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4416

‘ Candidatus Liberibacter asiaticus’, the putative causal agent of citrus greening disease, is transmitted by the Asian citrus psyllid, Diaphorina citri, in a propagative, circulative, and persistent manner. Unfortunately, ‘ Ca. L. asiaticus’ is not yet available in pure culture to carry out Koch's postulates and to confirm its etiology. When a pure culture is available, an assay to test its infectivity in both the insect vector and the plant host will be crucial. Herein, we described a transmission assay based on the use of phloem sap extracted from infected citrus plants and topical feeding to D. citri nymphs. Phloem sap was collected by centrifugation, diluted with 0.1 M phosphate buffer pH 7.4 containing 20% (wt/vol) sucrose and 0.1% ascorbic acid (wt/vol) as an antioxidant, and delivered to third through fifth instar nymphs by placing droplets on the mouthparts. Nymphs unfolded the stylets and acquired the phloem sap containing the bacterial pathogen. Nymphs were then placed onto Citrus macrophylla seedlings (10 nymphs per seedling) for an inoculation period of 2 weeks. A transmission rate of up to 80% was recorded at 6 months postinoculation. The method could be a powerful tool to test the transmissibility of the bacterial pathogen after various treatments to reduce the viability of the bacteria or to block its transmission. In addition, it might be a potent assay to achieve Koch's postulates if a pure culture of ‘ Ca. L. asiaticus’ becomes available.

The phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) is the putative causal pathogen of the severe Asiatic form of huanglongbing (citrus greening) and is most commonly transmitted by the Asiatic citrus psyllid Diaphorina citri. CLas severely affects many Citrus species and hybrids and has been recorded in the Citrus relative, orange jasmine, Murraya paniculata (L.) Jack (syn. M. exotica L.). In this study, 13 accessions of three Murraya species (M. paniculata, M. sumatrana Roxb., and M. lucida [G.Forst.] Mabb.) and the Papuan form of a putative hybrid (M. omphalocarpa Hayata) were identified morphologically and molecularly based on sequence identity of the matK-5′trnK region of the chloroplast genome, and infection on these plants under field conditions was determined by PCR and quantitative real-time PCR (qPCR) on two to four occasions over 14 months. CLas was repeatedly detected in leaflet midribs by PCR and qPCR on four and three accessions of M. paniculata and M. sumatrana, respectively. It was not detected in leaflet midribs of single accessions of M. lucida and M. omphalocarpa. The species identification of the CLas-positive accessions was further confirmed using all the molecular taxonomic markers consisting of the six fragments of the maternally inherited chloroplast genome and part of the nuclear-encoded internal transcribed spacer (ITS) region. The results indicated that natural infection of M. paniculata and M. sumatrana with CLas can occur in Java. To our knowledge, this is the first demonstration of the natural infection of M. sumatrana with CLas. Further studies are required to determine whether infections persist in the absence of D. citri.