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First report of southern blight of mung bean caused by Sclerotium rolfsii in China
Journal Pre-proof First report of southern blight of mung bean caused by Sclerotium rolfsii in China Suli Sun, Feifei Sun, Dong Deng, Xu Zhu, Canxing Duan, Zhendong Zhu PII:
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DOI:
https://doi.org/10.1016/j.cropro.2019.105055
Reference:
JCRP 105055
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Crop Protection
Received Date: 10 July 2019 Revised Date:
1 November 2019
Accepted Date: 17 December 2019
Please cite this article as: Sun, S., Sun, F., Deng, D., Zhu, X., Duan, C., Zhu, Z., First report of southern blight of mung bean caused by Sclerotium rolfsii in China, Crop Protection (2020), doi: https:// doi.org/10.1016/j.cropro.2019.105055. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
First report of southern blight of mung bean caused by Sclerotium rolfsii
2
in China
3 4
Suli Sun1, Feifei Sun1, Dong Deng1, Xu Zhu2, Canxing Duan1, Zhendong Zhu1*
5 6
1
7
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing,
8
100081, China
9
2
National Key Facility for Crop Gene Resources and Genetic Improvement,
Nanyang Academy of Agricultural Sciences, Nanyang, Henan, 473083, China
10 11
*Correspondening author: Dr. Zhendong Zhu
12
Institute of Crop Sciences
13
Chinese Academy of Agricultural Sciences
14
12 Zhongguancun South Street
15
Beijing, 100081
16
The Republic of China
17
Tel.: +86-10-82109609
18
Fax: +86-10-82109608
19
E-mail: [email protected]
20 21 22
1
1
Abstract
2
Mung bean (Vigna radiata) is an important legume crop in China. An emerging
3
mung bean disease with symptoms resembling southern blight was detected in
4
China. This study was conducted to confirm and characterize the responsible
5
pathogen through cultural and morphological characterization, and molecular
6
detection using multiple specific primers and multiple-clone sequence analysis
7
of the rDNA internal transcribed spacer (ITS) region and the translation
8
elongation factor 1α (EF1α) and RNA polymerase II subunit 2 (RPB2) genes.
9
All isolates obtained from mung bean were identified as Sclerotium rolfsii.
10
Interestingly, we found that rDNA ITS, EF1α, and RPB2 sequences of all
11
isolates consisted of two distinct types, which likely originated from different
12
nuclei. Our findings indicated that S. rolfsii isolates were heterokaryotic and
13
contain a heterozygous genome. Two trials of pathogenicity and host range
14
revealed that all S. rolfsii isolates were not only strongly virulent to mung bean
15
but also to several other important crops, indicating their potential threat to
16
future agricultural production. To our knowledge, this is the first report of S.
17
rolfsii causing southern blight on mung bean in China.
18
Keywords: Vigna radiata; Southern blight; Sclerotium rolfsii; Heterogeneity
2
1
1. Introduction
2
Mung bean [Vigna radiata (L.) R. Wilczek] is an important warm-season
3
legume crop that is mainly grown in tropical and subtropical regions in Asia,
4
including China, India, Pakistan, Thailand, Indonesia, and the Philippines (Nair
5
et al., 2013). In China, more than 20 mung bean diseases have been
6
documented to date (Cui et al., 2014; Li et al., 2016; Sun et al., 2016; Zhu and
7
Duan, 2012).
8
Sclerotium rolfsii Sacc. [teleomorph: Athelia rolfsii (Curzi) Tu & Kimbrough]
9
is a major soilborne plant pathogenic fungus with a worldwide distribution. It is
10
mainly present in warm-temperate, subtropical, and tropical regions, especially
11
under high humidity and warm conditions. S. rolfsii has a wide host range, and
12
can infect more than 500 monocotyledonous and dicotyledonous plant species,
13
commonly causing southern blight and leading to serious yield losses (Aycock,
14
1966; Punja, 1985; Punja, 1988). Recently, S. rolfsii has been continuously
15
detected on new plant hosts, including vegetables, ornamentals, grass, and
16
medicinal and leguminous crops (Galdames et al., 2010; Kwon et al., 2013;
17
Mahadevakumar et al., 2015; 2016; Shen et al., 2015; Pane et al., 2008). S.
18
rolfsii is usually characterized according to mycelial and sclerotial morphology
19
because its sexual stage (i.e., Athelia rolfsii) is not easily observed (Okabe et
20
al., 1998). However, S. rolfsii isolates originating from different hosts and
21
geographical areas frequently exhibit variable morphological characteristics
22
when cultured (Harlton et al., 1995; Okabe et al., 1998). Therefore, S. 3
1
rolfsii-specific primer pairs were recently designed based on the conserved
2
sequence within rDNA ITS regions (i.e., SCR-F/R and SRITSF/R) and within
3
the large-subunit (LSU) gene (i.e., D1–D2 region) (SRLSUF/R) (Jeeva et al.,
4
2010; Pravi et al., 2014; 2015; White et al., 1990). These three S.
5
rolfsii-specific primers could be used to accurately identify the pathogen to the
6
species level.
7
Several previous studies clarified the genetic variability in the rDNA ITS
8
regions of S. rolfsii and involved a comparison of the amplified ITS products,
9
analysis of the ITS-RFLP pattern, and construction of restriction maps (Harlton
10
et al., 1995; Okabe et al., 1998; Okabe et al., 2001; Okabe and Matsumoto,
11
2003). They indicated that field isolates of S. rolfsii may be heterokaryotic. The
12
ITS heterogeneity of S. rolfsii was also implied by the fact that the ITS regions
13
could not be directly sequenced (Nalim et al., 1995; Okabe et al., 2001).
14
The objective of the current study was to confirm the causal agent of an
15
emerging mung bean disease with the similar symptoms of southern blight,
16
using morphological and molecular characteristics of multiple genes (i.e.,
17
rDNA ITS, EF1α, and RPB2), and pathogenicity and host tests.
18 19
2. Material and methods
20
2.1. Disease survey
21
In August 2017, a field disease survey of mung bean was conducted by
22
our research team in Nanyang City of Henan Province, China. The survey
23
revealed the presence of southern blight symptoms similar to those caused by
24
S. rolfsii of mung bean plants in a field located in Xinye County. Incidence of 4
1
the diseased plants with symptoms is about 10% in the infected field. And the
2
diseased plants were distributed in a few spots in the field.
3
2.2. Isolation of pathogen from diseased plants
4
To isolate pathogen, samples of diseased mung bean plants with typical
5
southern blight symptoms or sclerotia were collected from Xinye County,
6
Henan Province. Diseased basal stem sections and sclerotia were surface
7
disinfected and placed on potato dextrose agar (PDA; AoBoXing Bio-tech,
8
Beijing, China). Plates were incubated at 25 °C under a 12-h photoperiod for 2
9
to 3 days. Fungal isolates were purified by transferring single hyphal tips to
10
fresh medium.
11
2.3. Identification of the pathogen
12
Five isolates were obtained and used to determine their identity. Mycelial
13
plugs (diameter: 5 mm) were inoculated to the center of PDA plates (diameter:
14
9 cm). Radial mycelial growth was assessed following the overgrowth of the
15
PDA plates. Young hyphae were stained with 0.03% safranin-O and 3% KOH
16
aqueous solution according to the protocol described by Bandoni (1979). The
17
nuclei in the stained hyphae were counted using an Olympus CX31
18
microscope (400× magnification) to determine whether cells were binucleate
19
or multinucleate. Sclerotial characteristics were also monitored after a 4-week
20
incubation, including form, size, and color. For each isolate, the diameters of
21
30 randomly selected mature sclerotia were measured. Each isolate was
22
analyzed using three replicates.
23
Genomic DNA was extracted from the five fungal isolates using a CTAB
24
method with minor modifications (Allen et al., 2006). The PCR detection was
25
conducted using three S. rolfsii-specific primer pairs (SCR-F/R, SRITSF/R and 5
1
SRLSUF/R) (Jeeva et al., 2010; Pravi et al., 2014; Pravi et al., 2015) (Table 1).
2
Next, PCR assays and partial sequence of three gene were conducted with
3
universal
4
EF1-983F/EF1-2218R primers in EF1α gene (Rehner and Buckley, 2005), and
5
fRPB2-5F/bRPB2-7R2 primers in the RPB2 gene (Liu et al., 1999). Then, we
6
applied a cloning method to generate sequence from the obtained amplicons
7
by
8
fRPB2-5F/bRPB2-7R2. The resulting sequences for each isolate were aligned
9
with
the
primers
three
Multalin
ITS1/ITS4
primer
pairs
in
ITS
region
ITS1/ITS4,
(White
et
al.,
EF1-983F/EF1-2218R
(http://multalin.toulouse.inra.fr/multalin/).
They
were
1990),
and
also
10
analyzed using the BLAST online tool in the GenBank database (National
11
Center for Biotechnology Information; NCBI) to compare sequence similarities.
12
2.4. Pathogenicity tests
13
Two pathogenicity trials were completed using mung bean seedlings (i.e.,
14
original host), and six other legumes [i.e., soybean (Glycine max), common
15
bean (Phaseolus vulgaris), cowpea (Vigna unguiculata), pea (Pisum sativum),
16
adzuki bean (Vigna angularis), and peanut (Arachis hypogaea)], and important
17
graminaceous species [i.e., maize (Zea mays) and wheat (Triticum aestivum)],
18
as well as one malvaceous species [i.e., cotton (Anemone vitifolia)]. All tested
19
crops were sown in 500-ml paper cups. The cups were randomly distributed on
20
a greenhouse bench and incubated at 25 ± 2 °C. The 10 to 12-day-old
21
seedlings were inoculated using the modified hypocotyl puncture technique
22
and root-drenching procedure. All plants inoculated with sterile medium were 6
1
used as negative controls. The treated plants were incubated in a misting room
2
at 25 °C under high humidity (> 90%) for 48 h and then transferred to a
3
greenhouse for an additional incubation at 25 ± 2 °C. Plants were monitored
4
every day for 4 weeks to detect symptoms caused by S. rolfsii. Pathogen was
5
re-isolated from inoculated plants to verify Koch’s postulates. All tests were
6
conducted twice.
7 8
3. Results and discussion
9
By the survey, the diseased plants of mung bean were observed with
10
symptoms similar to southern blight caused by S. rolfsii in the field. The first
11
observed above-ground symptoms on the infected plants involved the
12
yellowing of foliage (Fig. 1A) and the development of girdling stem lesions near
13
the soil line (Fig. 1B). Advanced disease development stages were
14
characterized by the growth of white mycelia on plants (Fig. 1C and D). During
15
the late disease stage, tan to brown sclerotia were produced on the basal stem
16
epidermis and on the soil surface surrounding infected plants at high moisture
17
levels (Fig. 1E). At last, infected plants gradually wilted and eventually died as
18
the disease progressed.
19
Five fungal isolates (i.e., MSB1 to MSB5) were obtained from diseased
20
mung bean plants. The mycelia of the five isolates were white in color with
21
even margins and a fluffy appearance (Fig. 2A). Colonies grew quickly and
22
covered the Petri dish (diameter: 9 cm) after 5 or 6 days. All five isolates
23
produced a considerable amount of calcium oxalate crystals, which formed a 7
1
wide halo (diameter: 5 - 6 mm) surrounding colonies (Fig. 2A). The shapes and
2
sizes of calcium oxalate crystals varied considerably, with prismatic, tetragonal,
3
druse, and irregular forms (Fig. 2B). Ansari and Agnihotri (2000) reported a
4
positive correlation between oxalic acid production and S. rolfsii virulence.
5
Their virulence may be associated with abundant oxalic acid production.
6
Colonies started to form sclerotia on the mycelia after 12 days incubation.
7
Sclerotia were initially white, but turned brown or dark brown at maturity.
8
Abundant spherical, subspherical, and irregularly shaped sclerotia were
9
observed after 28 days (Fig. 2C), with each plate containing 60 to 128 sclerotia
10
(diameter: 0.71 - 3.45 mm). Mycelia produced clamp connections, and were
11
observed to contain multiple nuclei (Fig. 2D). The observed morphology
12
characteristics were consistent with those of S. rolfsii Sacc. Previous studies
13
demonstrated a close affinity between S. rolfsii and S. delphinii (Okabe and
14
Matsumoto, 2003). In contrast, S. delphinii produces fewer (i.e., 20 to 30 per
15
plate) and larger (3 to 5 mm in diameter) sclerotia (Mahadevakumar et al.,
16
2016).
17
PCR products amplified by three specific primers (SCR-F/R, SRITSF/R
18
and SRLSUF/R) from all five isolates revealed consistent fragments of 540,
19
500, and 360 bp, respectively (Jeeva et al. 2010; Pravi et al. 2014; Pravi et al.
20
2015) (Fig. 3). The amplicons generated using the primers ITS1/ITS4,
21
EF1-983F/EF1-2218R, and fRPB2-5F/bRPB2-7R2 were approximately 700,
22
1,200, and 1,100 bp fragments, respectively (Fig. 3). Based on 10 clone 8
1
sequence alignments, all five isolates were determined to contain two distinct
2
sequence types (i.e., type 1 and type 2) for the ITS (type1: MN609999,
3
MN610001,
4
MN610002, MN610004, MN610006, MN610008), EF1α, and RPB2 regions
5
(Supplementary Fig. 1; Figs. 4 and 5). A BLASTn analysis revealed the
6
consensus ITS type 1 and type 2 sequences were 99% identical to dozens of
7
S. rolfsii (Athelia rolfsii) sequences in the NCBI GenBank database (e.g., type
8
1: HQ420816, KX186998, KU760984, JN017199, and KJ546416; type 2:
9
HM355751, GU4567776, JF966208, KT222899, and KJ552090). The BLASTn
10
analysis of the EF1α and RPB2 sequences indicated that obtained EF1α type
11
1 and type 2 sequences were highly homologous to two S. rolfsii sequences
12
(i.e., 99 and 96%) (Accession numbers: KP982854 and GU187681), and
13
obtained RPB2 type 1 and type 2 sequences were highly homologous to one S.
14
rolfsii sequence (i.e., 95 and 99%) (Accession number: GU187821). All
15
morphological and molecular characteristics confirmed that all five isolates
16
were S. rolfsii Sacc. Previous studies proved that S. rolfsii isolates contain two
17
sequence types only in the ITS region (Okabe, 2001; Okabe and Matsumoto,
18
2003). In contrast, our study firstly revealed that two sequence types are also
19
observed in the EF1α and RPB2 genes of S. rolfsii isolates based on multiple
20
cloned sequences (Figs. 4 and 5). Our results suggest that all five analyzed S.
21
rolfsii isolates contain two heterokaryotic nuclei, resulting from a distinct
22
phylogenetic history, which may have different origins via hybridization or
MN610003,
MN610005,
9
MN610007;
type2:
MN610000,
1
hyphae anastomosis between species or subgroups carrying different genome
2
types.
3
In two pathogenicity trials, all five isolates induced symptoms resembling
4
southern blight on the original host (i.e., mung bean) (Fig. 6) and other tested
5
crops, except for maize (Supplementary Fig. 2). Plants started wilting at 4 and
6
7 days after inoculation by hypocotyl puncture and mycelia root-drenching
7
procedures, respectively. Inoculated plants of all tested crops, except for
8
maize, seriously wilted and eventually died. In contrast, maize plants exhibited
9
mild symptoms, including dwarfism (Supplementary Fig. 2). Sclerotia were
10
produced at the base of stems and on the soil surface surrounding plants of all
11
inoculated crop species except for pea, adzuki bean, and maize plants (Fig. 6).
12
The five isolates were also re-isolated from symptomatic lesions in all tested
13
crops, which fulfilled Koch’s postulates. Control plants remained healthy and
14
no symptoms shown and pathogen isolation. Our pathogenicity tests indicate
15
that S. rolfsii isolates from mung bean will be a potential threat to other
16
important crops, especially legumes. It should be noted that our surveyed
17
mung bean fields represent peanut cultivation sites in China. Peanut is one of
18
the hosts of S. rolfsii and may have contributed to the accumulation of
19
pathogen inocula on mung bean (Fig. 6).
20
S. rolfsii is an important pathogenic fungus for legumes, which is the
21
causative agent of southern blight on several leguminous hosts, including
22
soybean (G. max) (Hartman et al., 1999), common bean (P. vulgaris) (Rusuku 10
1
et al., 1997), chickpea (C. arietinum) (Kokub et al., 2007), cowpea (V.
2
unguiculata) (Adandonon et al., 2004), lentil (Lens culinaris) (Shahid et al.,
3
1990), and peanut (A. hypogaea) (Bowen et al., 1992). In China, southern
4
blight caused by S. rolfsii has recently been detected on Canadian goldenrod
5
(Solidago canadensis) and Macleaya cordata (Tang et al., 2010; You et al.,
6
2015). However, there have been no reports of S. rolfsii on mung bean. To the
7
best of our knowledge, this is the first study of S. rolfsii causing southern blight
8
on mung bean in China. Our findings indicate that S. rolfsii may be a new
9
threat to mung bean and other crops, which should be carefully monitored in
10
China.
11 12
Acknowledgments
13
This study was supported by the Modern Agro-industry Technology Research
14
System (CARS-09), the Crop Germplasm Conservation and Utilization
15
Program (2019NWB036-12), the National Infrastructure for Crop Germplasm
16
Resources (NICGR2019-008), and the Scientific Innovation Program of the
17
Chinese Academy of Agricultural Sciences.
18
11
1
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Sclerotium rolfsii on Macleaya cordata in China. Plant Dis. 100, 530.
13
Zhu, Z.D., Duan, C.X., 2012. Identification and control of diseases and pests in
14
mung bean. Beijing, China Agriculture Science and Technology Press (in
15
Chinese).
16 17
16
1 2
Table 1. Details of six primer pairs used to detect Sclerotium rolfsii and clone fragments for subsequent sequence analyses. Primer name SCR-F/SCR-R SRITSF/SRITSR
Primer sequence (5′—3′) CGTAGGTGAACCTGCGGA / CATACAAGCTAGAATCCC TACACCTGTGAACCAACTG / CATACAAGCTAGAATCCC
Denature temperature
PCR product size
Target region gene
54℃
540bp
ITS
Jeeva et al. 2010
50℃
500bp
ITS
Pravi et al. 2014
D1/D2 domain LSU
or
SRLSUF/SRLSUR
AGTGTTTTCTGTGCTGGG / ACCTTCCTCTGGCCTTTTTC
62℃
360bp
ITS1/ITS4
TCCGTAGGTGAACCTGCGG / TCCTCCGCTTATTGATATGC
55℃
700bp
ITS
EF1-983F/EF1-2218R
GCYCCYGGHCAYCGTGAYTTYAT /ATGACACCRACRGCRACRGTYTG
60℃
1200bp
EF1α
fRPB2-5F/bRPB2-7R2
GAYGAYMGWGATCAYTTYGG /ACYTGRTTRTGRTCNGGRAANGG
62℃
1100bp
5-7 domain of RPB2
3 4
17
of
Reference
Pravi et al. 2015 White et al. 1990 Rehner and Buckley 2005; Matheny et al. 2007 Liu et al. 1999; Matheny et al. 2007
1
Figure Legends
2 3
Fig. 1. Southern blight symptoms caused by Sclerotium rolfsii on field-grown mung
4
bean plants. A, Overview of the occurrence of southern blight on mung bean plants.
5
The foliage of infected plants turned yellow. B, White mycelial initials near the soil line
6
of infected plants. C and D, Characteristic southern blight symptoms with abundant
7
white mycelial growth on basal stems. e, White and brown sclerotia on the surface of
8
basal stems.
9 10
Fig. 2. Morphology of Sclerotium rolfsii isolates from diseased mung bean plants. A,
11
Two-day-old colony with white and fluffy mycelia with even margins and a halo formed
12
by calcium oxalate crystals. B, A 4-week-old colony produced prismatic, tetragonal, or
13
irregular shaped calcium oxalate crystals. C, Tan to brown sclerotia were produced on
14
potato dextrose agar. D, Mycelia produced clamp connections, and multinucleate
15
cells were observed in the stained mycelia (scale bar: 10 µm).
16 17
Fig. 3. Polymerase chain reaction amplification patterns of five Sclerotium rolfsii
18
isolates. A, Amplification products for five S. rolfsii isolates using the SCR-F/R,
19
SRITSF/R, and SRLSUF/R three species-specific primer pairs. B, Amplification
20
products for five S. rolfsii isolates using three primer pairs for the rDNA ITS, EF1α,
21
and RPB2 sequences, which were used for sequencing by cloning. (M: marker ladder;
22
1: MSB1; 2: MSB2; 3: MSB3; 4: MSB4; 5: MSB5)
23 24
Fig. 4. Comparison of the sequences of 10 EF1α clone fragments from Sclerotium
25
rolfsii
isolate
MSB1
amplified
by
polymerase 18
chain
reaction
using
the
1
EF1-983F/EF1-2218R primer pair. The comparison revealed two distinct sequence
2
types, with three type 1 and seven type 2 sequences, respectively.
3 4
Fig. 5. Comparison of the sequences of 10 RPB2 clone fragments from Sclerotium
5
rolfsii
6
fRPB2-5F/bRPB2-7R2 primer pair. The sequences spanned domains 5 to 7 of RPB2
7
gene. The comparison revealed two distinct sequence types, with four type 1 and
8
six type 2 sequences.
isolate
MSB1
amplified
by
polymerase
chain
reaction
using
the
9 10
Fig. 6. Pathogenicity tests on mung bean (A) and peanut (B) seedlings inoculated
11
with Sclerotium rolfsii isolate MSB1 using a mycelia root-drenching procedure.
12
Inoculated mung bean and peanut plants wilted and died. Brown to dark brown
13
sclerotia were produced on the basal stems and on the soil surface surrounding
14
infected plants.
19
Fig. 1. Southern blight symptoms caused by Sclerotium rolfsii on field-grown mung bean plants. A, Overview of the occurrence of southern blight on mung bean plants. The foliage of infected plants turned yellow. B, White mycelial initials near the soil line of infected plants. C and D, Characteristic southern blight symptoms with abundant white mycelial growth on basal stems. e, White and brown sclerotia on the surface of basal stems.
Fig. 2. Morphology of Sclerotium rolfsii isolates from diseased mung bean plants. A, Two-day-old colony with white and fluffy mycelia with even margins and a halo formed by calcium oxalate crystals. B, A 4-week-old colony produced prismatic, tetragonal, or irregular shaped calcium oxalate crystals. C, Tan to brown sclerotia were produced on potato dextrose agar. D, Mycelia produced clamp connections, and multinucleate cells were observed in the stained mycelia (scale bar: 10 µm).
Fig. 3. Polymerase chain reaction amplification patterns of five Sclerotium rolfsii isolates. A, Amplification products for five S. rolfsii isolates using the SCR-F/R, SRITSF/R, and SRLSUF/R three species-specific primer pairs. B, Amplification products for five S. rolfsii isolates using three primer pairs for the rDNA ITS, EF1α, and RPB2 sequences, which were used for sequencing by cloning. (M: marker ladder; 1: MSB1; 2: MSB2; 3: MSB3; 4: MSB4; 5: MSB5)
Fig. 4. Comparison of the sequences of 10 EF1α clone fragments from Sclerotium rolfsii isolate MSB1 amplified by polymerase chain reaction using the EF1-983F/EF1-2218R primer pair. The comparison revealed two distinct sequence types, with three type 1 and seven type 2 sequences, respectively.
Fig. 5. Comparison of the sequences of 10 RPB2 clone fragments from Sclerotium rolfsii isolate MSB1 amplified by polymerase chain reaction using the fRPB2-5F/bRPB2-7R2 primer pair. The sequences spanned domains 5 to 7 of RPB2 gene. The comparison revealed two distinct sequence types, with four type 1 and
six type 2 sequences.
Fig. 6. Pathogenicity tests on mung bean (A) and peanut (B) seedlings inoculated with Sclerotium rolfsii isolate MSB1 using a mycelia root-drenching procedure. Inoculated mung bean and peanut plants wilted and died. Brown to dark brown sclerotia were produced on the basal stems and on the soil surface surrounding infected plants.
Highlights This study is the first report of S. rolfsii causing southern blight on mung bean in China. Characterization of S. rolfsii was confirmed by morphological and molecular identification. This study proved S. rolfsii isolates were heterokaryotic and contain a heterozygous genome. Pathogenicity and host range tests indicated S. rolfsii had a wide host range.
Conflict of interest: All authors declare that they have no conflict of interest.
S0261-2194(19)30401-6
DOI:
https://doi.org/10.1016/j.cropro.2019.105055
Reference:
JCRP 105055
To appear in:
Crop Protection
Received Date: 10 July 2019 Revised Date:
1 November 2019
Accepted Date: 17 December 2019
Please cite this article as: Sun, S., Sun, F., Deng, D., Zhu, X., Duan, C., Zhu, Z., First report of southern blight of mung bean caused by Sclerotium rolfsii in China, Crop Protection (2020), doi: https:// doi.org/10.1016/j.cropro.2019.105055. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
First report of southern blight of mung bean caused by Sclerotium rolfsii
2
in China
3 4
Suli Sun1, Feifei Sun1, Dong Deng1, Xu Zhu2, Canxing Duan1, Zhendong Zhu1*
5 6
1
7
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing,
8
100081, China
9
2
National Key Facility for Crop Gene Resources and Genetic Improvement,
Nanyang Academy of Agricultural Sciences, Nanyang, Henan, 473083, China
10 11
*Correspondening author: Dr. Zhendong Zhu
12
Institute of Crop Sciences
13
Chinese Academy of Agricultural Sciences
14
12 Zhongguancun South Street
15
Beijing, 100081
16
The Republic of China
17
Tel.: +86-10-82109609
18
Fax: +86-10-82109608
19
E-mail: [email protected]
20 21 22
1
1
Abstract
2
Mung bean (Vigna radiata) is an important legume crop in China. An emerging
3
mung bean disease with symptoms resembling southern blight was detected in
4
China. This study was conducted to confirm and characterize the responsible
5
pathogen through cultural and morphological characterization, and molecular
6
detection using multiple specific primers and multiple-clone sequence analysis
7
of the rDNA internal transcribed spacer (ITS) region and the translation
8
elongation factor 1α (EF1α) and RNA polymerase II subunit 2 (RPB2) genes.
9
All isolates obtained from mung bean were identified as Sclerotium rolfsii.
10
Interestingly, we found that rDNA ITS, EF1α, and RPB2 sequences of all
11
isolates consisted of two distinct types, which likely originated from different
12
nuclei. Our findings indicated that S. rolfsii isolates were heterokaryotic and
13
contain a heterozygous genome. Two trials of pathogenicity and host range
14
revealed that all S. rolfsii isolates were not only strongly virulent to mung bean
15
but also to several other important crops, indicating their potential threat to
16
future agricultural production. To our knowledge, this is the first report of S.
17
rolfsii causing southern blight on mung bean in China.
18
Keywords: Vigna radiata; Southern blight; Sclerotium rolfsii; Heterogeneity
2
1
1. Introduction
2
Mung bean [Vigna radiata (L.) R. Wilczek] is an important warm-season
3
legume crop that is mainly grown in tropical and subtropical regions in Asia,
4
including China, India, Pakistan, Thailand, Indonesia, and the Philippines (Nair
5
et al., 2013). In China, more than 20 mung bean diseases have been
6
documented to date (Cui et al., 2014; Li et al., 2016; Sun et al., 2016; Zhu and
7
Duan, 2012).
8
Sclerotium rolfsii Sacc. [teleomorph: Athelia rolfsii (Curzi) Tu & Kimbrough]
9
is a major soilborne plant pathogenic fungus with a worldwide distribution. It is
10
mainly present in warm-temperate, subtropical, and tropical regions, especially
11
under high humidity and warm conditions. S. rolfsii has a wide host range, and
12
can infect more than 500 monocotyledonous and dicotyledonous plant species,
13
commonly causing southern blight and leading to serious yield losses (Aycock,
14
1966; Punja, 1985; Punja, 1988). Recently, S. rolfsii has been continuously
15
detected on new plant hosts, including vegetables, ornamentals, grass, and
16
medicinal and leguminous crops (Galdames et al., 2010; Kwon et al., 2013;
17
Mahadevakumar et al., 2015; 2016; Shen et al., 2015; Pane et al., 2008). S.
18
rolfsii is usually characterized according to mycelial and sclerotial morphology
19
because its sexual stage (i.e., Athelia rolfsii) is not easily observed (Okabe et
20
al., 1998). However, S. rolfsii isolates originating from different hosts and
21
geographical areas frequently exhibit variable morphological characteristics
22
when cultured (Harlton et al., 1995; Okabe et al., 1998). Therefore, S. 3
1
rolfsii-specific primer pairs were recently designed based on the conserved
2
sequence within rDNA ITS regions (i.e., SCR-F/R and SRITSF/R) and within
3
the large-subunit (LSU) gene (i.e., D1–D2 region) (SRLSUF/R) (Jeeva et al.,
4
2010; Pravi et al., 2014; 2015; White et al., 1990). These three S.
5
rolfsii-specific primers could be used to accurately identify the pathogen to the
6
species level.
7
Several previous studies clarified the genetic variability in the rDNA ITS
8
regions of S. rolfsii and involved a comparison of the amplified ITS products,
9
analysis of the ITS-RFLP pattern, and construction of restriction maps (Harlton
10
et al., 1995; Okabe et al., 1998; Okabe et al., 2001; Okabe and Matsumoto,
11
2003). They indicated that field isolates of S. rolfsii may be heterokaryotic. The
12
ITS heterogeneity of S. rolfsii was also implied by the fact that the ITS regions
13
could not be directly sequenced (Nalim et al., 1995; Okabe et al., 2001).
14
The objective of the current study was to confirm the causal agent of an
15
emerging mung bean disease with the similar symptoms of southern blight,
16
using morphological and molecular characteristics of multiple genes (i.e.,
17
rDNA ITS, EF1α, and RPB2), and pathogenicity and host tests.
18 19
2. Material and methods
20
2.1. Disease survey
21
In August 2017, a field disease survey of mung bean was conducted by
22
our research team in Nanyang City of Henan Province, China. The survey
23
revealed the presence of southern blight symptoms similar to those caused by
24
S. rolfsii of mung bean plants in a field located in Xinye County. Incidence of 4
1
the diseased plants with symptoms is about 10% in the infected field. And the
2
diseased plants were distributed in a few spots in the field.
3
2.2. Isolation of pathogen from diseased plants
4
To isolate pathogen, samples of diseased mung bean plants with typical
5
southern blight symptoms or sclerotia were collected from Xinye County,
6
Henan Province. Diseased basal stem sections and sclerotia were surface
7
disinfected and placed on potato dextrose agar (PDA; AoBoXing Bio-tech,
8
Beijing, China). Plates were incubated at 25 °C under a 12-h photoperiod for 2
9
to 3 days. Fungal isolates were purified by transferring single hyphal tips to
10
fresh medium.
11
2.3. Identification of the pathogen
12
Five isolates were obtained and used to determine their identity. Mycelial
13
plugs (diameter: 5 mm) were inoculated to the center of PDA plates (diameter:
14
9 cm). Radial mycelial growth was assessed following the overgrowth of the
15
PDA plates. Young hyphae were stained with 0.03% safranin-O and 3% KOH
16
aqueous solution according to the protocol described by Bandoni (1979). The
17
nuclei in the stained hyphae were counted using an Olympus CX31
18
microscope (400× magnification) to determine whether cells were binucleate
19
or multinucleate. Sclerotial characteristics were also monitored after a 4-week
20
incubation, including form, size, and color. For each isolate, the diameters of
21
30 randomly selected mature sclerotia were measured. Each isolate was
22
analyzed using three replicates.
23
Genomic DNA was extracted from the five fungal isolates using a CTAB
24
method with minor modifications (Allen et al., 2006). The PCR detection was
25
conducted using three S. rolfsii-specific primer pairs (SCR-F/R, SRITSF/R and 5
1
SRLSUF/R) (Jeeva et al., 2010; Pravi et al., 2014; Pravi et al., 2015) (Table 1).
2
Next, PCR assays and partial sequence of three gene were conducted with
3
universal
4
EF1-983F/EF1-2218R primers in EF1α gene (Rehner and Buckley, 2005), and
5
fRPB2-5F/bRPB2-7R2 primers in the RPB2 gene (Liu et al., 1999). Then, we
6
applied a cloning method to generate sequence from the obtained amplicons
7
by
8
fRPB2-5F/bRPB2-7R2. The resulting sequences for each isolate were aligned
9
with
the
primers
three
Multalin
ITS1/ITS4
primer
pairs
in
ITS
region
ITS1/ITS4,
(White
et
al.,
EF1-983F/EF1-2218R
(http://multalin.toulouse.inra.fr/multalin/).
They
were
1990),
and
also
10
analyzed using the BLAST online tool in the GenBank database (National
11
Center for Biotechnology Information; NCBI) to compare sequence similarities.
12
2.4. Pathogenicity tests
13
Two pathogenicity trials were completed using mung bean seedlings (i.e.,
14
original host), and six other legumes [i.e., soybean (Glycine max), common
15
bean (Phaseolus vulgaris), cowpea (Vigna unguiculata), pea (Pisum sativum),
16
adzuki bean (Vigna angularis), and peanut (Arachis hypogaea)], and important
17
graminaceous species [i.e., maize (Zea mays) and wheat (Triticum aestivum)],
18
as well as one malvaceous species [i.e., cotton (Anemone vitifolia)]. All tested
19
crops were sown in 500-ml paper cups. The cups were randomly distributed on
20
a greenhouse bench and incubated at 25 ± 2 °C. The 10 to 12-day-old
21
seedlings were inoculated using the modified hypocotyl puncture technique
22
and root-drenching procedure. All plants inoculated with sterile medium were 6
1
used as negative controls. The treated plants were incubated in a misting room
2
at 25 °C under high humidity (> 90%) for 48 h and then transferred to a
3
greenhouse for an additional incubation at 25 ± 2 °C. Plants were monitored
4
every day for 4 weeks to detect symptoms caused by S. rolfsii. Pathogen was
5
re-isolated from inoculated plants to verify Koch’s postulates. All tests were
6
conducted twice.
7 8
3. Results and discussion
9
By the survey, the diseased plants of mung bean were observed with
10
symptoms similar to southern blight caused by S. rolfsii in the field. The first
11
observed above-ground symptoms on the infected plants involved the
12
yellowing of foliage (Fig. 1A) and the development of girdling stem lesions near
13
the soil line (Fig. 1B). Advanced disease development stages were
14
characterized by the growth of white mycelia on plants (Fig. 1C and D). During
15
the late disease stage, tan to brown sclerotia were produced on the basal stem
16
epidermis and on the soil surface surrounding infected plants at high moisture
17
levels (Fig. 1E). At last, infected plants gradually wilted and eventually died as
18
the disease progressed.
19
Five fungal isolates (i.e., MSB1 to MSB5) were obtained from diseased
20
mung bean plants. The mycelia of the five isolates were white in color with
21
even margins and a fluffy appearance (Fig. 2A). Colonies grew quickly and
22
covered the Petri dish (diameter: 9 cm) after 5 or 6 days. All five isolates
23
produced a considerable amount of calcium oxalate crystals, which formed a 7
1
wide halo (diameter: 5 - 6 mm) surrounding colonies (Fig. 2A). The shapes and
2
sizes of calcium oxalate crystals varied considerably, with prismatic, tetragonal,
3
druse, and irregular forms (Fig. 2B). Ansari and Agnihotri (2000) reported a
4
positive correlation between oxalic acid production and S. rolfsii virulence.
5
Their virulence may be associated with abundant oxalic acid production.
6
Colonies started to form sclerotia on the mycelia after 12 days incubation.
7
Sclerotia were initially white, but turned brown or dark brown at maturity.
8
Abundant spherical, subspherical, and irregularly shaped sclerotia were
9
observed after 28 days (Fig. 2C), with each plate containing 60 to 128 sclerotia
10
(diameter: 0.71 - 3.45 mm). Mycelia produced clamp connections, and were
11
observed to contain multiple nuclei (Fig. 2D). The observed morphology
12
characteristics were consistent with those of S. rolfsii Sacc. Previous studies
13
demonstrated a close affinity between S. rolfsii and S. delphinii (Okabe and
14
Matsumoto, 2003). In contrast, S. delphinii produces fewer (i.e., 20 to 30 per
15
plate) and larger (3 to 5 mm in diameter) sclerotia (Mahadevakumar et al.,
16
2016).
17
PCR products amplified by three specific primers (SCR-F/R, SRITSF/R
18
and SRLSUF/R) from all five isolates revealed consistent fragments of 540,
19
500, and 360 bp, respectively (Jeeva et al. 2010; Pravi et al. 2014; Pravi et al.
20
2015) (Fig. 3). The amplicons generated using the primers ITS1/ITS4,
21
EF1-983F/EF1-2218R, and fRPB2-5F/bRPB2-7R2 were approximately 700,
22
1,200, and 1,100 bp fragments, respectively (Fig. 3). Based on 10 clone 8
1
sequence alignments, all five isolates were determined to contain two distinct
2
sequence types (i.e., type 1 and type 2) for the ITS (type1: MN609999,
3
MN610001,
4
MN610002, MN610004, MN610006, MN610008), EF1α, and RPB2 regions
5
(Supplementary Fig. 1; Figs. 4 and 5). A BLASTn analysis revealed the
6
consensus ITS type 1 and type 2 sequences were 99% identical to dozens of
7
S. rolfsii (Athelia rolfsii) sequences in the NCBI GenBank database (e.g., type
8
1: HQ420816, KX186998, KU760984, JN017199, and KJ546416; type 2:
9
HM355751, GU4567776, JF966208, KT222899, and KJ552090). The BLASTn
10
analysis of the EF1α and RPB2 sequences indicated that obtained EF1α type
11
1 and type 2 sequences were highly homologous to two S. rolfsii sequences
12
(i.e., 99 and 96%) (Accession numbers: KP982854 and GU187681), and
13
obtained RPB2 type 1 and type 2 sequences were highly homologous to one S.
14
rolfsii sequence (i.e., 95 and 99%) (Accession number: GU187821). All
15
morphological and molecular characteristics confirmed that all five isolates
16
were S. rolfsii Sacc. Previous studies proved that S. rolfsii isolates contain two
17
sequence types only in the ITS region (Okabe, 2001; Okabe and Matsumoto,
18
2003). In contrast, our study firstly revealed that two sequence types are also
19
observed in the EF1α and RPB2 genes of S. rolfsii isolates based on multiple
20
cloned sequences (Figs. 4 and 5). Our results suggest that all five analyzed S.
21
rolfsii isolates contain two heterokaryotic nuclei, resulting from a distinct
22
phylogenetic history, which may have different origins via hybridization or
MN610003,
MN610005,
9
MN610007;
type2:
MN610000,
1
hyphae anastomosis between species or subgroups carrying different genome
2
types.
3
In two pathogenicity trials, all five isolates induced symptoms resembling
4
southern blight on the original host (i.e., mung bean) (Fig. 6) and other tested
5
crops, except for maize (Supplementary Fig. 2). Plants started wilting at 4 and
6
7 days after inoculation by hypocotyl puncture and mycelia root-drenching
7
procedures, respectively. Inoculated plants of all tested crops, except for
8
maize, seriously wilted and eventually died. In contrast, maize plants exhibited
9
mild symptoms, including dwarfism (Supplementary Fig. 2). Sclerotia were
10
produced at the base of stems and on the soil surface surrounding plants of all
11
inoculated crop species except for pea, adzuki bean, and maize plants (Fig. 6).
12
The five isolates were also re-isolated from symptomatic lesions in all tested
13
crops, which fulfilled Koch’s postulates. Control plants remained healthy and
14
no symptoms shown and pathogen isolation. Our pathogenicity tests indicate
15
that S. rolfsii isolates from mung bean will be a potential threat to other
16
important crops, especially legumes. It should be noted that our surveyed
17
mung bean fields represent peanut cultivation sites in China. Peanut is one of
18
the hosts of S. rolfsii and may have contributed to the accumulation of
19
pathogen inocula on mung bean (Fig. 6).
20
S. rolfsii is an important pathogenic fungus for legumes, which is the
21
causative agent of southern blight on several leguminous hosts, including
22
soybean (G. max) (Hartman et al., 1999), common bean (P. vulgaris) (Rusuku 10
1
et al., 1997), chickpea (C. arietinum) (Kokub et al., 2007), cowpea (V.
2
unguiculata) (Adandonon et al., 2004), lentil (Lens culinaris) (Shahid et al.,
3
1990), and peanut (A. hypogaea) (Bowen et al., 1992). In China, southern
4
blight caused by S. rolfsii has recently been detected on Canadian goldenrod
5
(Solidago canadensis) and Macleaya cordata (Tang et al., 2010; You et al.,
6
2015). However, there have been no reports of S. rolfsii on mung bean. To the
7
best of our knowledge, this is the first study of S. rolfsii causing southern blight
8
on mung bean in China. Our findings indicate that S. rolfsii may be a new
9
threat to mung bean and other crops, which should be carefully monitored in
10
China.
11 12
Acknowledgments
13
This study was supported by the Modern Agro-industry Technology Research
14
System (CARS-09), the Crop Germplasm Conservation and Utilization
15
Program (2019NWB036-12), the National Infrastructure for Crop Germplasm
16
Resources (NICGR2019-008), and the Scientific Innovation Program of the
17
Chinese Academy of Agricultural Sciences.
18
11
1
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16 17
16
1 2
Table 1. Details of six primer pairs used to detect Sclerotium rolfsii and clone fragments for subsequent sequence analyses. Primer name SCR-F/SCR-R SRITSF/SRITSR
Primer sequence (5′—3′) CGTAGGTGAACCTGCGGA / CATACAAGCTAGAATCCC TACACCTGTGAACCAACTG / CATACAAGCTAGAATCCC
Denature temperature
PCR product size
Target region gene
54℃
540bp
ITS
Jeeva et al. 2010
50℃
500bp
ITS
Pravi et al. 2014
D1/D2 domain LSU
or
SRLSUF/SRLSUR
AGTGTTTTCTGTGCTGGG / ACCTTCCTCTGGCCTTTTTC
62℃
360bp
ITS1/ITS4
TCCGTAGGTGAACCTGCGG / TCCTCCGCTTATTGATATGC
55℃
700bp
ITS
EF1-983F/EF1-2218R
GCYCCYGGHCAYCGTGAYTTYAT /ATGACACCRACRGCRACRGTYTG
60℃
1200bp
EF1α
fRPB2-5F/bRPB2-7R2
GAYGAYMGWGATCAYTTYGG /ACYTGRTTRTGRTCNGGRAANGG
62℃
1100bp
5-7 domain of RPB2
3 4
17
of
Reference
Pravi et al. 2015 White et al. 1990 Rehner and Buckley 2005; Matheny et al. 2007 Liu et al. 1999; Matheny et al. 2007
1
Figure Legends
2 3
Fig. 1. Southern blight symptoms caused by Sclerotium rolfsii on field-grown mung
4
bean plants. A, Overview of the occurrence of southern blight on mung bean plants.
5
The foliage of infected plants turned yellow. B, White mycelial initials near the soil line
6
of infected plants. C and D, Characteristic southern blight symptoms with abundant
7
white mycelial growth on basal stems. e, White and brown sclerotia on the surface of
8
basal stems.
9 10
Fig. 2. Morphology of Sclerotium rolfsii isolates from diseased mung bean plants. A,
11
Two-day-old colony with white and fluffy mycelia with even margins and a halo formed
12
by calcium oxalate crystals. B, A 4-week-old colony produced prismatic, tetragonal, or
13
irregular shaped calcium oxalate crystals. C, Tan to brown sclerotia were produced on
14
potato dextrose agar. D, Mycelia produced clamp connections, and multinucleate
15
cells were observed in the stained mycelia (scale bar: 10 µm).
16 17
Fig. 3. Polymerase chain reaction amplification patterns of five Sclerotium rolfsii
18
isolates. A, Amplification products for five S. rolfsii isolates using the SCR-F/R,
19
SRITSF/R, and SRLSUF/R three species-specific primer pairs. B, Amplification
20
products for five S. rolfsii isolates using three primer pairs for the rDNA ITS, EF1α,
21
and RPB2 sequences, which were used for sequencing by cloning. (M: marker ladder;
22
1: MSB1; 2: MSB2; 3: MSB3; 4: MSB4; 5: MSB5)
23 24
Fig. 4. Comparison of the sequences of 10 EF1α clone fragments from Sclerotium
25
rolfsii
isolate
MSB1
amplified
by
polymerase 18
chain
reaction
using
the
1
EF1-983F/EF1-2218R primer pair. The comparison revealed two distinct sequence
2
types, with three type 1 and seven type 2 sequences, respectively.
3 4
Fig. 5. Comparison of the sequences of 10 RPB2 clone fragments from Sclerotium
5
rolfsii
6
fRPB2-5F/bRPB2-7R2 primer pair. The sequences spanned domains 5 to 7 of RPB2
7
gene. The comparison revealed two distinct sequence types, with four type 1 and
8
six type 2 sequences.
isolate
MSB1
amplified
by
polymerase
chain
reaction
using
the
9 10
Fig. 6. Pathogenicity tests on mung bean (A) and peanut (B) seedlings inoculated
11
with Sclerotium rolfsii isolate MSB1 using a mycelia root-drenching procedure.
12
Inoculated mung bean and peanut plants wilted and died. Brown to dark brown
13
sclerotia were produced on the basal stems and on the soil surface surrounding
14
infected plants.
19
Fig. 1. Southern blight symptoms caused by Sclerotium rolfsii on field-grown mung bean plants. A, Overview of the occurrence of southern blight on mung bean plants. The foliage of infected plants turned yellow. B, White mycelial initials near the soil line of infected plants. C and D, Characteristic southern blight symptoms with abundant white mycelial growth on basal stems. e, White and brown sclerotia on the surface of basal stems.
Fig. 2. Morphology of Sclerotium rolfsii isolates from diseased mung bean plants. A, Two-day-old colony with white and fluffy mycelia with even margins and a halo formed by calcium oxalate crystals. B, A 4-week-old colony produced prismatic, tetragonal, or irregular shaped calcium oxalate crystals. C, Tan to brown sclerotia were produced on potato dextrose agar. D, Mycelia produced clamp connections, and multinucleate cells were observed in the stained mycelia (scale bar: 10 µm).
Fig. 3. Polymerase chain reaction amplification patterns of five Sclerotium rolfsii isolates. A, Amplification products for five S. rolfsii isolates using the SCR-F/R, SRITSF/R, and SRLSUF/R three species-specific primer pairs. B, Amplification products for five S. rolfsii isolates using three primer pairs for the rDNA ITS, EF1α, and RPB2 sequences, which were used for sequencing by cloning. (M: marker ladder; 1: MSB1; 2: MSB2; 3: MSB3; 4: MSB4; 5: MSB5)
Fig. 4. Comparison of the sequences of 10 EF1α clone fragments from Sclerotium rolfsii isolate MSB1 amplified by polymerase chain reaction using the EF1-983F/EF1-2218R primer pair. The comparison revealed two distinct sequence types, with three type 1 and seven type 2 sequences, respectively.
Fig. 5. Comparison of the sequences of 10 RPB2 clone fragments from Sclerotium rolfsii isolate MSB1 amplified by polymerase chain reaction using the fRPB2-5F/bRPB2-7R2 primer pair. The sequences spanned domains 5 to 7 of RPB2 gene. The comparison revealed two distinct sequence types, with four type 1 and
six type 2 sequences.
Fig. 6. Pathogenicity tests on mung bean (A) and peanut (B) seedlings inoculated with Sclerotium rolfsii isolate MSB1 using a mycelia root-drenching procedure. Inoculated mung bean and peanut plants wilted and died. Brown to dark brown sclerotia were produced on the basal stems and on the soil surface surrounding infected plants.
Highlights This study is the first report of S. rolfsii causing southern blight on mung bean in China. Characterization of S. rolfsii was confirmed by morphological and molecular identification. This study proved S. rolfsii isolates were heterokaryotic and contain a heterozygous genome. Pathogenicity and host range tests indicated S. rolfsii had a wide host range.
Conflict of interest: All authors declare that they have no conflict of interest.