Controlled replication of ‘Candidatus Liberibacter asiaticus‘ DNA in citrus leaf discs

Abstract

Summary ‘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.

Revisiting the Complex Pathosystem of Huanglongbing: Deciphering the Role of Citrus Metabolites in Symptom Development

“…Several previous studies have been conducted to investigate the population dynamics of ‘ Ca. L. asiaticus’ and its relation to root growth and flushing cycles of citrus and non-citrus hosts [ 138 , 139 , 140 , 141 , 142 ] as well as within the population of its vector, D. citri [ 140 , 143 ]. In addition, numerous studies focused on the association between citrus flushing cycles and the population dynamics and demography of D. citri [ 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 ].…”

“…Generally, root growth and flushing of citrus plants are cyclic [ 152 ] and the population dynamics of ‘ Ca. L. asiaticus’ and its vector are positively correlated with these cycles of root growth and flushing rhythm of citrus plants [ 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 ]. Interestingly, almost the same patterns of seasonal fluctuation were observed between flushing rhythm and the African citrus greening pathogen, ‘ Ca .…”

“…Seasonal flush cycles may differ based on location/region, varieties/cultivars, tree age and health, and environmental conditions [ 147 , 155 ]. Based on empiric observations and available published data on flush densities in Florida citrus, three major flush cycles are observed in citrus groves ( Figure 6 ), including one main flush cycle (Spring flush) and two minor flush cycles (Summer flush and Fall flush, respectively) [ 142 ]. The Spring flush is the major flushing period that follows the cool season of the year and starts in early February to May; the Summer flush extends from early June to August, and the Fall flush begins in September/October, but very few flushes are observed between November and late January [ 142 , 147 , 148 , 149 , 150 ].…”

“…Interestingly, populations of both ‘ Ca. L. asiaticus’ and D. citri were boosted during the flush cycles and dropped thereafter [ 138 , 140 , 141 , 142 , 147 , 148 , 149 , 150 , 151 ]. Briefly, both live and total ‘ Ca.…”

“…Briefly, both live and total ‘ Ca. L. asiaticus’ populations had almost the same patterns corresponding to flush cycles of citrus plants, with significantly lower bacterial titer in December, January, and early February ( Figure 6 ) [ 138 , 142 ]. Furthermore, ‘ Ca.…”