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SHORT COMMUNICATION Identification of Sugarcane White Leaf Phytoplasma in Fields and 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 Ó 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 Á Phytoplasma Á Intergenic spacer region Á SCWL Á 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 Pure TM 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 Á W.-F. Li Á Y.-K. Huang (&) Á R.-Y. Zhang Á H.-L. Shan Á Z.-M. Luo Á J. Yin Á 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

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Page 1: Identification of Sugarcane White Leaf Phytoplasma in Fields and Quaratine Sugarcane Samples in Yunnan Province, China

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

Page 2: Identification of Sugarcane White Leaf Phytoplasma in Fields and Quaratine Sugarcane Samples in Yunnan Province, China

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 –

86 Sugar Tech (Jan-Mar 2015) 17(1):85–88

123

Page 3: Identification of Sugarcane White Leaf Phytoplasma in Fields and Quaratine Sugarcane Samples in Yunnan Province, China

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

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Page 4: Identification of Sugarcane White Leaf Phytoplasma in Fields and Quaratine Sugarcane Samples in Yunnan Province, China

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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