA) of 90 mm diam. petri dishes. Three replicates for
each media. Dishes were sealed with laboratory film (Parafilm
M, West Berlin, NJ, USA), the isolate were cultured in the
dark at 15
o
C, colony diameters of were measured weekly for
8 wk. The color and the characteristics of the colony were
observed after 8 wk of cultivation. Mycelium and fruiting
bodies were transferred in a drop of water on a slide, pressed
gently under a cover glass and studied with light microscopy.
Growth rates were then expressed in mm/day.
Pathogenicity test. The pathogenicity of isolate was tested
on 10 R. palmatum plants. The plants previously grew in
the greenhouse in pots for 8 wk. Mycelium and spores were
liberated from 30-day-old cultures on PDA by dispensing
10 mL of a 0.1% Tween-80 solution onto each Petri plate
and brushing the surface of the culture with a sterile spatula.
The concentration of conidial inoculum was adjusted to
1×10
4
conidia/mL using a hemocytometer. Plants were
inoculated with 10 mL of the inoculum suspension using a
hand sprayer. Controls were sprayed with the same volume
of 1 µL Tween-80 and distilled water. Plants were incubated
by placing the pots in trays that were partially filled with
water inside the plastic bags to maintain high humidity.
The moist chambers were put at 20
o
C for 72 hr. The bags
were then removed and the plants were placed under diffused
room light in the laboratory. Symptoms were observed and
recorded at 7 days intervals and isolations were made from
lesions after 4 wk.
DNA extraction and sequencing of the internal
transcribed spacer region (ITS) of the ribosomal DNA.
Mycelium was scraped from cultures growing on PDA, put
into 100 mL potato dextrose liquid medium (PD; potato
200 g, dextrose 10 g, water 1,000 mL), agitated using an orbital
shaker set at 45 rpm and incubated at room temperature
under constant illumination by fluorescent lights. Mycelium
(100 mg) was removed after 7 days and transferred to a
mortar and pestle for grinding with liquid nitrogen. DNA
extraction was then carried out using the FastDNAkit
(SK1375; Sangon Biotech Co. Ltd., Shanghai, China) following
the manufacturer’s instructions. The presence of DNA was
verified by separation on a 0.8% agarose gel in 1 × TAE
(40 mM Tris-acetate, 1 mM EDTA, pH 8.0) stained with
ethidium bromide (0.5 mg/L) and visualized under UV light.
The ITS region was amplified using the universal primers,
ITS1 and ITS4 [22]. PCR was performed in 50 µL reaction
volumes containing 10~100 ng of genomic DNA, 2.5 µL of
each primer (10 µM), 0.25 µL dNTP (10 mM), 2.0 unit
Supertaq DNA polymerase and 5 µL 10 × PCR buffer
(Sangon Biotech Co. Ltd.). The amplification was performed
in a thermocycler (Biometra Tg PCR; Biometra, Gottingen,
Germany). The PCR thermal cycle program was as follows:
95
o
C for 3 min, followed by 30 cycles of denaturation at
94
o
C for 3 min, annealing at 52
o
C for 45 sec, and elongation
at 72
o
C for 1 min, with a final extension step of 72
o
C for
8 min. The PCR products, spanning approximately 500 bp
of ITS, were checked on 1% agarose electrophoresis gels
stained with ethidium bromide. The PCR product was
Septoria palmati Yan Wang, sp. nov., Novel Plathogen of Rheum palmatum Leaf Spot 95
purified and sequenced using the abovementioned primers
by Sangon. The novel sequence was deposited in GenBank
of National Center for Biotechnology Information (NCBI).
Sequence alignment and phylogenetic analyses.
Approximate phylogenetic placements were determined
with the Blastn search option of NCBI. The ITS sequences
from NCBI were aligned using MAFFT v. 7 (http://mafft.
cbrc.jp/alignment/server/index. html) [23] and manually
improved in BioEditv. 7.0.5.2 [24]. Dividiella tassiana (accession
No. JF911765) was set as the outgroup. Phylogenetic tree
was constructed with MEGA 5 using the neighbor-joining
method and the Tajima-Nei distance model.
RESULTS
Oberservation in the fields. Occurring on leaves. The
symptom initially appears as chlorotic, yellowish spots
scatter on the leaf, later the apots expanded to irrgular,
nearly round lesion, 8~12 mm, gray white in the center
and brown to red brown at the edge. The edge thin or
wide, a little upheaval, Finally, plenty of black granules
(pycnidia) separately scattered on the lesion. When it rains
or the humidity of environment is high, the spores expel
out of the pycnidia, the lesion were covered with a layer
pink to white mold and black empty pycnidia were buried
in it (Fig. 1A, B). The lesion is more solid, dry and thick
than healthy leaf tissue. Specimens are deposited in the
Herbarium of Gansu University of Traditional Chinese
Medicine, Lanzhou, China (HTCM).
Tax onomy.
Septoria palmati Yan Wan g , sp. nov. (Plate 2)
MycoBank: MB 817262.
Differs from all other Septoria species by its Polygonaceae
host and large spores.
Typ e : China, Gansu Province, Weiyuan county, on leaves
of Rheum palmatum L. (Polygonaceae), 8 Jul 2008, Yan
Wang (holotype, Herbarium specimen in HTCM: 11001215;
GenBank: KC874676).
Etymology: Palmati (meaning “of [Rheum] palmatum”].
Conidiomata pycnidia, semi-immersed, separate, spherical to
subglobose, medium brown, (174.9~) 188.1~277.7 (~349.4)
(mean, 228.6 µm) in diam. (Fig. 2D). Walls pseudoparen-
chymatous, 4~6 cells thick, 5~10 µm wide, composed of
texture angular cells. Ostile single, circular, central, 14~
21 µm in diam. Conidiophores absent. Conidiogeous cells
hyaline, single cell. Conidia hyaline, thick rope, nematode,
straight or slightly curved, 2~5 septate, 61.2~134.1 × 3.53~
5.3 µm (mean, 89.8 × 4.3 µm) (Fig. 2E and 2F).
Colony grows very slowly (0.33 mm/day), the upper surface
of the isolate growing on PDA is powdery and velvet in
pink, slightly uplift, conidia form directly from hypha
and assembled together to appear yeast-like growth type,
mycelium assembl to yellow-brownish to black dormancy
organization structure when cultivated for more than 3 mo
(Fig. 2A). Colony on OA is white, velvet and slightly uplift
(Fig. 2B). The colony cannot grow on MEA, only some
mycelium assemble around the previous disk (Fig. 2C).
Pycnidia not formed on three tested media.
Pathogenicity. Septoria isolate TCM-6 was inoculated
on Rheum plants and symptom can be observed in all
repetitions. Initial symptom was small yellow dots that
appeared approximately 20 days after inoculation. These
dots enlarged to typical necrotic lesions, round or irregular
spots, pale gray with red purple margins on the leaves, no
symptoms developed on non-inoculated control plants in
any of the experiments. The pathogen were isolated again
from the lesions, the Koch’s regulation were confirmed.
Fig. 1. A, B, Symptoms of leaf spot on Rheum palmatum caused by Septoria.
96 Wang et al.
Phylogenetic analysis. Amplification was successfully
carried out with the ITS1/ITS4 primers, and fragmentof
approximately 489 bp was obtained. The ITS alignment
contains 13 taxa including the outgroup and the high
similarity strains through Blastn search using the ITS
sequence in Genbank. The tree was rooted to D. tassiana
and two sister clades were revealed from the analysis.
One clade includes S. cucubali (GU214698), S. matthiolae
voucher (KM975409), S. create (KF251233), S. lavandulae,
S. polygonorum (KF373078), S. polygonorum (KF251505),
S. rumicum (KF251529). The other clade includes three
strains of Mycosphaerella brassicicola (AF297227, EU167607,
and AF297223) and TCM-6 as well (Fig. 3). The strain has
high similarity with M. brassicicola, we assume that the
sexual stage of TCM-6 is Mycosphaerella sp.
Based on morphological features, pathogenicity test and
amplification of ribosomal nuclear sequences, we supposed
that the Septoria isolate: TCM-6 is a newly-described plant
pathogenic species.
DISCUSSION
The cultural characters of S. palmati is hemi-hypha and
yeast-like. In Septoria, the cultural characters are complex,
Fig. 3. Bootstrap consensus tree constructed using the neighbor-joining method from 489 bp of ribosomal internal transcribed
spacer DNA obtained from the TCM-6 and Septoria population. The tree was rooted to Dividiella tassiana. The isolate TCM-6
are indicated in bold font.
Fig. 2. Septoria palmatum. A~C, Colony on potato dextrose agar, oatmeal agar, and malt extract agar; D, Pycnidia; E, F,
Conidia (scale bars: D = 100 µm, E = 20 µm, F = 25 µm).
Septoria palmati Yan Wang, sp. nov., Novel Plathogen of Rheum palmatum Leaf Spot 97
asexual and sexual stage are possible be the yeast-like or
mycelium type [25]. Kema and Annone’s study [26] on 26
strains of S. tritici from different part of the world and
found nutrition and environment has minor effects on the
cultural characters, the strain which is yeast-like style will
not form pycnidia on any kind of medias, and in liquid
cultural condition, only form spores and short hypha
suspension unlike the mycelium balls. To date, only small
groups of Septoria have been revised with cultural studies
[27], the function of this characters is not unclear in the
identification of Septoria species. So, the relationship between
cultural characters and species need to be further studied.
The telemorph stage of S. palmati was not observed in
vitro or in vivo. Mycosphaerella Johanson is one of the largest
genera of ascomycetes [19]. Von Arx [18] accepted 23 genera
as known or suspected anamorphs of Mycospherella, including
Septoria Sacc. Through Blastn search and sequence alignment
S. palmati was aligned with several strains of Mycosphaerella
brassicicola. We suppose that the telemorph of the new
species is also Mycospherella sp.
Beach [28] invested a wide range of species and
demonstrated both host and biological specificity in all
taxa, he proposed that species of Septoria do not have a
broad host range, and infectivity rarely beyond two or
three related genera. Verkley and Starink-Willemse [15]
indicate that the Septoria species from Asteraceae are all
closely related, and share an evolutionary history within
the Mycosphaerellaceae through studies on 14 Septoria
species pathogenic to Asteraceae by analysing 5.8S ribosomal
RNA gene and flanking ITS (ITS1 and ITS2) sequences
sequence analyses [15]. So, it is reasonable to identify the
taxa according to the host in this genus. Seventeen species
have been reported can infect Polygonaceae plants [29]. S.
palmatum exhibited all of the morphological traits attributed
to Septoria and conidia 2~5 septate, size 61.2~134.1 µm×
3.53~5.3 µm which is much larger than the other Septoria
species pathogenic to Polygonaceae, even double size of
conidia of S. polygonorum (100~150 µm diam., 3~4-septate,
hyaline, smooth, 26~46 [38] × 2.0~2.4 µm), which was
reported as a pathogen of rhubarb leaf spot (S. polygonorum)
[4], so here we described the isolate S. palmati as a new taxa.
The distribution of S. palmati thus, may be restricted to
the areas where it has been found. However, further surveys
are necessary to determine exactly where this new leaf spot
pathogen occurs. The occurrence area of the pathogen in
Gansu, China is at an narrow area apparently. It might
also be present in other locations where the rhubarb
were cultivated. Further research work will focus on the
pathogenicity rang, the disease circle and control methods,
including manipulation of agronomic factors such as plant
density and irrigation frequency and timing, to decrease
the disease intensity.
ACKNOWLEDGEMENTS
The authors are grateful to reviewers for critical comments,
corrections and pre-submission reviews. This study has been
supported by the National Natural Science Foundation of
China, project 31460013; Gansu Nature Science Fund, project
1312RJZA092 and Gansu College Scientific Research, project
2013A-089.
REFERENCES
1. Chinese Pharmacopoeia Commission. Pharmacopoeia of the
People’s Republic of China, Vol. 1. Beijing: Medical Science
and Technology Press; 2010. p. 17.
2. Kang SC, Lee CM, Choi H, Lee JH, Oh JS, Kwak JH, Zee OP.
Evaluation of oriental medicinal herbs for estrogenic and
antiproliferative activities. Phytother Res 2006;20:1017-9.
3. Lee EH, Kim JM, Kim C. Pharmacokinetic analysis of rhein
in Rheum undulatum L. J Ethnopharmacol 2003;84:5-9.
4. Bai JK. Flora Fungorum Sinicorum. Vol. 17. Sphaeropsidales,
Ascochyta, Septoria. Beijing: Science Press; 2003.
5. Guo L. Flora Fungorum Sinicorum. Vol. 12. Ustilaginaceae.
Beijing: Science Press; 2000.
6. Zhuang JY. Flora Fungorum Sinicorum. Vol. 19. Uredinales
(II). Beijing: Science Press; 2003.
7. Bai JK. Flora Fungorum Sinicorum. Vol. 15. Sphaeropsidales,
Phoma, Phyllosticta. Beijing: Science Press; 2000. p. 162-3.
8.Zhuang WY. Fungi of northwestern China. Ithaca (NY):
Mycotaxon, Ltd.; 2005.
9. Wang Y, Chen X, Li Y. Survey and pathogen identification of
rhubarb diseases in Gansu Province. Zhongguo Zhong Yao
Za Zhi 2009;34:953-6.
10. Liu W, Yan LY, Wen ZH, Chen XR. Survey of virus diseases
on 18 species of medical herbs in Gansu Province and
identification of two virus disease. Plant Prot 2014;40:133-7.
11. Donk MA. Nomina conservanda proposita I. Proposals in
fungi: Deuteromycetes. Regnum Veg 1964;34:7-15.
12. Sutton BC. The Coelomycetes: fungi imperfecti with pycnidia,
acervuli and stromata. Kew: Commonwealth Mycological
Institute; 1980.
13. Constantinescu O. Taxonomical revision of Septoria-like fungi
parasitic on Betulaceae. Trans Br Mycol Soc 1984;83:383-98.
14. Farr DF. Species of Septoria on the Fabaceae, subfamily
Faboideae, tribe Genisteae. Sydowia 1992;44:13-31.
15. Verkley GJ, Starink-Willemse M. A phylogenetic study of
some Septoria species pathogenic to Asteraceae based on ITS
ribosomal DNA sequences. Mycol Prog 2004;3:315-23.
16. Verkley GJ, Starink-Willemse M, van Iperen A, Abeln EC.
Phylogenetic analyses of Septoria species based on the ITS
and LSU-D2 regions of nuclear ribosomal DNA. Mycologia
2004;96:558-71.
17. Priest MJ. Fungi of Australia: Septoria. Melbourne: CSIRO
Publishing; 2006.
18. von Arx JA. Mycosphaerella and its anamorphs. Proc K Ned
Akad Wet C 1983;86:15-54.
19. Crous PW, Aptroot A, Kang JC, Braun U, Wingfield MJ. The
genus Mycosphaerella and its anamorphs. Stud Mycol 2000;
45:107-21.
20. Crous PW, Kang JC, Braun U. A phylogenetic redefinition of
anamorph genera in Mycosphaerella based on ITS rDNA
sequence and morphology. Mycologia 2001;93:1081-101.
98 Wang et al.
21. Farr DF, Rossman AY. Fungal Databases. Washington, DC:
Systematic Mycology and Microbiology Laboratory, ARS,
USDA; 2015.
22. White TJ, Bruns TD, Lee S, Taylor J. Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics.
In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors.
PCR protocols, a guide to methods and applications. San
Diego (CA): Academic Press; 1990. p. 315-22.
23. Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel
method for rapid multiple sequence alignment based on fast
Fourier transform. Nucleic Acids Res 2002;30:3059-66.
24. Hall TA. BioEdit: a user-friendly biological sequence alignment
editor and analysis program for Windows 95/98/NT. Nucleic
Acids Symp Ser 1999;41:95-8.
25. Pierobom CR. Studies on pycnidial and ascocarpic fungi
which cause leaf spots on wheat in Washington [dissertation].
Washington, DC: Washington State University; 1983.
26. Kema GH, Annone JG. In vitro production of pycnidia by
Septoria tritici. Neth J Plant Pathol 1991;97:65-72.
27. Verkley GJ, Priest MJ. Septoria and similar coelomycetous
anamorphs of My co sphaerel la. Stud Mycol 2000;45:123-8.
28. Beach WS. Biologic specialization in the genus Septoria. Am J
Bot 1919;6:1-33.
29. Farr DF. Septoria species on Cornus. Mycologia 1991;83:611-
23.
