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Huanglongbing (HLB), considered to be the most serious insect-vectored bacterial disease of citrus, is transmitted in nature by the Asian citrus psyllid Diaphorina citri and the African citrus psyllid Trioza erytreae. D. citri was discovered in southern Florida in 1998 and the HLB disease in 2005. Both have become established throughout citrus-producing areas of Florida. Murraya species are widely grown in southern Florida as ornamental hedges and are readily colonized by D. citri vectors. Colonies of D. citri, isolates of 'Candidatus Liberibacter asiaticus' from Taiwan and Florida, and the Murraya species were established in the BSL-3 biosecurity facility at Fort Detrick. In controlled inoculation experiments, D. citri transmitted 'Ca. L. asiaticus' into M. paniculata (34/36 plants) and M. exotica (22/23 plants), but not into Bergera (Murraya) koenigu. Disease symptoms rarely developed in Murraya plants; however, positive infections were deter- mined by conventional and real-time polymerase chain reaction (PCR). Back-inoculations of 'Ca. L. asiaticus' from M. paniculata to Madam Vinous sweet orange resulted in disease devel- opment in 25% of the inoculated plants. Considerable variability was observed in infection rates, titer, and persistence of 'Ca. L. asiaticus' in infected Murraya. [ABSTRACT FROM AUTHOR]

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 μm, 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. [ABSTRACT FROM AUTHOR]

Polymerase chain reaction/electrospray ionization-mass spectrometry (PCR/ESI-MS, previously known as "TIGER") utilizes PCR with broad-range primers to amplify products from a wide array of organisms within a taxonomic group, followed by analysis of PCR amplicons using mass spectrometry. Computer analysis of precise masses allows for calculations of base compositions for the broad-range PCR products, which can then be compared to a database for identification. PCR/ESI-MS has the benefits of PCR in sensitivity and high-throughput capacity, but also has the distinct advantage of being able to detect and identify organisms with no prior characterization or sequence data. Existing broad range PCR primers, designed with an emphasis on human pathogens, were tested for their ability to amplify DNA of well characterized phytobacterial strains, as well as to populate the existing PCR/ESI-MS bacterial database with base counts. In a blinded panel study, PCR/ESI-MS successfully identi- fied 93% of unknown bacterial DNAs to the genus level and 73% to the species/subspecies level. Additionally, PCR/ESI-MS was capable of detecting and identifying multiple bacteria within the same sample. The sensitivity of PCR/ESI-MS was consistent with other PCR based assays, and the specificity varied depending on the bacterial species. Preliminary tests with real life samples demonstrate a high potential for using PCR/ESI-MS systems for agricultural diagnostic applications. [ABSTRACT FROM AUTHOR]

The article discusses report on Citrus leprosis virus nuclear type in Sweet Orange in Colombia which includes types of Citrus leprosis virus, by researchers including Avijit Roy from Citrus Research and Education Center (CREC),University of Florida in Lake Alfred, Florida.

Many RNA viruses have genetically diverse populations known as quasispecies. Important biological characteristics may be related to the levels of diversity in the quasispecies (quasispecies cloud size), including adaptability and host range. Previous work using Tobacco mosaic virus and Cucumber mosaic virus indicated that evolutionarily related viruses have very different levels of diversity in a common host. The quasispecies cloud size for these viruses remained constant throughout serial passages. Inoculation of these viruses on a number of hosts demonstrated that quasispecies cloud size is not constant for these viruses but appears to be dependent on the host. The quasispecies cloud size remained constant as long as the viruses were maintained on a given host. Shifting the virus between hosts resulted in a change in cloud size to levels associated with the new host. Quasispecies cloud size for these viruses is related to host-virus interactions, and understanding these interactions may facilitate the prediction and prevention of emerging viral diseases.

The levels of population diversity of three related Sindbis-like plant viruses, Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), and Cowpea chlorotic mottle virus (CCMV), in infections of a common host, Nicotiana benthamiana, established from genetically identical viral RNA were examined. Despite probably having a common evolutionary ancestor, the three viruses maintained different levels of population diversity. CMV had the highest levels of diversity, TMV had an intermediate level of diversity, and CCMV had no measurable level of diversity in N. benthamiana. Interestingly, the levels of diversity were correlated to the relative host range sizes of the three viruses. The levels of diversity also remained relatively constant over the course of serial passage. Closer examination of the CMV and TMV populations revealed biases for particular types of substitutions and regions of the genome that may tolerate fewer mutations.