A new medium designated Liber A has been designed and used to successfully cultivate all three 'Candidatus Liberibacter spp.,' the suspect causative agents of huanglongbing (HLB) in citrus. The medium containing citrus vein extract and a growth factor sustained growth of 'Ca. Liberibacter spp.' for four or five single-colony transfers before viability declined. Colonies, positive for 'Ca. L. asiaticus' by a 16s-based rDNA real-time polymerase chain reaction (RT-PCR) assay and sequencing, were irregular-shaped, convex, and 0.1 to 0.3 mm after 3 to 4 days. Suspect 'Ca. L. asiaticus' and 'Ca. L. americanus' cells were observed in infected tissue and on agar culture by scanning electron microscopy. The cells were ovoid to rod shaped, 0.3 to 0.4 by 0.5 to 2.0 microm, often with fimbriae-like appendages. Two strains of 'Ca. L. asiaticus' and one of 'Ca. L. americanus' grown on Liber A medium were pathogenic on citrus and could be isolated from noninoculated tissues of inoculated trees and seedlings 9 and 2 months later, respectively. The identity was confirmed by RT-PCR and 16s rDNA sequencing. This is the first report of the cultivation and pathogenicity of 'Ca. L. asiaticus' and 'Ca. L. americanus' associated with symptoms of HLB.

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive diseases of citrus worldwide. HLB is associated with three species of ‘Candidatus Liberibacter’ with ‘Ca. L. asiaticus’ (Las) being the most widely distributed around the world, and the only species detected in Thailand. To understand the genetic diversity of Las bacteria in Thailand, we evaluated two closely-related effector genes, lasA I and lasA II, found within the Las prophages from 239 infected citrus and 55 infected psyllid samples collected from different provinces in Thailand. The results indicated that most of the Las-infected samples collected from Thailand contained at least one prophage sequence with 48.29% containing prophage 1 (FP1), 63.26% containing prophage 2 (FP2), and 19.38% containing both prophages. Interestingly, FP2 was found to be the predominant population in Las-infected citrus samples while Las-infected psyllids contained primarily FP1. The multiple banding patterns that resulted from amplification of lasA I imply extensive variation exists within the full and partial repeat sequence while the single band from lasA II indicates a low amount of variation within the repeat sequence. Phylogenetic analysis of Las-infected samples from 22 provinces in Thailand suggested that the bacterial pathogen may have been introduced to Thailand from China and the Philippines. This is the first report evaluating the genetic variation of a large population of Ca. L. asiaticus infected samples in Thailand using the two effector genes from Las prophage regions.

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Characterization of strains of Ralstonia solanacearum, the causal agent of potato bacterial wilt disease from Nepal and Thailand was performed based on pathogenicity, biochemical/physiological and serological tests. Fifteen R. solanacearum strains isolated from wilt infected potato plants and tubers grown in Nepal were characterized as race 3, biovar II based on the pathogenicity on different host plants, hypersensitive reaction on tobacco leaf and utilization of some sugars. Results of pathogenicity test show that all strains from Nepal had limited host range. Degree of virulence of all strains varied from high to medium in potato and tomato and medium to low in eggplant. They did not cause wilting in tobacco, pepper and peanut plants. Six strains from Thailand were characterized as biovar II and III. Additionally, comparisons on the physiological, biological and serological characters of seven strains from Nepal and six from Thailand revealed similar characters. Race 3 and biovar II of the pathogen was widely spread over potato growing areas of mid and high hills of Nepal. Both biovars II and III were prevalent in the potato growing areas of Thailand but biovar III was the most dominating one.Key words: Bacterial wilt; Potato; Pseudomonas solanacearum; Ralstonia solanacearumDOI: http://dx.doi.org/10.3126/narj.v4i0.4868Nepal Agriculture Research Journal Vol. 4&5, 2001/2002Page: 42-47Uploaded date: 9 June, 2011

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