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Blood disease in bananas caused by Ralstonia syzygii subsp. celebesensis is a bacterial wilt disease that causes major yield losses of banana in Indonesia and peninsular Malaysia. The disease has significantly increased its geographic distribution in the past decade. Diagnostic methods are an important component of disease management in vegetatively propagated crops such as banana to constrain incursions of plant pathogens. Therefore, the objectives of this study were (i) to design and rigorously validate a novel banana Blood disease (BBD) real-time PCR assay with a high level of specificity and sensitivity of detection and (ii) to validate published PCR-based diagnostic methods targeting the intergenic region in the megaplasmid (“121 assay” with primer set 121) or the phage tail protein-coding sequence in the bacterial chromosome (“Kubota assay” and “BDB2400 assay” with primer set BDB2400). Assay validation included 339 samples (174 Blood disease bacteria, 51 bacteria associated with banana plants, 51 members of the Ralstonia solanacearum species complex, and 63 samples from symptomatic and healthy plant material). Validation parameters were analytical specificity (inclusivity and exclusivity), selectivity, limit of detection, accuracy, and ruggedness. The 121 assay and our newly developed BBD real-time PCR assay detected all R. syzygii subsp. celebesensis strains with no cross-specificity during validation. Two different PCR assays using the primer set BDB2400 lacked specificity and selectivity. This study reveals that our novel BBD real-time PCR assay and the conventional PCR 121 assay are reliable methods for Blood disease diagnostics, as they comply with all tested validation parameters.

Blood disease in bananas caused by Ralstonia syzygii subsp. celebesensis is a bacterial wilt causing significant crop losses in Indonesia and Malaysia. Disease symptoms include wilting of the plant and red-brown vascular staining, internal rot, and discoloration of green banana fruit. There is no known varietal resistance to this disease in the Musa genus, although variation in susceptibility has been observed, with the popular Indonesian cooking banana variety Kepok being highly susceptible. This study established the current geographic distribution of Blood disease in Indonesia and confirmed the pathogenicity of isolates by Koch’s postulates. The long-distance distribution of the disease followed an arbitrary pattern indicative of human-assisted movement of infected banana materials. In contrast, local or short-distance spread radiated from a single infection source, indicative of dispersal by insects and possibly contaminated tools, water, or soil. The rapid expansion of its geographical range makes Blood disease an emerging threat to banana production in Southeast Asia and beyond.

Banana Blood disease is a bacterial wilt caused by Ralstonia syzygii subsp. celebesensis and is an economically important disease in Indonesia and Malaysia. Transmission of this pathogen is hypothesized to occur through insects mechanically transferring bacteria from diseased to healthy banana inflorescences and other pathways involving pruning tools, water movement, and root-to-root contact. This study demonstrates that the ooze from the infected male bell and the sap from various symptomatic plant parts are infective, and the cut surfaces of a bunch peduncle, petiole, corm, pseudostem, and the rachis act as infection courts for R. syzygii subsp. celebesensis. In addition, evidence is provided that R. syzygii subsp. celebesensis is highly tool transmissible, that the bacterium can be transferred from the roots of a diseased plant to the roots of a healthy plant and transferred from the mother plant to the sucker. We provide evidence that local dispersal of Blood disease occurs predominantly through mechanical transmission by insects, birds, bats, or human activities from diseased to healthy banana plants and that long-distance dispersal occurs through the movement of contaminated planting material. Disease management strategies to prevent crop losses associated with this emerging disease are discussed based on our findings.

Okuda, M., Matsumoto, M., Tanaka, Y., Subandiyah, S., and 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. Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to amplify the uncharacterized regions adjacent to the nusG-rplKAJL-rpoB gene cluster of citrus greening organism (GO) isolates from different locations in Japan and Indonesia. Conventional PCR was used to amplify the internal nusG-rplKAJL-rpoB gene cluster of these isolates, and the complete sequence of this 6.1-kb fragment was determined. Comparisons with other bacterial sequences showed that the fragment is the tufB-secE-nusG-rplKAJL-rpoB gene cluster. The organization of this gene cluster is similar to that of the homologous cluster found in Escherichia coli. Except for three nucleotide changes, the sequence was identical among Japanese and Indonesian isolates. A loop-mediated isothermal amplification (LAMP) assay based on the conserved sequence of the nusG-rplKAJL-rpoB gene cluster was developed for the detection of the GO. The LAMP product was rapidly detected on nylon membranes by staining with AzurB. LAMP could detect as low as about 300 copies of the nusG-rplKAJL-rpoB fragment of the Japanese and Indonesian isolates of GO. The LAMP-based detection method, which does not depend upon a thermal cycler and electrophoresis apparatus, will be useful for under-equipped laboratories, including those found in extension centers and quarantine offices. Additional keywords: Diaphorina citri, quantitative PCR

Subandiyah, S., Iwanami, T., Tsuyumu, S., and Ieki, H. 2000. Comparison of 16S rDNA and 16S/23S intergenic region sequences among citrus greening organisms in Asia. Plant Dis. 84:1518. Polymerase chain reaction was used to amplify and sequence the 16S ribosomal RNA gene (rDNA) and 16S/23S intergenic region of several isolates of citrus greening organism (GO) from Japan, the Philippines, Indonesia, and Thailand. The sequences of 16S rDNA were identical among all the isolates studied, very similar to the published sequences of Thai (99.4 to 100% identity), Nepalese (100% identity), and Indian (98.8% identity) strains, and less similar to an African strain (97.5% identity). The sequences of the intergenic region between 16S and 23S rDNA were also identical among the isolates examined as well as the reported Nepalese and Thai isolates. They were close to the sequences of reported strains of India and China (99.2%) and apart from those of the African strain (85.5%). These results suggested that some isolates of GO from Japan, the Philippines, Indonesia, Thailand, and Nepal cons titute one strain, which is similar to Indian and Chinese strains and distinct from the African strain.