Sequence homogeneity of the ψ ser A- trm U- tuf B sec E- nus G- rp lKAJL- rpo B gene cluster and...

BACTERIAL AND PHYTOPLASMA DISEASES

Sequence homogeneity of the wserA-trmU-tufB-secE-nusG-rplKAJL-rpoB gene cluster and the flanking regionsof ‘Candidatus Liberibacter asiaticus’ isolates aroundOkinawa Main Island in Japan

Noriko Furuya • Keiichiro Matsukura •

Kenta Tomimura • Mitsuru Okuda •

Shin-ichi Miyata • Toru Iwanami

Received: 25 June 2009 / Accepted: 24 December 2009 / Published online: 2 March 2010

� The Phytopathological Society of Japan and Springer 2010

Abstract In Japan and Southeast Asia, ‘Candidatus

Liberibacter asiaticus’ (Las) is the dominant causal agent

of citrus greening (huanglongbing) disease. Using PCR

techniques, we determined the 11168-nucleotide sequence

of the wserA-trmU-tufB-secE-nusG-rplKAJL-rpoB gene

cluster and the flanking regions for 51 Japanese, four

Taiwanese, four Indonesian, and three Vietnamese isolates

of Las. The sequence is identical in 62 isolates collected

from Japan, Taiwan, Indonesia, and Vietnam, except for

nucleotide substitutions at 11 positions. Some Las isolates

from Sakishima Islands near Taiwan had unique nucleotide

mutations, but all Las isolates around Okinawa Main Island

were homologous. On the basis of the pattern of single

nucleotide polymorphisms (SNPs) of the 11 nucleotide

substitutions, the 62 Las isolates from Japan, Taiwan,

Indonesia, and Vietnam could be divided into 12 pattern

groups, and the 51 Japanese isolates consisted of six pat-

terns. The results suggested that one unique genetic group

is dominant around Okinawa Main Island, whereas several

different are commonly distributed around islands near

Taiwan.

Keywords Citrus greening � Genetic diversity �Huanglongbing � Japan � Okinawa � Kagoshima

Introduction

In Asia as well as Africa and America, citrus greening

(huanglongbing) is a destructive disease affecting citrus

trees, particularly mandarin and sweet orange. This disease

was first noted in southern China at the end of the nine-

teenth century and known as yellow shoot disease in this

region (Zhao 1981). By the 1920s, similar diseases were

recorded in Taiwan (likubin or drooping disease) (Otake

1990), Philippines (mottle-leaf disease) (Lee 1921), and

India (citrus die-back) (Capoor 1963). In the late 1920s, a

similar disease was observed in South Africa (van der

Merwe and Andersen 1937). In Indonesia, the disease was

first recorded in the 1940s and was described as vein

phloem degeneration (Tirtawidjaja et al. 1965). In Viet-

nam, the high rate of infection was a serious problem,

especially in the southern part of the country (Gottwald

et al. 2007; Ichinose et al. 2004).

The causal agents, which are phloem-limited, noncul-

turable, Gram-negative bacteria, belong to the genus

‘Candidatus Liberibacter’. Thus far, three species have

been identified. ‘Candidatus Liberibacter africanus’ (Laf) is

mainly found in African countries, and ‘Candidatus Libe-

ribacter americanus’ (Lam) is present in Brazil (Bove

2006). In Asian countries as well as in Sao Paulo, Brazil,

and Florida (USA), ‘Candidatus Liberibacter asiaticus’

(Las) is widespread and causes serious economic damage in

many citrus-producing areas (Bove 2006; Da Graca 1991).

The nucleotide sequence data reported in this paper will appear in the

DDBJ, EMBL, and GenBank nucleotide sequence databases with the

following accession numbers AB490292 and AB490682–AB490691.

N. Furuya � S. Miyata � T. Iwanami (&)

National Institute of Fruit Tree Science, Fujimoto, Tsukuba,

Ibaraki 305-8605, Japan

e-mail: [email protected]

K. Matsukura � M. Okuda

National Agricultural Research Center for Kyushu Okinawa

Region, Koshi, Kumamoto 861-1192, Japan

K. Tomimura

Kuchinotsu Citrus Research Station,

National Institute of Fruit Tree Science, Kuchinotsu,

Minami-shimabara, Nagasaki 859-2501, Japan

123

J Gen Plant Pathol (2010) 76:122–131

DOI 10.1007/s10327-010-0223-8

In Japan, citrus greening was first identified in Iriomote

Island, Okinawa, in 1988 (Miyakawa and Tsuno 1989), and

the disease is apparently moving northward through the

Ryukyu Islands. Thus far, the disease has been confirmed

in more than 15 islands of the Ryukyu Islands; the current

northern limit is Kikai Island (Shinohara et al. 2006). In

Kikai Island, the local government is vigorously imple-

menting an emergency eradication program to prevent the

spread of this disease and its invasion into the Japanese

mainland. Currently, only Las occurs in Japan. The bac-

terium is transmitted by the citrus psyllid Diaphorina citri

Kuwayama, which is common in the Ryukyu Islands

(Miyatake 1965); however, information about the disper-

sion of the bacterium between fields and between islands is

too sparse to devise an effective countermeasure.

Studies of greening have been impeded because Las

isolates have never been cultured on artificial nutrient

media and their titers in citrus are very low (Ghosh et al.

1978). Recently, Davis et al. (2008) and Sechler et al.

(2009) independently succeeded in cultivating Las by ‘‘co-

cultivation’’ with actinobacteria or on Liber A agar med-

ium that included citrus vein extract. Although Duan et al.

(2009) determined the complete genome sequence of Las,

obtaining genomic information about the pathogen remains

challenging because pure pathogenic DNA is lacking.

In early attempts, random cloning from partially purified

DNA was successfully used to obtain a few specific frag-

ments from the bacterium (Villechanoux et al. 1992).

These fragments comprised the rplKAJL-rpoBC gene

cluster and a partial bacteriophage-type DNA polymerase

gene. Subsequently, using random primers to screen DNA

from infected periwinkle plants and healthy plants,

Hocquellet et al. (1999) identified another fragment that

included nusG, pgm, omp, and a hypothetical protein gene.

In the early stages of Las sequence analysis, the rplKAJL-

rpoBC gene cluster, 16S rRNA gene, and 16S/23S rRNA

intergenic space did not reveal any differences among

various Las isolates (Garnier et al. 2000; Jagoueix et al.

1994, 1997; Planet et al. 1995; Subandiyah et al. 2000;

Villechanoux et al. 1993); however, Okuda et al. (2005)

and Bastianel et al. (2005) showed that the Las species may

have several variants, based on the sequence in the

rplKAJL-rpoBC gene cluster region and omp gene,

respectively.

After the sequence of the rplKAJL-rpoBC gene cluster

was reported (Villechanoux et al. 1992), identification of

the fragment was extended to the tufB-secE-nusG-

rplKAJL-rpoBC gene cluster and the flanking regions,

using thermal asymmetric interlace polymerase chain

reaction (TAIL-PCR, Liu and Whittier 1995; Okuda et al.

2005). In addition, recently, by the genomic walking

technique, the sequence of this gene cluster and the

flanking regions was determined to be 2920 bp at the 50 end

and 2329 bp at the 30 end (Lin et al. 2008).

The purpose of this study was to further characterize the

tufB-secE-nusG-rplKAJL-rpoBC gene cluster and the

flanking regions by identifying the adjacent sequences

using TAIL-PCR and to reveal the genetic diversity of Las,

especially in Japan. The information obtained in this study

will provide a better understanding of the epidemiology

and population structure of Las.

Materials and methods

Infected plant materials

Leaves infected with Las were collected from 62 citrus

trees in Taiwan, Indonesia and Vietnam as well as in 13 of

the Ryukyu Islands, which are located in the southernmost

region of Japan (Fig. 1; Tables 1, 2). In the case of isolates

OK-901, Miyako-13, and KIN-1, infected source plants

were introduced into our greenhouses, in compliance with

plant quarantine regulations, and total DNA was extracted

from the infected rough lemon leaves. For the other iso-

lates, leaves were collected from citrus gardens of Kago-

shima Prefecture, Okinawa Prefecture, Taiwan, Vietnam,

and Indonesia. Most Japanese isolates were taken from

unidentified local cultivars including lines of Shiikuwasha

(Citrus depressa). In each collaborator’s laboratory, total

DNA was extracted from the leaves, and the extracted

DNA was sent to our laboratory in the form of pellets or

solutions. Total DNA was extracted from 0.1 g of midribs

from Japanese leaf samples, using a DNeasy Plant Mini kit

(Qiagen, Hilden, Germany), and the DNA was suspended

in 100–1000 ll TE buffer (10 mM Tris–HCl, 1 mM

EDTA, pH 8.0). After PCR screening using a Las-specific

primer set of MHO353 (50-GTG TCT CTG ATG GTC

CGT TTG CTT CTT TTA-30) and MHO354 (50-GAA CCT

TCC ACC ATA CGC ATA GCC CCT TCA-30), as

reported by Hoy et al. (2001), all Las-positive samples

were subjected to sequencing analysis.

TAIL-PCR of the regions adjacent to the tufB-secE-

nusG-rplKAJL-rpoB gene cluster and the flanking

regions

TAIL-PCR (Liu and Whittier 1995) was performed to

determine the nucleotide sequences of the regions adjacent

to the tufB-secE-nusG-rplKAJL-rpoBC gene cluster and

the flanking regions, essentially as previously reported by

Okuda et al. (2005), using the new primers shown in Fig. 2

and Table 3. The typical Japanese isolate KIN1 was used

for the TAIL-PCR experiments.

J Gen Plant Pathol (2010) 76:122–131 123

123

PCR amplification and DNA sequencing

After TAIL-PCR, a 10-kbp fragment of the gene cluster and

the flanking regions was obtained. The sequence had high

homology with a slightly longer sequence reported by Lin

et al. (2008), and we concluded that our newly obtained

fragment of the trmU-tufB-secE-nusG-rplKAJL-rpoB gene

cluster and the flanking regions was a part of the fragment

previously reported by Lin et al. (2008). Therefore, in the

subsequent analysis, we used both our newly obtained

sequence and the sequence by Lin et al. (2008) as references

to design the PCR primer pairs FW-902/RV78 and

FW8990/RV10370 (Table 4) to further extend our sequence

at the 50 and 30 termini. The total nucleotide sequence

obtained was 11 kbp. To amplify each approximately 2-kbp

fragment (amplicons Z and A–F; Fig. 3) of the 11-kbp gene

cluster and the flanking regions, seven sets of specific

primers were designed (Table 4). PCR was conducted using

20-ll reaction mixtures (10 mM Tris–HCl (pH 8.3), 50 mM

KCl, 1.5 mM MgCl2), 0.2 mM of each dNTP, 0.5 units of

the Hot Start version of Ex Taq polymerase (TaKaRa, Otsu,

Shiga, Japan), 1 ll of extracted DNA, and 0.2 lM of each

primer. The PCR protocol was as follows: 40 cycles of 94�C

for 1 min, 55�C for 1 min, and 72�C for 2 min after an

initial denaturation for 5 min in a thermal cycler (Gene

Amp PCR System 9600, Perkin Elmer, Waltham, MA,

USA). The amplified 2-kbp (approximately) products were

separated on a 1% agarose gel in Tris-boric acid-EDTA

(TBE) buffer and stained with ethidium bromide. Each

target band was cut from the gel, and the PCR products were

extracted using a QIAquick Gel Extraction kit (Qiagen).

The nucleotide sequence of the recovered PCR products

was determined by direct sequencing, using an Applied

Biosystems 3130xl Genetic Analyzer with a BigDye Ter-

minator v3.1 Cycle Sequencing Kit (Applied Biosystems,

Foster City, CA, USA) and sequence primers designed for

each 500 bp of the 11-kbp fragment of the gene cluster and

the flanking regions (Table 4). The sequence of each DNA

fragment was combined and compared using a SeqMan

Genome Assembler (DNASTAR, Madison, WI, USA).

Miyako Is.

Tarama Is.

Ishigaki Is. Iriomote Is.

Hateruma Is.

Kohama Is. Yonaguni Is. Okinawa Main Is.

Kikai Is.

Tokunoshima Is.

Iheya Is.

Yoron Is.

Irabu Is.

Fig. 1 Map of collection sites

of ‘Candidatus Liberibacter

asiaticus’ isolates in the Ryukyu

Islands and Satsunan Islands.

The Sakishima Islands are

located southwest of Ryukyu

Islands

124 J Gen Plant Pathol (2010) 76:122–131

123

Table 1 Details of ‘Candidatus Liberibacter asiaticus’ isolates collected in Japan used in this study

Collection island Location Year of collection Code Sample name and the synonyma

Iriomote Is. Iriomote, Taketomi-town, Okinawa 1988 Iw1 OK-901, OK901

Yonaguni Is. Yonaguni-town, Okinawa 2007 K11 Higawa-1

Ishigaki Is. Ishigaki-city, Okinawa 2005 Iw3 Ishigaki-1, Ishi1

Ishigaki-city, Okinawa 2007 Iw6 Ishigaki-16

Hirakubo, Ishigaki-city, Okinawa 2007 K5 Hirakubo-5

Hirano, Ishigaki-city, Okinawa 2007 K6 Hirano-4

Kawahara, Ishigaki-city, Okinawa 2007 K7 Kawahara-2

Hirae, Ishigaki-city, Okinawa 2007 K8 Hirae-1

Hateruma Is. Hateruma, Taketomi-town, Okinawa 2007 K9 Hateruma-1

Kohama Is. Kohama, Taketomi-town, Okinawa 2007 K10 Kohama-4

Tarama Is. Tarama-village, Okinawa 2007 K12 Tarama-12

Shiogawa, Tarama-village, Okinawa 2008 Mt3 MT-3




Miyako Is. Miyakojima-city, Okinawa 2007 Iw4 Miyako-13

Miyakojima-city, Okinawa 2007 K1 S-2ˆ

Miyakojima-city, Okinawa 2007 K3 H-3

Miyakojima-city, Okinawa 2007 K4 U-4

Irabu Is. Miyakojima-city, Okinawa 2007 K2 I-1

Okinawa Main Is. Kin-town, Okinawa 1994 Iw2 KIN-1, KIN1

Okinawa 2005 Iw5 I-4

Kin-town, Okinawa 2007 Ns1 KIN-3

Ishikawa, Uruma-city, Okinawa 2007 Ns2 Ishi-2

Nago-city, Okinawa 2007 K14 Nago-Nc-1

Higashi-village, Okinawa 2007 K19 HigashiA-3

Ogimi-village, Okinawa 2007 K20 OgimiA-3

Gushikawa, Uruma-city, Okinawa 2007 K21 Uruma-1-�

Tomigusuku-city, Okinawa 2007 K26 A-11

Itoman-city, Okinawa 2007 K27 C-3

Naha-city, Okinawa 2007 K28 A-3

Haebaru-town, Okinawa 2007 K29 Hae-5

Kochinda, Yaese-town, Okinawa 2007 K30 KO-7

Iheya Is. Iheya-village, Okinawa 2007 K13 Iheya-2

Yoron Is. Yoron-town, Kagosima 2002 H1 Yoron-57, Y02-57

Yoron-town, Kagosima 2002 H2 Yoron-83

Tokunoshima Is. Kinen, Isen-town, Kagosima 2006 Hm8 Toku-225

Kinen, Isen-town, Kagosima 2006 Hm9 Toku-228

Kinen, Isen-town, Kagosima 2006 Hm10 Toku-229

Nishimetegu, Isen-town, Kagosima 2006 Hm15 Toku-234

Higashimetegu, Isen-town, Kagosima 2006 Hm16 Toku-235

Higashimetegu, Isen-town, Kagosima 2006 Hm18 Toku-237

Saben, Isen-town, Kagosima 2006 Hm21 Toku-240

Saben, Isen-town, Kagosima 2006 Hm22 Toku-241

Kikai Is. Oasato, Kikai-town, Kagosima 2006 Hm1 Kikai-130

Oasato, Kikai-town, Kagosima 2006 Hm2 Kikai-145






a Tomimura et al. (2010)


123

Results

Extension of the tufB-secE-nusG-rplKAJL-rpoB gene

cluster and the flanking regions by TAIL-PCR

Utilizing the TAIL-PCR depicted in Fig. 2, we further

extended the sequencing of the tufB-secE-nusG-rplKAJL-

rpoB gene cluster and the flanking regions by 2018 bp at the

50 end and by 1892 bp at the 30 end. The combined sequence

of this region was 10055 bp. The sequence appears in the

DNA database of the DNA Data Bank of Japan (DDBJ)

under the accession number AB490292. When the DNA

database was searched for homologous sequences, we found

that the 50 region of our sequence was similar to trmU of

Escherichia coli (Fig. 2). Also, our sequence had the

highest overall homology with another Las fragment

(wserA-trmU-tufB-secE-nusG-rplKAJL-rpoB gene cluster

and the flanking regions) reported by Lin et al. (2008).

Sequence comparison of the wserA-trmU-tufB-secE-

nusG-rplKAJL-rpoB gene cluster and the flanking

regions

Based on a new report by Lin et al. (2008), we modified the

target size to approximately 11 kbp, and then performed

direct sequencing after PCR amplification with the primers

Table 2 Details of ‘Candidatus Liberibacter asiaticus’ isolates collected in Indonesia, Vietnam, and Taiwan used in this study

Country Collection island or region Location Year of collection Code Sample namea

Indonesia Java Island Ngaglik, Sleman, Yogyakarta 2003 IDN5 IDN5

Java Island Ngaglik, Sleman, Yogyakarta 2003 IDN6 IDN6

Java Island Kemili, Purworejo 2003 IDN7 IDN7

Timor Island Tesbatan, Kupang 2003 IDN17 IDN17

Vietnam Southland Vinh Long 2004 V2 V2

Mid-southland Khanh Hoa 2004 VN50 VN50

Northland Ha Giang 2004 V62 V62

Taiwan – Chiayi Unknown T1 TW1

– Hsinchu Unknown T2 TW2

– Chiayi-Hualien Unknown T3 TW3

– Chiayi-Hualien Unknown T5 TW5

a Tomimura et al. (2009)

secE nusG rplK rplA rplJ rplL rpoBtufB

(Okuda et al., 2005)

secE nusG rplK rplA rplJ rplL rpoBtufB

tufBRRV3

This study

6.1 kbp

1.7 kbp 1.5 kbp

trmU

tufBRRV2tufBRRV1

tufBupRRV1tufBupRRV2

tufBupRRV3

rpoBFW1rpoBFW2

rpoBFW3

Tail3

Tail2 Tail4

secE nusG rplK rplA rplJ rplL rpoBtufBtrmU

(Lin et al., 2008)

serA

2.2 kbp 2.9 kbp

Fig. 2 Deduced gene organization of ‘Candidatus Liberibacter

asiaticus’ in the vicinity of the tufB-secE-nusG-rplKAJL-rpoB gene

cluster and the flanking regions amplified by thermal asymmetric

interlaced (TAIL)-PCR. Primers (Tail2, tufBRRV1, tufBRRV2,

tufBRRV3, Tail3, tufBupRRV1, tufBupRRV2, tufBupRRV3) were

used to specifically amplify the upstream region. Primers Tail4,

rpoBFW1, rpoBFW2, and rpoBFW3 were used to amplify the

downstream region


123

listed in Table 4. The sequence of this gene cluster and the

flanking regions was compared among 51 isolates collected

from Japan and 11 isolates collected from Indonesia,

Vietnam, and Taiwan.

The sequence of this gene cluster and the flanking

regions was 11168 bp, and it was highly conserved in all

isolates from Japan, Indonesia, Vietnam, and Taiwan, in

accordance with the report by Okuda et al. (2005). How-

ever, there were 11 nucleotide substitutions at nucleotide

positions 958, 1082, 1268, 4001, 4149, 4677, 5176, 7883,

7929, 8978, and 9335 in the 62 isolates used in this study

(Table 5, Fig. 3). Most of these 11 nucleotide substitutions

were in the second half of wserA, around secE-nusG, and

at the 50 end of rpoB. Fewer nucleotide substitutions were

observed in the noncoding regions.

In the Japanese isolates collected from Ishigaki Island

(Iw6), Miyako Island (K3), Irabu Island (K2), and Hate-

ruma Island (K9) near Taiwan, unique nucleotide substi-

tutions in each isolate were confirmed at nucleotide

positions 958 (G to T), 9335 (C to T), 1268 (G to A), and

4149 (T to A), respectively (Table 5).

These nucleotide substitutions were single nucleotide

polymorphisms (SNPs). Based on the SNPs pattern of the

11 nucleotide substitutions, our 62 isolates were divided

into 12 patterns (No. 1–12), as shown in Table 5. The four

Indonesian isolates had only one pattern (No. 9), while

various patterns were observed in the 51 Japanese isolates,

four Taiwanese isolates, and three Vietnamese isolates

(Table 5).

The 51 Japanese isolates were highly homologous in the

wserA-trmU-tufB-secE-nusG-rplKAJL-rpoB gene cluster

and the flanking regions; however, based on two nucleotide

substitutions at nucleotide positions 1082 and 8978, these

isolates were divided into two groups: the 1082A/8978G

group containing isolates from Miyako Island (Iw4 of the

pattern No. 5 group) and the 1082G/8978A group con-

taining isolates from Iriomote Island (Iw1 of the pattern

No. 1 group) (Table 5). Although the 1082G/8978A group

comprised isolates that were from 11 islands from the

Ryukyu Islands, the 1082A/8978G group included only

isolates from Sakishima Islands (Miyako, Kohama, Yon-

aguni, Irabu, and Hateruma Islands) near Taiwan. In

addition, three Taiwanese isolates (T1, T2 and T5), three

Vietnamese isolates (V2, VN50, and V62), and four

Indonesian isolates (IDN5, IDN6, IDN7, IDN17) belonged

to the 1082A/8978G group, but, one Taiwanese isolate (T3)

belonged to the 1082G/8978A group.

Discussion

The wserA-trmU-tufB-secE-nusG-rplKAJL-rpoB gene

cluster and the flanking regions in the bacterium that causes

Asian citrus greening disease, ‘Candidatus Liberibacter

asiaticus’, was successfully characterized using 62 isolates

from Japan, Taiwan, Vietnam, and Indonesia, and the

cluster was then compared to that in a Chinese isolate (Lin

et al. 2008). Comparison of the nucleotide sequence

revealed changes at 11 nucleotide positions. A previous

study reported that the rplKAJL-rpoBC gene cluster region

in Japanese and Indonesian isolates differed at three

nucleotide positions (Okuda et al. 2005). This study indi-

cated that there are more nucleotide substitutions around

this gene cluster and the flanking regions. However, this

wserA-trmU-tufB-secE-nusG-rplKAJL-rpoB gene cluster

and the flanking regions have only 11 nucleotide substi-

tutions in the 11-kbp sequence, and we confirmed that this

long sequence was conserved among various isolates from

Asian countries. This conserved sequence might be useful

for developing a molecular diagnosis tool to detect Las

isolates in the future.

Based on the SNPs pattern of the 11 nucleotide substi-

tutions observed in the wserA-trmU-tufB-secE-nusG-

rplKAJL-rpoB gene cluster and the flanking regions in the

62 isolates, these isolates were classified into 12 pattern

groups. Interestingly, the three Vietnamese isolates used

for analysis showed three patterns. Based on the genetic

diversity of phage-type DNA polymerase, these three

Vietnamese isolates belonged to three different genetic

groups (Tomimura et al. 2009). These results suggest that

in Vietnam, Las would be very diverse. Further molecular

study of Las isolates from Vietnam would be very

interesting.

The 51 Japanese isolates were divided into six pattern

groups (No. 1, 2, 4, 5, 6, and 7) based on the SNPs patterns.

Table 3 The primers used for TAIL-PCR in this study

Primer name Primer sequence (50-30)

For Tail2

tufBRV1 CATAGCTAACATGCGCAGTCGC

tufBRV2 TATAGCACCATCAGCCTGCG

tufBRV3 CTTAGGACCATCCTCTGCAG

Tail2 GTNCGASWCANAWGTT

For Tail3

tufBupRV1 AAAGAGGCTCACCCATCGCC

tufBupRV2 CCAAGACCTCGTCTTTGTCC

tufBupRV3 ATCCCATTGTGACGCCCTAG

Tail3 WGTGNAGWANCANAGA

For Tail4

rpoBFW1 CGACTGTTGGTCGTGTCAAG

rpoBFW2 GCGTTTGAATCTGGATACGCC

rpoBFW3 CGTTCTGTTGGGGAGATGTTG

Tail4 GTCGASWGANAWGAAN


123

Of these six patterns, five (No. 1, 2, 4, 6, and 7) were

unique to Japan, and only one (No. 5) was present in a

Vietnamese isolate (V2). This No. 5 pattern group included

one Vietnamese and five Japanese isolates, but the Tai-

wanese isolate (T2) that belonged to the pattern No. 8

group apparently is also closely related to the No. 5 pattern

Table 4 The primers used for PCR and sequencing in this study

Primer name Primer sequence (50–30) PCR primerb

rpl10055-FW-902 – GTCATGAATACTCCTTTCGGC Z-FW

rpl10055-FW38a 38–58 GAACTGATAGTCAAGACAGTG A-FW

rpl10055-FW501 501–520 TCCAAAAGATACGCGGGTCG –

rpl10055-FW981 981–1002 GTTCGCTACTACTCAGCAACAG –

rpl10055-FW1514 1514–1535 GTGAAGTTGGCGTTGCATCTGG –

rpl10055-FW1991 1991–2011 GGAGAGTCTTGGTCTCAGTAC B-FW

rpl10055-FW2461 2461–2479 GTGGTTCTGCTCTTTGTGC –

rpl10055-FW3001 3001–3023 AAGCGGTAATGCCTGGTGATAGG –

rpl10055-FW3531 3531–3552 CAGTCAATAGGATGGCTGATGC –

rpl10055-FW3988 3988–4008 GTGTCTCTGATGGTCCGTTTG C-FW

rpl10055-FW4501 4501–4519 CTGGTAAGGAGTCTTGCGG –

rpl10055-FW5002 5002–5021 GCTACACCGGATATGATGCC –

rpl10055-FW5611 5611–5627 GGGAATTAGTGTTGCGC –

rpl10055-FW5978 5978–5999 GGTACGCCACAGACTCAAGTTG D-FW

rpl10055-FW6448 6448–6468 GGTGCAACCGTAGAATTACGC –

rpl10055-FW6980 6980–7000 GGGCGATCTTCCTCTTATGAC –

rpl10055-FW7521 7521–7541 GTCAATGGCGAAACTGGTGAG –

rpl10055-FW8016 8016–8036 GGGTTATTGCGTATGGAGCG E-FW

rpl10055-FW8524 8524–8541 GTCGTTGTGCAGGAGAAG –

rpl10055-FW8990 8990–9009 AGGAGATCTGGCTCTTGGTC F-FW

rpl10055-FW9561 9561–9580 CAGAATGCAGTATCTGGCCC –

rpl10055-RV78 60–78 TGTTCCCGCTACCGAATAC Z-RV

rpl10055-RV596 573–596 CCAATTACATCATATCCATCACGC –

rpl10055-RV1073 1051–1073 CCCATCTCTCTAGCCAAATCTC –

rpl10055-RV1620 1601–1620 ATCCGAACGCTTAGAACCAC –

rpl10055-RV2127 2106–2127 CCCGTAATTTCTCTTCTGGAGC A-RV

rpl10055-RV2620 2600–2620 CCTTCAATCCCACAAGAACCC –

rpl10055-RV3118 3101–3118 AAACCAGCCCCTACCGTC –

rpl10055-RV3622 3604–3622 ACTATATACCAGCGAGGCG –

rpl10055-RV4101 4083–4101 ACTCTACTGGTGTGACACG B-RV

rpl10055-RV4620 4602–4620 CGAACCTTCCACCATACGC –

rpl10055-RV5135 5116–5135 GCACCACTCTTAGACTCTCG –

rpl10055-RV5668 5648–5668 TCCACCAGCTTCCCGCATCTT –

rpl10055-RV6199 6181–6199 CGCAGAAGCAGAAACACCC C-RV

rpl10055-RV6614 6594–6614 GAGACCATTGAACACAACGCC –

rpl10055-RV7112 7092–7112 GCCCGATAAACTAGCTCTTCC –

rpl10055-RV7613 7596–7613 ATCCCTGATCTCACTGTG –

rpl10055-RV8120 8101–8120 AGCAGACACAACAGGTTTGG D-RV

rpl10055-RV8623 8602–8623 GGAATGAGAGAAGCCGCTATTG –

rpl10055-RV9174 9153–9174 CACGAGTGATTTCTTCTGGTCC –

rpl10055-RV9659 9639–9659 AGCGAACTGCCACCATTGAG E-RV

rpl10055-RV10370 – CTTCAACATGTAGATATACCCAAC F-RV

a The numbers correspond to the nucleotide positions in the sequence with the accession number AB490292 in the DNA sequence database

DDBJb The prefixes A–F, Z indicate amplicons depicted in Fig. 3


123

Ta

ble

5V

aria

bil

ity

of

SN

Ps

pat

tern

inth

ew

serA

-trm

U-t

ufB

-sec

E-n

usG

-rplK

AJL

-rp

oB

gen

ecl

ust

eran

dth

efl

ank

ing

reg

ion

Pat

tern

no

.C

oll

ecti

on

cou

ntr

yS

amp

len

ame

Nu

cleo

tid

ep

osi

tio

na

95

81

08

21

26

84

00

14

14

94

67

75

17

67

88

37

92

98

97

89

33

5

1Ja

pan

(Iri

om

ote

,Is

hig

aki,

Tar

ama,

Miy

ako

,O

kin

awa

Mai

n,

Ihey

a,Y

oro

n,

Kik

ai,

To

ku

no

shim

aIs

lan

ds)

Iw1

,Iw

2,Iw

3,Iw

5,N

s1,N

s2,H

1,H

2,

K5

,K

6,

K7

,K

8,

K1

2,

K1

3,

K1

4,

K1

9,

K2

0,

K2

1,

K2

6,

K2

7,

K2

8,

K2

9,

K3

0,

Hm

1,

Hm

2,

Hm

3,

Hm

4,

Hm

5,

Hm

6,

Hm

7,

Hm

8,

Hm

9,

Hm

10

,H

m1

5,

Hm

16

,H

m1

8,

Hm

21

,

Hm

22

,M

t3,

Mt1

0,

Mt1

1,

Mt1

2

GG

GG

TG

CC

TA

C

2Ja

pan

(Ish

igak

iIs

lan

d)

Iw6

T�

��

��

��

��

�3

Tai

wan

T3

��

�A

��

��

��

�4

Jap

an(M

iyak

oIs

lan

d)

K3

��

��

��

��

��

T

5Ja

pan

(Miy

ako

,K

oh

ama,

Yo

nag

un

i

Isla

nd

s)an

dV

ietn

am

Iw4

,K

1,

K4

,K

10

,K

11

,V

2�

A�

��

��

��

G�

6Ja

pan

(Ira

bu

Isla

nd

)K

2�

AA

��

��

��

G�

7Ja

pan

(Hat

eru

ma

Isla

nd

)K

9�

A�

�A

��

��

G�

8T

aiw

anT

2�

A�

��

�T

��

G�

9In

do

nes

iaan

dV

ietn

amID

N5

,ID

N6

,ID

N7

,ID

N1

7,

V6

2�

A�

��

T�

T�

G�

10

Vie

tnam

VN

50

�A

��

��

�T

�G

�1

1T

aiw

anT

1�

A�

��

��

TC

G�

12

Tai

wan

T5

�A

��

��

��

CG

�a

Nu

mb

ers

ver

tica

lly

po

siti

on

edab

ov

eth

ese

qu

ence

sal

ign

men

tin

dic

ate

po

siti

on

so

fn

ucl

eoti

des

inth

eL

asg

ene

clu

ster

(acc

essi

on

nu

mb

erA

B4

90

68

2–A

B4

90

69

1in

the

DN

Ase

qu

ence

dat

abas

eD

DB

J).

Bas

esth

atar

ed

iffe

ren

tfr

om

the

tho

seo

fp

atte

rnN

o.

1ar

ein

dic

ated

,w

her

eas

bas

esid

enti

cal

toth

ose

of

pat

tern

No

.1

are

mar

ked

wit

hd

ots


123

group because only one nucleotide differs between the

No. 5 and 8 pattern groups. The southern part of Vietnam,

where Vietnamese isolate V2 was collected, is 2300 km

away from the three islands of Japan, where the pattern

No. 5 isolates were collected. However, Okinawa has his-

torically had close economic and cultural ties with Viet-

nam, as well as with Taiwan and China through sea trade.

The Las isolates belonging to the pattern No. 5 group and

similar pattern groups might be widely distributed in Asian

countries bordering the East China Sea. The origin of

isolates that are genetically unique to Japan, especially

those in the pattern No. 1 group, is still unknown. It is

possible that we might find similar isolates in Taiwan if

more isolates are collected and studied.

Another study showed that the phage-type DNA poly-

merase gene region contains several variable regions

(Tomimura et al. 2009). However, this region was not

successfully amplified from many isolates used in this

study. We assume that some Japanese isolates lack these

variable phage-type DNA polymerase gene regions. Fur-

ther analyses are underway, and the results will be pub-

lished elsewhere.

In conclusion, our nucleotide sequence analysis revealed

that Las is genetically homogenous in the northern part of

the Ryukyu Islands, including Okinawa Main Island,

whereas Las varies in the southernmost islands in Japan.

The difference in the population structure of Las between

the north and south in Ryukyu Islands would support the

field observation that Las in the Ryukyu Islands has dis-

persed northward throughout the Ryukyu Islands.

Acknowledgments We thank Ms. Akiko Hamashima (Kagoshima

Prefectural Institute for Agricultural Development) and Drs. Shinji

Kawano (Okinawa Prefectural Agricultural Research Center), Kuni-

masa Kawabe (Japan International Research Center for Agricultural

Sciences), Hong-Ji Su (Taiwan University, Taiwan), and Siti

Subandiyah (Gadjah Mada University, Indonesia) for providing DNA

samples of Las.

References

Bastianel C, Garnier-Semancik M, Renaudin J, Bove JM, Eveillard S

(2005) Diversity of ‘‘Candidatus Liberibacter asiaticus’’, based

on the omp gene sequence. Appl Environ Microbiol 71:6473–

6478

Bove JM (2006) Huanglongbing: a destructive, newly-emerging,

century-old disease of citrus. J Plant Pathol 88:7–37

Capoor SP (1963) Decline of citrus trees in India. Bull Natl Inst Sci

India 24:48–64

Da Graca JV (1991) Citrus greening disease. Annu Rev Phytopathol

29:109–136

Davis MJ, Mondal SN, Chen H, Rogers ME, Brlansky RH (2008) Co-

cultivation of ‘Candidatus Liberibacter asiaticus’ with actino-

bacteria from citrus with huanglongbing. Plant Dis 92:1547–

1550

Duan Y, Zhou L, Hall DG, Li W, Doddapaneni H, Lin H, Liu L,

Vahling CM, Gabriel DW, Williams KP, Dickerman A, Sun Y,

Gottwald T (2009) Complete genome sequence of citrus

huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’

obtained through metagenomics. Mol Plant Microbe Interact

22:1011–1020

Garnier M, Jagoueix-Eveillard S, Cronje P, Le Roux H, Bove JM

(2000) Genomic characterisation of a liberibacter present in an

ornamental rutaceous tree, Calodendrum capense, in the Western

Cape province of South Africa. Proposal for a ‘‘CandidatusLiberibacter africanus subsp. capensis’’. Int J System Evol

Microbiol 50:2119–2125

Ghosh SK, Giannotti J, Louis C (1978) Intense multiplication of

prokaryotes associated with greening disease of citrus in phloem

cells of dodder. Ann Phytopathol 9:525–530

Gottwald TR, Da Graca JV, Bassanezi RB (2007) Citrus huanglong-

bing: the pathogen and its impact. Plant Health Progr. doi:

10.1094/PHP-2007-0906-01-RV

Hocquellet A, Toorawa P, Bove JM, Garnier M (1999) Detection and

identification of the two Candidatus Liberobacter species

associated with citrus huanglongbing by PCR amplification of

ribosomal protein genes of the b operon. Mol Cell Probes

13:373–379

secE nusG rplK rplA rplJ rplL rpoBtufBtrmUserA

1004 8621 8594149

3887 77647929 1082 5176

9335

1 kbp

ZA

B

C

D

E

F

8978

Fig. 3 Gene organization of the wserA-trmU-tufB-secE-nusG-

rplKAJL-rpoB gene cluster and the flanking regions in the ‘Candid-atus Liberibacter asiaticus’ isolates and the nucleotide positions that

differed among the 62 isolates. Vertical arrows and the number

indicate the position of the nucleotide substitution. Shaded double-ended arrows (Z and A–F) indicate the amplicons produced by PCR

performed using primer pairs indicated by prefixes Z and A–F in

Table 4


123

http://dx.doi.org/10.1094/PHP-2007-0906-01-RV

Hoy MA, Jeyaprakash A, Nguyen R (2001) Long PCR is a sensitive

method for detecting Liberobacter asiaticum in parasitoids

undergoing risk assessment in quarantine. Biol Control 22:278–

287

Ichinose K, Dien QL, Onuki M, Kawabe K (2004) Occurrence of

citrus greening disease in southern Vietnam and effectively

controlling factors on the disease (Abstract in Japanese). Jpn J

Trop Agric 48:101–102

Jagoueix S, Bove JM, Garnier M (1994) The phloem-limited

bacterium of greening disease of citrus is a member of the asubdivision of the proteobacteria. Int J Syst Bacteriol 44:386–

397

Jagoueix S, Bove JM, Garnier M (1997) Comparison of the 16S/23S

ribosomal intergenic regions of ‘‘Candidatus Liberobacter

asiaticum’’ and ‘‘Candidatus Liberobacter africanum’’, the two

species associated with citrus huanglongbing (greening) disease.

Int J Syst Bacteriol 47:224–227

Lee HA (1921) The relation of stocks to mottled leaf of citrus leaves.

Phil J Sci 18:85–95

Lin H, Doddapaneni H, Bai X, Yao J, Zhao X, Civerolo EL (2008)

Acquisition of uncharacterized sequences from CandidatusLiberibacter, an unculturable bacterium, using an improved

genomic walking method. Mol Cell Probes 22:30–37

Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR:

automatable amplification and sequencing of insert end frag-

ments from P1 and YAC clones for chromosome walking.

Genomics 25:674–681

Miyakawa T, Tsuno K (1989) Occurrence of citrus greening disease

in the southern islands of Japan. Ann Phytopathol Soc Jpn

55:667–670

Miyatake Y (1965) Notes on the Psyllidae from the Ryukyu Islands.

Konchu 33:171–189

Okuda M, Matsumoto M, Tanaka Y, Subandiyah S, Iwanami T (2005)

Characterization of the tufB-secE-nusG-rplKAJL-rpoB gene

cluster of the citrus greening organism and detection by loop-

mediated isothermal amplification. Plant Dis 89:705–711

Otake A (1990) Bibliography of citrus greening disease and its

vectors attached with indices and a critical review on the ecology

of the vectors and their control. Japanese International Cooper-

ative Agency, Tokyo, p 161

Planet P, Jagoueix S, Bove JM, Garnier M (1995) Detection and

characterization of the African citrus greening liberobacter by

amplification, cloning and sequencing of the rplKAJL-rpoBC

operon. Curr Microbiol 30:137–141

Sechler A, Schuenzel EL, Cooke P, Donnua S, Thaveechai N,

Postnikova E, Stone AL, Schneider WL, Damsteegt VD, Schaad

NW (2009) Cultivation of ‘Candidatus Liberibacter asiaticus’,

‘Ca. L. africanus’, and ‘Ca. L. americanus’ associated with

huanglongbing. Phytopathology 99:480–486

Shinohara K, Yuda T, Nishimoto H, Hamashima A, Hashimoto S,

Tokimura K, Satou T (2006) Survey of citrus huanglongbing

(greening disease) on the Amami Islands. 1. Characteristics of

distribution in the Amami Islands (in Japanese with English

summary). Kyushu Plant Protect Res 52:6–10

Subandiyah S, Iwanami T, Kondo Y, Kobayashi M, Tsuyumu S, Ieki

H (2000) Comparison of 16S rDNA and 16S/23S intergenic

region sequences among citrus greening organisms in Asia. Plant

Dis 84:15–18

Tirtawidjaja S, Hadewidjaja T, Lasheen AM (1965) Citrus vein

phloem degeneration virus, a possible cause of citrus chlorosis in

Java. Proc Am Soc Hort Sci 86:235–243

Tomimura K, Miyata S, Furuya N, Kubota K, Okuda M, Subandiyah

S, Hung TH, Su HJ, Iwanami T (2009) Evaluation of genetic

diversity of ‘Candidatus Liberibacter asiaticus’ isolates collected

in Southeast Asia. Phytopathology 99:1062–1069

Tomimura K, Furuya N, Miyata S, Hamashima A, Torigoe H,

Murayama Y, Kawano S, Okuda M, Subandiyah S, Su HJ,

Iwanami T (2010) Distribution of two distinct genotypes of

citrus greening organism in the Ryukyu Islands of Japan. Jpn

Agric Res Quart (in press)

Van der Merwe AJ, Andersen FG (1937) Chromium and manganese

toxicity. Is it important in transvaal citrus greening? Farming S

Afr 12:439–440

Villechanoux S, Garnier M, Renaudin J, Bove JM (1992) Detection of

several strains of the bacterium-like organism of citrus greening

disease by DNA probes. Curr Microbiol 24:89–95

Villechanoux S, Garnier M, Laigret F, Renaudin J, Bove JM (1993)

The genome of the non-cultured, bacterial-like organism asso-

ciated with citrus greening disease contains the nusG-rplKAJL-

rpoBC gene cluster and the gene for a bacteriophage type DNA

polymerase. Curr Microbiol 26:161–166

Zhao XY (1981) Citrus yellow shoot disease (huanglonbing)—a

review. In: 4th International Citrus Congress, Tokyo. Interna-

tional Society of Citriculture, Tokyo, pp 466–469


123

Sequence homogeneity of the ψ ser A- trm U- tuf B sec E- nus G- rp lKAJL- rpo B gene cluster and...

Documents

Transcript of Sequence homogeneity of the ψ ser A- trm U- tuf B sec E- nus G- rp lKAJL- rpo B gene cluster and...