identification of sugarcane white leaf phytoplasma in fields and quaratine sugarcane samples in...
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SHORT COMMUNICATION
Identification of Sugarcane White Leaf Phytoplasma in Fieldsand Quaratine Sugarcane Samples in Yunnan Province, China
Xiao-Yan Wang • Wen-Feng Li • Ying-Kun Huang • Rong-Yue Zhang •
Hong-Li Shan • Zhi-Ming Luo • Jiong Yin • Ke Shen
Received: 15 August 2014 / Accepted: 9 October 2014 / Published online: 22 October 2014
� Society for Sugar Research & Promotion 2014
Abstract Sugarcane white leaf (SCWL) phytoplasma
was identified from sugarcane varieties in fields of Lincang
and Baoshan, and imported sugarcane varieties from
Myanmar, Philippines and Thailand in quarantine glass-
house at Yunnan Province, China. The associated phy-
toplasmas were characterized as strains of 16SrXI group by
using MLOX/MLOY and P1/P2 primer pairs, 16Sr
sequence identity and phylogenetic relationships.
Keywords Sugarcane white leaf disease � Phytoplasma �Intergenic spacer region � SCWL � Phylogenetics
Sugarcane white leaf (SCWL) disease is one of the most
important sugarcane diseases of Asia and is caused by a
phytoplasma (Hanboonsong et al. 2006; Nakashima et al.
1994; Rao et al. 2005, 2012). SCWL was first observed in
Lumpang Province in the northern part of Thailand in 1954
(Marcone 2002). Currently, SCWL has been reported from
India, Sri Lanka, Bangladesh, Pakistan, China, Sudan, Laos
and Vietnam (Thein et al. 2012; Rao et al. 2012; Li et al.
2013), where it causes significant damage to sugarcane
crops. In Thailand, a 5–35 % incidence of SCWL has been
recorded, resulting in recurrent losses to sugarcane farmers
amounting to US$20 million every year (Hanboonsong
et al. 2006).
In 2011 and 2012, SCWL phytoplasmas suspected
symptoms were observed in some sugarcane genotypes
imported by the Sugarcane Research Institute, Yunnan
Academy of Agricultural Science (YSRI) from Myanmar,
the Philippines and Thailand. These genotypes were plan-
ted in the Kunming Sugarcane Quarantine Greenhouse at
YSRI. In 2013, SCWL phytoplasma was reported in sug-
arcane fields at Lincang, Yunnan Province, China (Li et al.
2013). Hence, in the present study we attempted to identify
the association of phytoplasmas with symptomatic white
leaf samples collected from natural sugarcane fields at
Lincang and Baoshan, Yunnan Province and quarantine
samples maintained in glasshouse to further study the
identity and diversity among the identified isolates.
In this study, 16S-23S intergenic spacer region (ISR)
sequences of 10 Yunnan SCWL samples collected from
sugarcane fields and 23 SCWL quarantine samples
imported from Myanmar, the Philippines and Thailand
were analyzed to determine the relative levels of homol-
ogy, and the genetic diversity among them (Table 1).
The Easy PureTM plant Genomic DNA Kit (TransGen
Biotech Co., Ltd., Beijing, China) was used to extract total
DNA from 0.2 g of leaf tissue from each sample. DNA
extracted from SCWL-infected sugarcane cultivar UT3
was used as a positive control (provided by the Yunnan
Key Laboratory of Sugarcane Genetic Improvement,
Kaiyuan, China), and healthy non-symptomatic sugarcane
plants were used as the negative control.
Two sets of primers, MLOX/MLOY and P1/P2, were
used for the first and second PCR amplifications in a nes-
ted-PCR reaction that targeted a 210 bp fragment of 16S-
23S ISR of SCWL phytoplasmas (Hanboonsong et al.
2006). The first round PCR (20 lL) reaction mixture
contained 1 lL of total DNA template, 2.5 lL
of10 9 PCR buffer, 1 lL of each primer (20 lmol/L),
X.-Y. Wang � W.-F. Li � Y.-K. Huang (&) � R.-Y. Zhang �H.-L. Shan � Z.-M. Luo � J. Yin � K. Shen
Sugarcane Research Institute, Yunnan Academy of Agricultural
Science, Yunnan Key Laboratory of Sugarcane Genetic
Improvement, Kaiyuan 661699, People’s Republic of China
e-mail: [email protected]
123
Sugar Tech (Jan-Mar 2015) 17(1):85–88
DOI 10.1007/s12355-014-0351-0
0.5 lL of MgCl2 (25 mmol/L), 1 lL of dNTP mixture
(10 mmol/L), 0.2 lL of Taq DNA polymerase (5 U/lL)
and 12.8 lL of ddH2O. The first round PCR reaction was
carried out for 25 cycles (denaturation at 94 �C for 1 min;
annealing at 55 �C for 1 min; extension at 72 �C for 1 min;
and final extension at 72 �C for 10 min) in a thermocycler.
Nested PCR with P1/P2 primers was performed for 35
cycles (denaturation at 94 �C for 1 min; annealing at 62 �C
for 1 min; extension at 72 �C for 1 min; and final extension
at 72 �C for 10 min) with 1 lL PCR product from the first
cycle (diluted 1:100 in sterile deionized water). PCR pro-
ducts were electrophoresed in a 2 % agarose gel containing
ethidium bromide in 1 9 TAE buffer and visualized with a
UV transilluminator.
The TIANgel Midi Purification kit (TIANGEN, Bei-
jing, China) was used to purify the nested PCR products,
which were ligated to the plasmid vector pMD18-T (Ta-
kara, Dalian, China). The recombinant plasmids were
introduced into Escherichia coli strain DH5a (Sangon,
Shanghai, China). Six positive clones from each sample
were selected and the inserts were sequenced at BGI
Sequencing Co. Ltd. (Beijing, China). The software
DNAMAN version 6.0 performed multiple alignments of
the16S-23S ISR sequences. For phylogenetic analysis,
other reference phytoplasma sequences retrieved from
GenBank were compared with SCWL phytoplasmal
sequences in the present study and the neighbor-joining
method in software MEGA version 4.0 was used to
construct the phylogeny (Tamura et al. 2007). Achole-
plasma laidlawii (JQ390342) was used as outgroup to root
the tree.
An approximately 210 bp nested-PCR product was
amplified from all the ten Yunnan SCWL samples col-
lected from natural sugarcane fields and the 23 foreign
SCWL samples collected from quarantine greenhouse of
YSRI and no amplification product was produced from the
negative control (Fig. 1). Two of the amplified nested PCR
products from the sugarcane varieties, viz. Yuetang 86–368
from Baoshan and ROC22 from Lincang and one imported
variety each from Myanmar (K98-282), Philippines
(PSR03-48) and Thailand (UT6) were purified, cloned and
sequenced (Table1).
The multiple sequence alignment showed that the 16S-
23S ISR sequence of the Lincang SCWL phytoplasma
strain (KF431837) shared 96.2 to 96.7 % sequence identity
with that of the Baoshan SCWL phytoplasma strain
(KC662509), and other SCWL phytoplasma strains repor-
ted from other countries(KC662511, KC662512,
KF431838, HQ917068, JF754446). Baoshan SCWL phy-
toplasma strain (KC662509) had sequence similarities of
100 % with SCWL phytoplasmas identified SCWL posi-
tive from quarantine samples (KC662511, KC662512 and
KF431838) and Thailand SCWL phytoplasma
strain(HQ917068); 99.5 % with Sri Lanka SCWL strain
(JF754446); and 96.2 % with Lincang SCWL phytoplasma
strain(KF431837).
Table 1 Tested sugarcane white leaf (SCWL) samples and their 16S-23S intergenic spacer region (ISR) sequences obtained in this study
No. Source Variety GenBank
accession No.
No. Source Variety GenBank
accession no.
BS1 Baoshan, Yunnan, China PY3120 – V6 Imported from Myanmar VN99-838 –
BS2 Baoshan, Yunnan, China Yuetang 86–368 KC662509 g9 Imported from Myanmar 09-02 –
BS3 Baoshan, Yunnan, China Yunzhe 89–161 – F3 Imported from Philippines PSR03-48 KC662512
BS4 Baoshan, Yunnan, China Yunzhe 03–194 – F7 Imported from Philippines PSR03-128 –
BS5 Baoshan, Yunnan, China Yuetang 86–368 – f10 Imported from Philippines PSR02-237 –
LC1 Lincang, Yunnan, China ROC25 – f1 Imported from Philippines PSR05-180 –
LC2 Lincang, Yunnan, China ROC25 – f2 Imported from Philippines PSR05-102 –
LC3 Lincang, Yunnan, China ROC22 KF431837 f3 Imported from Philippines PSR05-09 –
LC4 Lincang, Yunnan, China ROC22 – f4 Imported from Philippines PSR01-51 –
LC5 Lincang, Yunnan, China Yuetang 86–368 – f5 Imported from Philippines PSR05-203 –
g2 Imported from Myanmar K98-282 KC662511 f6 Imported from Philippines PSR05-191 –
g3 Imported from Myanmar k95-89 – f7 Imported from Philippines PSR05-197 –
g4 Imported from Myanmar k91-2-056 – T2 Imported from Thailand KK3 –
g5 Imported from Myanmar k95-87 – T5 Imported from Thailand UT6 KF431838
V3 Imported from Myanmar VN85-1427 – T6 Imported from Thailand SP50 –
V4 Imported from Myanmar VN99-716 – T7 Imported from Thailand SP72 –
V5 Imported from Myanmar VN84-4137 –
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123
Fig. 1 Nested-PCR amplification of 16S-23S intergenic spacer
region (ISR) sequences from selected sugarcane white leaf (SCWL)
samples. M: DNA marker. Lanes 1–2 ROC25 collected from Lincang
sugarcane field. Lanes 3–4 ROC22 collected from Lincang sugarcane
field. Lane 5 Yuetang 86–368 collected from Lincang sugarcane field.
Lanes 6–9 PY3120, Yuetang 86–368, Yunzhe 89–161, Yunzhe
03–194 collected from Baoshan sugarcane field respectively. Lane 10
Yuetang 86–368 collected from Baoshan sugarcane field. Lanes
11–12: K92-282 and VN85-1427 imported from Myanmar. Lanes
13–14 PSR03-48 and PSR 03-128 imported from the Philippines.
Lanes 13–16 KK3 and UT6 imported from Thailand. PC positive
control; NC negative control; CK blank control
KC662512 (the Philippines)HQ917068JF754446KF431837 (Lincang, Yunnan, China)KC662511 (Myanmar)KF431838 (Thailand)KC662509 (Baoshan, Yunnan, China)
AY139875 (16Sr V)AY139871(16Sr VIII)
AY139868 (16Sr II)AF028814 (16Sr IX)
EU168781 (16Sr X)
AF035361 (16Sr XII)AF025428 (16Sr XIII)
AF363066 (16Sr VI)L27010 (16Sr III)
AF105317 (16Sr VII)
AF421542 (16Sr I)EU168770 (16Sr IV)
AF100412 (16Sr XIV)
JQ390342 (A. laidlawii)
8499
97
71
66
65
58
36
15
23
29
41
96
0.02
16Sr XI
Fig. 2 Phylogenetic tree showing relationships between 16S-23S
intergenic spacer region (ISR) sequences of sugarcane white leaf
(SCWL) phytoplasma obtained in the present study with those of
other representative phytoplasma 16S-23S ISR sequences
downloaded from GenBank. The sequence from A. laidlawii was
used as the outgroup. The tree was constructed by the neighbor-
joining method as implemented in MEGA version 4.0; numbers at the
branch nodes are bootstrap percentages (1,000 replicates)
Sugar Tech (Jan-Mar 2015) 17(1):85–88 87
123
A phylogenetic tree was constructed using the16S-23S
ISR sequences of SCWL strains and other typical phy-
toplasmas of the 16Sr group. The phylogenetic analyses of
the identified SCWL phytoplasma isolates in the present
study revealed that all the SCWL isolates clustered with
members of 16SrXI group (Fig. 2). Our results confirmed
that the SCWL phytoplasma isolates identified from
Yunnan agricultural fields and samples from imported
varieties from other countries in Yunnan were sequence
similarity of over 96 % and belonged to 16Sr XI group of
phytoplasmas.
The identification of SCWL phytoplasma at two places
in Yunnan Province of China and its similarity with the
phytoplasmas identified from imported sugarcane varieties
from Myanmar, Philippines and Thailand poses a serious
threat for spreading SCWL phytoplasma to other parts of
Yunnan and other Provinces of China. Hence an immediate
concerned is required to develop infrastructure and facility
to screen the imported sugarcane varieties before release
under field condition in China. The possible natural sources
for secondary spread of SCWL in China also needs
immediate attention.
Acknowledgments This work was supported by Grants from the
Earmarked Fund for China Agriculture Research System (CARS-20-
2-2) and the Earmarked Fund for Yunnan Province Agriculture
Research System.
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