AWARD

Studies on early and highly sensitive detection of citrus greeningpathogenic bacterium

Takashi Fujikawa

Received: 22 May 2014 / Accepted: 22 May 2014

� The Phytopathological Society of Japan and Springer Japan 2014

Introduction

Citrus greening (huanglongbing; HLB) is a devastating

disease of citrus trees with high economic costs to the

world’s citrus industry. In Japan, infected trees have been

confirmed throughout Okinawa Prefecture and on the

Amami Islands with the exception of Amami-Oshima and

Kikai Islands of Kagoshima Prefecture. This disease is

caused by a phloem-limited fastidious bacterium, ‘Can-

didatus Liberibacter asiaticus’ (Las) in Japan (Hamashima

et al. 2003) and is transmitted by grafting and by the sap-

sucking psyllid Diaphorina citri (Bove 2006). When

infected trees develop symptoms, tree vigor declines rap-

idly, and the tree soon dies. All major commercial citrus

cultivars are susceptible to the bacterium, and no effective

control is known other than the removal of infected trees.

Therefore, in areas where the bacterium has not become

established, rapid identification and culling of infected

trees and quarantine measures for budwoods are the most

important control strategies. Various DNA amplification

methods, including polymerase chain reaction (PCR) have

been used to test greening-infected plants (National

Research Council 2010), but obtaining stable results is

often difficult at an early stage of infection when the bac-

terial density in trees is low. Therefore, in this study, a

highly sensitive method for the early detection of the citrus

greening bacterium was developed.

Highly sensitive and accurate PCR

In a re-examination of the specificity of primers used in

PCR tests for greening-infected plants, we found a Las-

specific sequence region after aligning and comparing the

various bacterial 16S rDNA sequences and were able to

design new primers with high sensitivity and accuracy

(Fujikawa and Iwanami 2012). The PCR using the new

primer set was more sensitive than with any other known

primer sets, and we confirmed the high specificity for Las

(Fujikawa and Iwanami 2012).

Development of methods to simplify extraction

and visualize the pathogenic bacterium

Next, to facilitate detection of the pathogenic bacterium by

PCR using the new primer set described, we tried to

develop a direct-PCR method that does not require DNA

extraction from infected trees. Since the pathogenic bac-

teria are localized in the phloem of infected trees, we were

able to obtain extracts with the bacteria after grinding the

midribs of the infected leaves and centrifuging the col-

lected solutions. Thus, we were able to detect the pathogen

at sufficiently high sensitivity with PCR using the extracted

solutions as templates (Fujikawa et al. 2013). Further, we

could see the pathogenic bacteria in the extract solutions by

microscopic observation after in situ hybridization against

Las-specific RNAs (Fujikawa et al. 2013). With this tech-

nique, we were able to observe host-free and living bacteria

for the first time; until then, fixed bacteria in infected tree

This article is an abstract of the paper presented by a winner of the

Young Scientist Award at the 2014 Annual Meeting of the

Phytopathological Society of Japan in Sapporo.

T. Fujikawa (&)

NARO Institute of Fruit Tree Science, Tsukuba,

Ibaraki 305-8605, Japan

e-mail: ftakashi@affrc.go.jp

123

J Gen Plant Pathol

DOI 10.1007/s10327-014-0545-z

samples had been observed only with the electron

microscope.

Study on metabolic pathways of the pathogenic

bacterium

Development of the technique described above enabled us

to extract and to handle the living bacterium. We then tried

to grow the bacteria, even though culturing had only been

attempted occasionally. First, we used genomic informa-

tion for the bacterium to predict metabolic pathways (e.g.,

amino acid synthesis and energy metabolism). On the basis

of our predictions, we included some amino acids that the

bacteria cannot synthesize and developed a selective

medium for the bacterium (Fujikawa et al. 2014). By using

various methods such as PCR, RT-PCR and in situ

hybridization, we confirmed that the bacteria on the med-

ium were multiplying and viable. Now we are testing this

cultivation method for detecting the pathogen in greening-

infected plants.

Acknowledgments I am grateful to Drs. Toru Iwanami and Shinichi

Miyata (NARO Institute of Fruit Tree Science) and our laboratory

members for their generous support and valuable suggestions. Also I

sincerely appreciate Prof. Emeritus Shinji Tsuyumu and Prof. Yuichi

Takikawa (Graduate School of Science and Technology, Shizuoka

University), Drs. Hisatoshi Kaku (Sakata Seed Corp.), Marie Ni-

shimura (National Institute of Agrobiological Sciences), Mamoru

Satou (NARO Institute of Floricultural Science) for their valuable

suggestions and encouragement.

References

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

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

Fujikawa T, Iwanami T (2012) Sensitive and robust detection of citrus

greening (huanglongbing) bacterium ‘‘Candidatus Liberibacter

asiaticus’’ by DNA amplification with new 16S rDNA-specific

primers. Mol Cell Probes 26:194–197

Fujikawa T, Miyata S, Iwanami T (2013) Convenient detection of the

citrus greening (huanglongbing) bacterium ‘Candidatus Liberib-

acter asiaticus’ by direct PCR from the midrib extract. PLoS One

8:e57011

Fujikawa T, Miyata S, Iwanami T (2014) Cultivation of citrus

greening disease (huanglongbing) pathogenic bacteria based on

metabolic pathway prediction (abstract in Japanese). Jpn J

Phytopathol 80:25

Hamashima A, Hashimoto S, Nagamatsu K, Nuta T (2003) First

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National Research Council (2010) Strategic planning for the Florida

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Academies Press, Washington DC. http://www.nap.edu/catalog/

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