Heat treatment eliminates 'Candidatus Liberibacter asiaticus' from infected citrus trees under controlled conditions.

Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide. The three known causal agents of HLB are species of α-proteobacteria: 'Candidatus Liberibacter asiaticus', 'Ca. L. africanus', and 'Ca. L. americanus'. Previous studies have found distinct variations in temperature sensitivity and tolerance among these species. Here, we describe the use of controlled heat treatments to cure HLB caused by 'Ca. L. asiaticus', the most prevalent and heat-tolerant species. Using temperature-controlled growth chambers, we evaluated the time duration and temperature required to suppress or eliminate the 'Ca. L. asiaticus' bacterium in citrus, using various temperature treatments for time periods ranging from 2 days to 4 months. Results of quantitative polymerase chain reaction (qPCR) after treatment illustrate significant decreases in the 'Ca. L. asiaticus' bacterial titer, combined with healthy vigorous growth by all surviving trees. Repeated qPCR testing confirmed that previously infected, heat-treated plants showed no detectable levels of 'Ca. L. asiaticus', while untreated control plants remained highly infected. Continuous thermal exposure to 40 to 42°C for a minimum of 48 h was sufficient to significantly reduce titer or eliminate 'Ca. L. asiaticus' bacteria entirely in HLB-affected citrus seedlings. This method may be useful for the control of 'Ca. Liberibacter'-infected plants in nursery and greenhouse settings.

'Ca. Liberibacter asiaticus' carries an excision plasmid prophage and a chromosomally integrated prophage that becomes lytic in plant infections.

Huanglongbing (HLB), also known as citrus greening, is a lethal disease of citrus caused by several species of 'Candidatus Liberibacter', a psyllid-transmitted, phloem-limited, alpha proteobacteria. 'Ca. Liberibacter asiaticus' is widespread in Florida citrus. The recently published 'Ca. L. asiaticus' psy62 genome, derived from a psyllid, revealed a prophage-like region of DNA in the genome, but phage have not been associated with 'Ca. L. asiaticus' to date. In the present study, shotgun sequencing and a fosmid DNA library of curated 'Ca. L. asiaticus' UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in 'Ca. L. asiaticus' and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. Phage particles associated with 'Ca. L. asiaticus' were found in the phloem of infected periwinkles by transmission electron microscopy. In psyllids, both SC1 and SC2 were found only as prophage.