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Controlled replication of 'Candidatus Liberibacter asiaticus' DNA in citrus leaf discs.

ABSTRACT: 'Candidatus Liberibacter asiaticus' is a fastidious bacterium and a putative agent of citrus greening disease (a.k.a., huanglongbing, HLB), a significant agricultural disease that affects citrus fruit quality and tree health. In citrus, 'Ca. L. asiaticus' is phloem limited. Lack of culture tools to study 'Ca. L. asiaticus' complicates analysis of this important organism. To improve understanding of 'Ca. L. asiaticus'-host interactions including parameters that affect 'Ca. L. asiaticus' replication, methods suitable for screening pathogen responses to physicochemical and nutritional variables are needed. We describe a leaf disc-based culture assay that allows highly selective measurement of changes in 'Ca. L. asiaticus' DNA within plant tissue incubated under specific physicochemical and nutritional conditions. qPCR analysis targeting the hypothetical gene CD16-00155 (strain A4) allowed selective quantification of 'Ca. L. asiaticus' DNA content within infected tissue. 'Ca. L. asiaticus' DNA replication was observed in response to glucose exclusively under microaerobic conditions, and the antibiotic amikacin further enhanced 'Ca. L. asiaticus' DNA replication. Metabolite profiling revealed a moderate impact of 'Ca. L. asiaticus' on the ability of leaf tissue to metabolize and respond to glucose.

SUBMITTER: Attaran E

PROVIDER: S-EPMC7111093 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Controlled replication of 'Candidatus Liberibacter asiaticus' DNA in citrus leaf discs.

Attaran Elham E Berim Anna A Killiny Nabil N Beyenal Haluk H Gang David R DR Omsland Anders A

Microbial biotechnology 20200120 3

'Candidatus Liberibacter asiaticus' is a fastidious bacterium and a putative agent of citrus greening disease (a.k.a., huanglongbing, HLB), a significant agricultural disease that affects citrus fruit quality and tree health. In citrus, 'Ca. L. asiaticus' is phloem limited. Lack of culture tools to study 'Ca. L. asiaticus' complicates analysis of this important organism. To improve understanding of 'Ca. L. asiaticus'-host interactions including parameters that affect 'Ca. L. asiaticus' replicati ...[more]

PMID: 31958876

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Project description:BackgroundHuanglongbing (HLB) is one of the most destructive citrus diseases in the world. The disease is associated with the presence of a fastidious, phloem-limited α- proteobacterium, 'Candidatus Liberibacter asiaticus', 'Ca. Liberibacter africanus' or 'Ca. Liberibacter americanus'. HLB-associated Liberibacters have spread to North America and South America in recent years. While the causal agents of HLB have been putatively identified, information regarding the worldwide population structure and epidemiological relationships for 'Ca. L. asiaticus' is limited. The availability of the 'Ca. L. asiaticus' genome sequence has facilitated development of molecular markers from this bacterium. The objectives of this study were to develop microsatellite markers and conduct genetic analyses of 'Ca. L. asiaticus' from a worldwide collection. Two hundred eighty seven isolates from USA (Florida), Brazil, China, India, Cambodia, Vietnam, Taiwan, Thailand, and Japan were analyzed.ResultsA panel of seven polymorphic microsatellite markers was developed for 'Ca. L. asiaticus'. Microsatellite analyses across the samples showed that the genetic diversity of 'Ca. L. asiaticus' is higher in Asia than Americas. UPGMA and STRUCTURE analyses identified three major genetic groups worldwide. Isolates from India were genetically distinct. East-southeast Asian and Brazilian isolates were generally included in the same group; a few members of this group were found in Florida, but the majority of the isolates from Florida were clustered separately. eBURST analysis predicted three founder haplotypes, which may have given rise to three groups worldwide.ConclusionsOur results identified three major genetic groups of 'Ca. L. asiaticus' worldwide. Isolates from Brazil showed similar genetic makeup with east-southeast Asian dominant group, suggesting the possibility of a common origin. However, most of the isolates recovered from Florida were clustered in a separate group. While the sources of the dominant 'Ca. L. asiaticus' in Florida were not clearly understood, the less-pervasive groups may have been introduced directly from Asia or via Brazil. Notably, the recent outbreak of HLB in Florida probably occurred through multiple introductions. Microsatellite markers developed in this study provide adequate discriminatory power for the identification and differentiation of closely-related isolates, as well as for genetic studies of 'Ca. L. asiaticus'.

Project description:Huanglongbing (HLB) is a citrus disease of worldwide importance, associated with the presence of Candidatus Liberibacter asiaticus (Las) and vectored by the psyllid Diaphorina citri in Asia and the Americas. To properly manage HLB, removal of inoculum sources and control of the psyllid are undertaken. We evaluated the percentage of the psyllid population with Las, sampled from yellow sticky traps over a three-year period and its relationship with insect population, regions, season of the year, and HLB management in citrus areas in the southwestern, central, and northern regions of São Paulo (SP) and southwestern region of Minas Gerais states, Brazil. In each reading, up to 50 psyllids per region were collected and detection of Las in individual psyllids were made by quantitative polymerase chain reaction. The percentage of psyllids with Las-an average of 65.3%-was constant throughout the year in the southwestern region of SP state, while showing an increase from spring to autumn when sampled from central to northern regions. The proportion of psyllids carrying Las from each region and year period were compared by a proportion test and spectral density analysis. The proportion of psyllids carrying Las evaluated in the same region in different seasons presented statistical differences in central (Araraquara) and southwestern (Santa Cruz do Rio Pardo) regions in 2015, with higher values in the first semester (summer and autumn) than in the second semester (winter and spring). Orchards with poor HLB management had higher incidence of psyllids with Las. Spectral density analysis indicated that good management areas had 50% less relevant peaks of psyllids with Las than in areas with poor HLB management practices. The relationship between the percentage of psyllids carrying Las and the number of captured psyllids in the region in a given time denotes the most critical intake time for HLB spread in citrus orchards. The reduction in the population of psyllids carrying Las is a direct benefit from the use of good management practices.

Dataset Information

Controlled replication of 'Candidatus Liberibacter asiaticus' DNA in citrus leaf discs.

Publications

Controlled replication of 'Candidatus Liberibacter asiaticus' DNA in citrus leaf discs.

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