2009-12-24

In recent decades, citrus greening disease or huanglongbing (HLB) has been causing major damage and economic losses to the citrus industry in the Asian and Pacific (ASPAC) region. The identification of HLB disease infection is extremely difficult because of its similarity to the symptoms of such nutrient deficiencies of Zn and Fe, and its pathogen, Diaphorina citri, cannot be cultivated on an artificial culture medium. In addition, no effective agrochemicals to control this disease are yet available. The only countermeasure to cope with HLB infection is to cut down the infected citrus trees. Therefore, early detection is very important to mitigate the damage caused by HLB infection. In view of the urgency and seriousness of this problem, FFTC implemented a research project to develop a rapid, less-costly and accurate detection of the HLB pathogen in partnership with the National Taiwan University (NTU) and the National Institute of Fruit Tree Science (NIFTS), Japan.

Polymerase chain reaction (PCR), which is sensitive and reproducible, is the most common method used for detection of HLB pathogen. However, this method requires a thermal cycler and other basic apparatus for molecular biological experiments, as well as well-trained personnel. Meanwhile, the loop-mediated isothermal amplification (LAMP) is a newly developed method for DNA amplification in Japan. This method is considered to have the following advantages over PCR: 1) DNA amplification is catalyzed by only one enzyme; 2) Highly efficient amplification (within 30 minutes using a simple, affordable water bath); 3) Highly specific detection of HLB pathogen by four different kinds of primer; and 4) Large quantity of the product amplified suitable for under equipped laboratories of extension centers and local quarantine offices.

In the second-year implementation of this project, the following research results were obtained:

• 1) Nucleotide sequence analysis of the tufB-secE-nusG-rplKAJL-rpoB region of Candidatus Liberibacter asiaticus (Las) for Southeast Asian isolates
• The Asian causal agent has been designated as Candidatus Liberibacter asiaticus (Las). The fragment of the tufB-secE-nusG-rplKAJL-rpoB of Las was sequenced, and various isolates from Southeast Asian countries were compared. The sequence of about 9 kbp was identical with each other among all the isolates tested except for six nucleotide positions (Fig. 1). The results suggested that the sequence of the tufB-secE-nusG-rplKAJL-rpoB gene cluster was highly conserved in most of the Las, except for the six mutation points. Taking advantage of this conserved sequence, Loop-mediated Isothermal Amplification (LAMP) of Las was developed to detect most, if not all of Southeast Asian isolates.
• 2) Designing specific primers of LAMP for detection of Southeast Asian Las isolates
• Specific primers were designed based on the highly conserved part of the tufB-secE-nusG-rplKAJL-rpoB gene cluster for detecting Las. Positive reaction in LAMP was visualized by adding fluorescent dye in the reaction mixture (Fig. 1). Direct fluorescent visualization without opening the reaction tubes has the merit of reducing a risk of cross contamination among samples.
• Three strains of Las with different pathotypes (I, II, III) are spread in Taiwan. As expected, LAMP utilizing the conserved sequence of tufB-secE-nusG-rplKAJL-rpoB gene cluster could detect isolates from all three Taiwanese strains, as well as isolates from Japan, Vietnam and Cambodia. The results suggested that the LAMP can be widely applicable for detecting Southeast Asian Las isolates of Las.
• 3) Comparison of cost, accuracy and efficiency in HLB infection detection between LAMP and PCR
• Loop-mediated isothermal amplification (LAMP) is a newly developed method for DNA amplification in Japan. This method is considered to have some advantages over PCR (polymerase chain reaction) for under-equipped laboratories of extension centers and local quarantine offices.

In the laboratory test using Taiwanese HLB infected leaf samples, the LAMP method proved to more rapid and simpler than PCR in terms of HLB-detection; the sensitivity of the LAMP method was almost the same as PCR. However, more efficient and reliable extraction method of HLB pathogen from infected leaves should be developed to increase the sensitivity of the LAMP method.

International Collaborative Project on

"A rapid, less-costly and accurate detection of citrus greening (HLB) pathogen in the ASPAC region" (Year 2)

This two-year (April 2007 - March 2009) collaborative research project was carried out in Taiwan ROC and Japan.

Cooperating organizations: National Taiwan University (NTU), Taiwan ROC; National Institute of Fruit Tree Science (NIFTS), Japan

Sponsor: Japan Interchange Association, Taipei Office

For further information, contact:

Dr. Hong-Ji Su, FFTC Technical Consultant

Index of Images

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  Figure 1 Schematic representation of gene organization of the tufB-secE-nusG-rplKAJL-rpoB gene cluster and an example of LAMP reaction with direct fluorescent visualization. Position of nucleotide changes among Southeast Asian isolates of Las are indicated with black triangles. A region with highly conserved sequence, which is applied for LAMP, is shown with a thick line. Fluorescent greenish color in reaction tubes (five tubes on left) indicate positive results in LAMP.

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