Botanical Studies (2008) 49: 49-56.

*

Corresponding author: E-mail: hjsu@ntu.edu.tw; Fax:

+886-2-23625717; Tel: +886-2-33664095.

INTRODUCTION

Citrus greening was first reported in South Africa in

1947, while a similar disease known as "Huanglongbing"

(HLB) was already found in China in 1943. This psyllid-

borne virus-like disease also known as Likubin caused by

Candidatus Liberibacter asiaticus has been devastating

the citrus production in Taiwan since 1951 (Matsumoto et

al., 1961). The Asian heat-tolerant form of Huanglongbing

bacteria (HLBB) has seriously affected citrus trees in

the tropical and subtropical regions of Asia, and recently

in North and South America as well. The HLB caused

great damage to the citrus industry by shortening tree

lifespan and lowering fruit yield and quality. The HLBB

was transmitted by the Asian psyllid (Diaphorinia citri)

in a persistent manner without transovarial passage.

Chinese box orange (Severinia buxifolia) was found to

be the alternative host of the HLB pathogen and vector

psyllid. No HLBB multiplication was detected in Jasmine

orange (Murraya paniculata) or Curly leaf (Murraya

koenigii) (Hung et al., 2000b). Most citrus cultivars,

except pummelo, were susceptible to the Asian form of

HLB before 1971. However, pummelo became infected

by a new HLB strain in Taiwan and Southeast Asia in

1970s. The pummelo cultivars grown in Philippines,

Malaysia, Southern China, Vietnam, Thailand, Sri Lanka,

Bangladesh, and Cambodia have become susceptible to

HLB in recent decades. It is caused by a nonculturable

fastidious bacteria inhabiting the sieve tube and is referred

to as Candidatus Liberibacter (Jagoueix et al., 1994).

The HLB disease retards growth of the plant and causes

serious yellowing and decline of citrus trees, atrophy, and

incomplete colouring of mature fruit. The pathogen can

be categorized into two forms, Asian and African, based

on the influence of temperature on symptom expression.

The Asian form caused by "Ca. Liberibacter asiaticus,"

the symptoms of which can occur at temperatures above

30¢XC, is heat-tolerant, and the African form caused by

"Ca. Liberibacter africanus," the symptoms of which

Strain identification and distribution of citrus

Huanglongbing bacteria in Taiwan

Chia-Hsin TSAI, Ting-Hsuan HUNG, and Hong-Ji SU*

Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 106, Taiwan

(Received July 30, 2007; Accepted September 6, 2007)

ABSTRACT.

The 48 representative Huanglongbing bacteria (HLBB) isolates were selected from 457 disease

samples collected from Huanglongbing (HLB)-diseased citrus trees grown in seven main citrus-producing

areas, including tropical and subtropical regions of Taiwan. After indexing and eliminating the citrus viruses,

the selected HLBB isolates, free from the viruses, were used for identification of HLBB strains based on

a pathogenicity and virulence test with the following differential citrus cultivars: Ponkan mandarin (Citrus

reticulata Blanco), Liucheng sweet orange (C. sinensis Osb.), Wentan pummelo (C. grandis f. buntan Hay.),

and Eureka lemon (C. limon Burm.). Four strains of HLBB were identified. Strain I showed pathogenicity

on mandarin and sweet orange by inducing typical HLB symptoms. Strain II showed high virulence on all

differential cultivars and multiplied fast in all cultivars. Strain III caused intermediate symptoms on mandarin

and sweet orange and mild symptoms on pummelo, but did not infect Eureka lemon. Mild strain IV infected

mandarin and sweet orange without causing symptoms and was rarely isolated. Strain II, which attacked all

citrus cultivars grown in Taiwan, was found to dominate over the other strains in the field. Strains III and I

were second in quantity. One-third (32.6%) of the diseased samples showing HLB-like symptoms in the field

survey were found to be infected by HLB pathogen in PCR detection. Only 1.9% of healthy-looking citrus

trees surveyed, including Wentan pummelo (5%) and Eureka lemon (5.7%), were infected by HLBB. The

HLBB-isolates collected from mandarin, tangor, sweet orange, and kumquat (Fortunella margarita [Lour.]

Swingle) were commonly co-infected with Citrus tristeza closterovirus (CTV) and/or Citrus tatter leaf

capillovirus (CTLV). About 32% of the HLBB-infected trees examined were infected with HLBB only. Most

HLB-affected mandarin (66.7%) and tangor (69%) trees were also infected by CTV while CTV was rarely

detected in HLBB-infected pummelo (5%) or lemon (0%).

Keywords: Huanglongbing; HLBB strain; Pathogenicity.

PLANT PATHOLOGy

50

Botanical Studies, Vol. 49, 2008

appear above 30¢XC, is heat-sensitive (Bove et al., 1974). In

Taiwan, severe leaf yellowing was first noticed in Ponkan

mandarin (PM), Tankan tangor (Tan), and Liucheng sweet

orange (LSO), but not in Wentan pummelo (WP) in the

field in 1951 (Matsumoto et al., 1961). The pummelo

cultivar formerly resistant to HLB eventually became

infected and displayed HLB symptoms about 30 years

after HLB first appeared (Su and Wu, 1979; Huang and

Chang, 1980). The kumquat (Fortunella margarita (Lour.)

Swingle), which was formerly resistant to HLB, recently

became infected and displayed yellow mottling symptoms

in 2006 (Tsai et al., 2006). It was assumed that the change

of host range may have been due to the evolution of HLB

strains in pathogenicity. The present research work was

mainly aimed at isolation and differentiation of HLB

pathogen stains in Taiwan by pathogenicity and virulence

characterization. In this study, the adequate differential

cultivars were selected for identifying strains of HLBB

isolates collected from tropical and subtropical regions.

MATERIALS AND METHODS

HLBB isolates and sources

Totally 457 diseased samples showing HLB-like

symptoms were collected from seven main citrus

producing areas over Taiwan island, i.e., 85 from the

Hsinchu area growing Ponkan mandarin (PM) and

Tankan tangor (Tan); 49 from the Miaoli, Tan area; 77

from Chiayi, cultivating Wentan pummelo (WP); 93

from the Hualien WP area; 42 from the Tainan Liucheng

sweet orange (LSO) producing area; 53 from Pingtung,

cultivating Eureka lemon (EL), and 42 from the Yilan

kumquat producing area (Figure 1). The HLB isolates

were selected as representative ones after indexing of

HLB, CTV and CTLV with PCR, ELISA and RT-PCR,

respectively. Many healthy-looking samples were also

collected from all citrus producing areas to detect the

latent HLBB, CTV, and CTLV infections.

Pathological characterization of HLBB isolates

for strain identification

To avoid interaction between CTLV and CTV, the

HLBB-infected samples without CTLV and CTV, or with

only mild CTV, were selected as HLBB isolates for strain

identification. The HLBB isolates with CTV infection

became free from CTV by passage through filter plants

like Trifoliate orange (Poncirus trifoliate (L.) Raf.), which

is immune to CTV (Yoshida et al., 1983), or Pummelo,

which is hypersensitive to most CTV strains except the

pummelo stem-pitting strain (Tsai et al., 1993). The 48

Taiwanese representative isolates of HLBB were selected

from 457 diseased samples. Among many citrus cultivars,

Ponkan mandarin (PM) and Liucheng sweet orange (LSO)

were found to be most susceptible to HLBB by showing

distinct yellow mottling symptoms. Wentant pummelo

(WP), formerly immune to HLBB became infected in 1971

in Taiwan. The Eureka lemon (EL) cultivar, originally

quite resistant to HLBB, became susceptible and displayed

classic HLB symptoms in recent years (Matsumoto et al.,

1961). Therefore, PM, LSO, WP and EL were used as

differential cultivars of the HLBB strain. HLBB isolates

were subjected to pathogenicity tests with these four

differential citrus cultivars. Healthy seedlings about 15

cm tall were inoculated by grafting with diseased buds

or leaf petioles. More than two trial buds were grafted

onto each differential plant. For each HLBB isolate, four

replicates of each differential cultivar were used. The test

citrus seedlings were grown in an insect-proof greenhouse

Figure 1. Agroecological areas of HLB epidemic in Taiwan

island. PM and Tan are grown in 1 (Hsinchu) and 2 (Miaoli).

WP and other pummelo are grown in areas 3 (Chiayi) and 5

(Hualien). LSO is grown in area 6 (Chiayi) EL is grown in area

7 (Pingtung) and Kq is centralized in area 4 (Yilan). Bold line

crossing area 3 and 5 is Tropic of Cancer. Region above bold

line is subtropical, and region below bold line is tropical. ^

denotes the Central Mountain Range.

TSAI et al. ¡X Strain identification of Huanglongbing bacteria in Taiwan

51

with an air-cooling system. The symptom development

on each test plant was observed and recorded each month

after inoculation. Multiplication dynamics of HLB

pathogen in test plants was analyzed by PCR each month

until 12 months after inoculation. Disease severity was

graded as follows: 0 = no symptom; 1 = mild chlorosis

without dwarfing; 2 = intermediate chlorotic mottling with

moderate dwarfing; 3 = severe HLB symptoms, including

yellow mottle, leaf hardening, and curling with distinct

dwarfing.

Genomic DNA isolation and HLBB-PCR

detection

The nucleic acid extracts to be used as the templates

for PCR detection were prepared by using the method

described by Hung et al. (1999). Leaf midrib (500 mg)

was powdered in liquid nitrogen, and each sample

was suspended in 1.5 ml of DNA extraction buffer

[1 M Tris-HCl (pH 8.0) 0.5 M EDTA, 5 M NaCl, 1%

N-Lauroylsarcosine] and transferred to a 1.5 ml Eppendorf

tube. After incubation at 55¢XC for 1 h, the sample was

centrifuged at 4,000 g for 5 min. The supernatanat (800

£gl) was collected, and 100 £gl 5 M NaCl and 100 £gl 10%

CTAB (hexadecyl-trimethyl-ammonium-bromide) in

0.7 M NaCl were added. The mixture was incubated at

65¢XC for 10 min. The sample was subjected to one cycle

of chloroform/isoamyl alcohol (24:1) extraction, and the

aqueous supernatant was then reextracted by an additional

cycle of phenol/chloroform/isoamyl alcohol (25:24:1).

The nucleic acids were precipitated by mixing 600 £gl

of the supernatant with 360 £gl isopropanol followed by

centrifugation at 12,000 g for 10 min. The pellets were

washed with 70% ethanol, dried, and resuspended in 150

£gl TE buffer as template solution.

Two £gl of the extract as template was pipeted into 25 £gl

tube containing PCR mixture. The amplification of HLBB-

DNA in PCR cycle with the primer pairs, consisted of the

forward primer 5¡¦-CAC CGA AGA TAT GGA CAA CA-3¡¦

and the reverse primer 5¡¦-GAG GTT CTT GTG GTT TTT

CTG-3¡¦ (Hung et al., 1999). The amplification of HLBB

specific DNA fragment (226-bp) in the PCR thermal cycle

consisted of 94¢XC for 3 min, 30 cycles at 94¢XC for 1 min,

60¢XC for 1 min, and 72¢XC for 2 min followed by a 72¢XC

extension for 10 min. Reactions were carried out in PCR

Thermal Cycler AB 2720 followed by electrophoresis

analysis. The PCR products were quantified using the

densitometer supplied by AlphaEase

R

FC Image Analysis

Software. The density of the PCR products was determined

and represented by a pixel (picture element) value with a

range from 0 to 255.

Genomic RNA extraction and RT-PCR for CTLV

detection

RNeasy Mini Kit (Qiagen, Hilden, Germany) was used

for preparing the total RNA from diseased samples. Two

c-DNA primers for the RT-PCR-based detection of CTLV

were designed from GenBank (accession No. AY646511)

(Lin and Hung, 2004). The primer pair, consisting of the

forward primer 5¡¦-GGA AGA CTC ACA TAG ACC CG

-3¡¦and the reverse primer 5¡¦-TAC TCT CCG AAC CTG

CCT C-3¡¦, was used for proceeding one-step RT-PCR to

amplify a CTLV-specific 636-bp fragment (Hung et al.,

1999a; Hung et al., 2000; Lin and Hung, 2004).

ELISA tests for CTV detection

Monoclonal antibody (3E10) recognizing a common

epitope of the CTV strain was used for indexing CTV

in all diseased samples by a double antibody sandwich

enzyme-linked immunosorbent assay (DAS-ELISA) (Tsai

and Su, 1991; Tsai et al., 1993).

RESULTS

Identification of HLBB strains by pathogenicity

The HLBB strains were differentiated based on the

criteria of pathological characterization including host

range, pathogenicity and virulence. Multiplication of

HLBB was shown with PCR test by pixel value. Taiwan

HLBB isolates were categorized into four strains. They

were identified according to the disease index in symptom

expression on the four differential citrus cultivars and

PCR detection of HLBB multiplication 12 month after

inoculation (Table 1). Strain I caused severe yellow-

mottling and typical HLB symptoms on susceptible

Ponkan mandarin (PM) and Liucheng sweet orange (LSO)

cultivars but did not infect Wentan pummelo (WP) or

Eureka lemon (EL) cultivars (Figure 2). No multiplication

of HLBB in the latter two cultivars was confirmed by PCR

detection. Strain II showed high virulence of pathogenicity

in all cultivars. It induced severe HLB symptoms,

including leaf yellow-mottling and curling, vein-yellowing

Table 1. The pathogenicity characterization of HLBB isolates

according to disease index and PCR detection of HLBB infec-

tion.

Strain

Differential cultivars of citrus

a

PM

LSO

WP

EL

I

3 /+++

a

3 / +++ 0 / - 0 / -

II

3 / +++ 3 / +++ 3 / +++ 3 / +++

III

2 / ++ 2 / ++ 1 / + 0 / -

IV

0 / + 0 / + 0 / - 0 / -

a

Disease index / PCR index. Disease index was graded

12 mont hs a fter i nocula tion: 0, hea lthy l ooking wit hout

s ym ptom s ; 1, m ild c hlorot ic s ym ptom s; 2, int erm edia te

symptoms including chlorosis, mottling with intermediate

dwarfing; 3, typical greening symptoms including leaf yellow

m ottle and curling, vein-yellowing with dis tinct dwarfing.

Pixe l va lue (de ns ity c ount) in dex of t he HL BB-s pec ific

band on agarose gel electrophoresis: -, pixel value< 50; +,

50 ~ <110; ++, 110 ~ < 170; +++, 170 ~ < 230. Differential

cultivars were Ponkan mandarin (PM), Liucheng sweet orange

(LSO), Wentan pummelo (WP), and Eureka lemon (EL).

52

Botanical Studies, Vol. 49, 2008

and distinct dwarfing. Multiplication of the strain II was

demonstrated by high pixel value in PCR detection of

HLBB. Strain III, the intermediate virulent strain, infected

PM, LSO and WP, but not EL, and caused leaf-chlorosis

with mild vein enation. No yellow mottling or typical HLB

symptoms were induced by strain III. Strain IV was a mild

strain infecting PM and LSO without symptoms and did

not infect WP or EL. This mild strain was not commonly

isolated.

Detectable duration of HLB pathogen and

incubation period of symptom appearance

The detectable duration, indicated by the multiplication

rate, and the incubation period, indicated by the symptom

expression after infection, were affected by pathogen

strains and host cultivars. The multiplication rate of the

pathogen in each differential cultivar was monitored by

PCR. The first HLBB detection by PCR and incubation

period of the first symptom appearance were shown in

Table 2. Strain I propagated faster in PM than LSO as

indicated by detectable durations of 2 and 3 months,

respectively, determined by PCR analysis after inoculation.

A 3-month inoculation period was demonstrated in both

cultivars. No infection of strain I was detected in WP or

EL. The multiplication rate of strain II was most rapid in

all the tested cultivars. The strain II HLBB was detected

within 2 months in mandarin (PM) and 3 months in the

other test cultivars. It induced symptoms in 3 months on

susceptible PM and LSO cultivars and in 4 months on

tolerant WP and EL cultivars. Strain III was detected in

PM within 3 months and in LSO and WP within 4 months.

Its incubation period was 5 months in PM and LSO, and

Figure 2. Different types and severities of the symptoms induced by the four HLBB strains (I-¢¼) in four differential citrus cultivars of

Ponkan mandarin (PM), Liucheng sweet orange (LSO), Wentan pummelo (WP) and Eureka lemon (EL) under greenhouse conditions

during 2005-2006.

TSAI et al. ¡X Strain identification of Huanglongbing bacteria in Taiwan

53

6 months in WP. The EL plant was immune to strain III.

The mild strain IV showed latent infection in mandarin

(PM) and sweet orange (LSO) and multiplied slowly

with a detectable duration of 3~5 months. However, it

disappeared in some test plants 1~2 years after inoculation.

Based on the multiplication rate and incubation period,

strain II was the most virulent strain, and it dominated

over the other strains in Taiwan. Strains I and III, showing

moderate virulence, were detected at a lower rate.

Generally speaking the incubation period of susceptible

cultivars such as PM and LSO was shorter than those of

tolerant cultivars such as WT and EL.

Distribution of HLBB strains on Taiwan island

A total of 457 diseased samples were collected from

seven major citrus-greening areas on the island of Taiwan,

and 48 representative HLBB isolates were selected for

strain identification by pathogenicity assay within 12

months. The infection and replication of HLBB were

confirmed by PCR (Table 3). The strain II, detected in

68.8% of the samples, was isolated most commonly

from all cultivars grown in Taiwan, including mandarin

(PM), tangor (Tan), sweet orange (LSO), pummelo (WP),

kumquat, and lemon (EL). Strain III, with an 18.3%

detection rate, was isolated mainly from WP and rarely

from PM and Tan grown in northern, eastern, and central

parts of Taiwan. Strain I, with an 8.3% detection rate,

was isolated from PM and Tan from old citrus-growing

Hsinchu and Miaoli areas in northern Taiwan. Mild strain

IV, with a 4.2% detection rate, was rarely isolated from

tangor grown in Hsinchu. Only strain II was detected in

EL in the tropical Pingtong area while all four strains were

found in the subtropical areas.

HLB incidence and complex infection of HLBB

with viruses in the field

The citrus trees were commonly infected by HLBB,

citrus tristeza closterovirus (CTV), and tatter leaf

capillovirus (CTLV) (Su and Cheon, 1984; Tsai et al.,

1991). One third (32.6%) of the suspected citrus trees

with HLB-like symptoms were indexed to be infected by

HLBB. About 32% of HLBB-infected trees were infected

by HLBB only, and the other HLBB-infected trees

(68%) were also mix-infected with CTV (24.8%), CTLV

(27.5%) or CTV+CTLV (15%) (Table 4). Over half of the

suspected Eureka lemon trees (52%) grown in the tropical

Pingtung area were infected by HLBB while about 30%

of the other suspected citrus trees were infected by HLBB

on the whole island. The samples of HLBB-infected trees

free from the viruses were used for the isolate sources

of HLBB strains. The frequency of HLBB/viruses mix-

infection was varied in citrus cultivars. All the citrus trees

of mandarin (PM), tangor (Tan), sweet orange (LSO), and

kumguat were infected by HLBB in combination with

CTV and/or CTLV. No viruses were detected in half of

the HLBB-infected pummelo (WP) (50.8%) or lemon

(EL) (61.5%) trees. The HLB-affected mandarin (66.7%)

and tangor (69%) trees were frequently mix-infected with

CTV. However, CTV was rarely detected in pummelo

(4.8%) and lemon (0%). The CTLV was found commonly

infecting WP (44.4%), kumquat (30%), and EL (38.5%)

Table 2. Detectable duration of pathogen by PCR-monitoring

and incubation period of symptom appearance by HLBB

s trains in four differential cultivars, inspected m onthly after

graft-inoculation.

Strain

Months after inoculation

PM LSO WP EL

¢¹

PCR / Sym

a

2 / 3 3 / 3 - / - - / -

II

PCR / Sym. 2 / 3 3 / 3 3 / 4 3 / 4

III PCR / Sym. 3 / 5 4 / 5 4 / 6 - / -

IV

PCR / Sym. 3 / N 5 / N - / - - / -

a

First positive detection of PCR / First month of symptom ap-

pearance (incubation period) Differential citrus cultivars men-

tioned in Table 1.

N: symptomless.

Table 3. The proportion and distribution of HLBB strains over the island of Taiwan via pathogenicity tests with 48 representative

HLBB isolates obtained from various localities.

Locality (Prefecture)

Citrus cultivar No. of HLBB isolates

Strains of HLBB

Strain I Strain II Strain III Strain IV

Hsinchu (N)

PM

5

1

3

1

0

Tan

7

1

4

0

2

Miaoli (N)

Tan

5

2

2

1

0

Chiayi (M)

WP

9

0

6

3

0

Yilan (E)

Kq

4

0

4

0

0

Hualien (E)

WP

7

0

3

4

0

Tainan (S)

LSO

5

0

5

0

0

Pingtung (S)

EL

6

0

6

0

0

Total/percentage

48

4 (8.3%) 33 (68.8%) 9 (18.8%) 2 (4.2%)

N, northern; M, central; S, southern; E, eastern part of Taiwan.

54

Botanical Studies, Vol. 49, 2008

while PM (33.3%), Tan (31.2%), and LSO (66.7%) were

frequently infected by HLBB with a complex infection of

CTV plus CTLV.

Latent infection of HLBB, CTV, and CTLV in

citrus trees in the field

The infection of HLBB and citrus viruses was detected

in citrus samples collected from healthy-looking citrus

trees of different cultivars grown in seven counties in

Taiwan (Table 5). In total, only 1.9% trees of healthy-

looking 305 samples¡Xincluding 4 of 80 WP-samples (5%)

grown in Chiayi and Hualien and 2 of 35 EL-samples

(5.7%) grown in tropical Pingtung area¡Xwere infected by

HLBB without a mix-infection of CTV and CTLV. HLBB

was not detected in the 30 healthy-looking PM-trees from

Hsinchu, 70 Tan-trees from Miaoli, 50 kumquat trees

from Yilan, or 40 LSO-trees from Tainan. About 32.7% of

healthy-looking citrus trees were infected by CTV. A high

percentage of PM trees (73.3%, Hsinchu) and Tankan trees

(68.6%, Miaoli) were commonly infected by CTV without

visible symptoms. CTV was not detected in healthy WP

and EL trees. A single infection of CTLV was detected

frequently in WP (52.5%, Chiayi and Hualien) and EL

trees (54.3%, Pingtong) while about 20% of surveyed

trees were infected singly with CTLV. A mixed-infection

of CTLV plus CTV was commonly detected in LSO

(70%, Tainan) and kumquat (64%, Yilan), and in a certain

amount of Tan (31.4%, Miaoli) and PM (26.7%, Hsinchu).

DISCUSSION

Citrus Huanglongbing was first found in China in

1943. The virus-like disease was first noticed in Taiwan in

1951, six years after the Second World War (Matsumoto

et al., 1961). The HLB disease became epidemic after

introduction, possibly from China circa 1945. HLB has

been devastating the citrus production and causing great

damage to the citrus industry by shortening the tree

Table 5. Incidence of latent infection of HLBB, CTV, and CTLV in symptomless citrus trees.

Citrus cultivars

a

Locality (Prefecture) Sample no.

HLBB and citrus viruses in symptomless trees

HLBB

CTV

CTLV

CTV+CTLV

PM

Hsinchu

30

0/30 (0%) 22/30 (73.3%) 0/30 (0%) 8/30 (26.7%)

Tan

Miaoli

70

0/70 (0%) 48/70 (68.6%) 0/70 (0%) 22/70 (31.4%)

WP

Chiayi, Hualien

80

4/80 (5.0%)

0/80 (0%) 42/80 (52.5%)

0/80 (0%)

Kq

Yilan

50

0/50 (0%) 18/50 (36.0%) 0/50 (0%) 32/50 (64.0%)

LSO

Tainan

40

0/40 (0%) 12/40 (30.0%) 0/40 (0%) 28/40 (70.0%)

EL

Pingtung

35

2/35 (5.7%)

0/35 (0%) 19/35 (54.3%)

0/35 (0%)

Total/percentage

305 6/305 (1.9 %) 100/305 (32.7%) 61/305 (20%) 90/305 (29.5%)

a

The test samples collected from the healthy-looking citrus trees of different cultivars including Ponkan mandarin (PM), Tankan

tangor (Tan), Wentan pummelo (WP), kumquat (Kq), Liucheng sweet orange (LSO), and Eureka lemon (EL).

Table 4. Complex infection of HLBB with CTV and CTLV in the main citrus-growing areas.

Locality

(Prefecture)

Citrus

cultivars

HLBB-infected plants /

No. of indexed plants

a

(%)

Complex infection of citrus viruses in HLBB-positive samples (%)

HLBB HLBB+CTV HLBB+CTLV HLBB+CTV+CTLV

Hsinchu

PM

9/29 (31.0%)

0/9 (0%) 6/9 (66.7%)

0/9 (0%) 3/9 (33.3%)

Ta n

16/56(28.6%)

0/16 (0%) 11/16 (68.9%) 0/16 (0%) 5/16 (31.2%)

Miaoli

Ta n

13/49 (26.5%)

0/13 (0%) 9/13 (69.2%) 0/13 (0%) 4/13 (31.8%)

Chiayi

WP

30/77 (39.0%) 14/30 (46.7%) 2/30 (6.7%) 14/30 (46.7%)

0/25 (0%)

Yilan

Kq

10/60 (16.7%)

0/10 (0%) 4/10 (40.0%) 3/10 (30.0%) 3/10 (30.0%)

Hualien

WP

33/93 (35.5%) 18/33 (54.5%) 1/33 (3.0%) 14/33 (42.4%)

0/29 (0%)

Tainan

LSO

12/42 (28.6%)

0/12 (0%) 4/12 (33.3%) 0/12 (0%) 8/12 (66.7%)

Pingtung

EL

26/50 (52.0%) 16/26 (61.5%) 0/26 (0%) 10/26(38.5%)

0/26 (0%)

Total

149/457 (32.6%) 48/149 (32.2%) 37/149 (24.8%) 41/149 (27.5%) 23/149 (15.4%)

a

The citrus trees showing HLB-like symptoms including chlorosis, vein-yellowing, yellowing and yellow-mottling which may be

induced by HLBB and/or the other causes.

TSAI et al. ¡X Strain identification of Huanglongbing bacteria in Taiwan

55

lifespan and lowering fruit yield and quality in Taiwan.

The heat-tolerant form of HLBB has severely affected

citrus trees in the tropical and subtropical regions of Asia

and recently in South and North America (Coletta et al.,

2004; Zhou et al., 2007). The systemic HLB disease is

caused by non-culturable fastidious bacteria. It easily

becomes epidemic because of common transmission

through vegetative-propagated seedlings and is rapid

spread by psyllid vector.

Taiwan has both tropical and subtropical regions. The

Central Mountain Range separates the island into west and

east regions. This special topography and geographical

feature causes diversity in climates. Due to this diversity,

different citrus cultivars were planted in different areas.

For example, PM and Tan were concentrated in northwest

region, and WP was concentrated in southwest and

southeast region while EL was only planted in southern

Taiwan. HLBB-infected leaves collected from PM, Tan,

and LSO were all mix-infected with CTV or CTLV. This

may be why these HLB-infected trees showed severe

symptoms and declined quickly in the field. Among the

457 HLB-like trees surveyed, only approximately 30%

were caused by HLBB. Other factors, including root rot,

stem borer (Anoplophora macularia [Thomson]), and

nutrient deficiency, also caused HLB-like symptoms

including chlorosis of leaves, vein-yellowing, and tree

decline.

In this report, HLBB isolates were separated into

different strains for the first time. Strain I was specific to

PM, Tan and LSO cultivars in our pathogenicity tests. This

pathogenicity character was similar to the HLB observed

from 1951 to 1970 in Taiwan (Su and Wu, 1979; Huang

and Chang, 1980). Severe HLB symptoms were only found

in PM, Tan, and LSO, but not in WP. Therefore, strain I

was similar to the original type observed in the field in

1951. HLB was a virus-like and systemic disease. The

cultivar limitation of strain was also found in CTV. CTV

pummelo stem-pitting strain (Pum/SP) found in southeast

Taiwan caused severe symptoms, including bunchy twigs

and curly leaves and stem-pitting on twigs, trunk, and

roots in WP, but it could not infect LSO and PM. Sweet

orange stem-pitting strain (SO/SP), Mandarin stem-

pitting strain (Mand/SP), and ordinary seedling-yellow

strain (SY) could not persist in WP trees (Tsai et al.,

1993). HLBB strain II, a highly virulent strain, was able

to infect the formerly immune WP and the other cultivars,

and it rapidly propagated in all seedlings to cause severe

yellowing symptoms in pathogenicity tests. In the field,

this predominant strain usually existed in trees showing

severe symptoms, especially in tolerant cultivars, and

could therefore be easily located. Strain II was assumed to

have evolved from strain I. Strain III was an intermediate

strain that could be found in trees which had been infected

by HLB for several years, but still maintained their

growth and capacity to produce fruit. Trees in the field

infected with strain III usually showed intermediate or

mild symptoms, and this observed phenomenon was in

accordance with our pathogenicity tests. Strain IV was a

mild strain and only found in Tan producing areas. The

trees in the field infected with strain IV were usually

symptomless and differential cultivar tests also showed

the same degree of symptoms 12 months after inoculation.

The mild strain IV was used to test its ability to cross-

protect citrus plants, but it did not show a protective effect

against the severe strains in our preliminary trials. In view

of HLBB strain evolutions, molecular characterization

of Taiwanese strains is expected to be investigated in

comparison with foreign strains through international

collaboration.

Acknowledgements. The authors thank Y.C. Feng at

the Department of Plant Pathology and Microbiology,

National Taiwan University for assistance with inspection

of disease samples in this study.

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