Detection of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and Its Importance in the Management of Citrus Huanglongbing in Florida

Abstract

Citrus huanglongbing (HLB or citrus greening), is a highly destructive disease that has been spreading in both Florida and Brazil. Its psyllid vector, Diaphorina citri Kuwayama, has spread to Texas and Mexico, thus threatening the future of citrus production elsewhere in mainland North America. Even though sensitive diagnostic methods have been developed for detection of the causal organisms, Candidatus Liberibacter spp., the pathogen cannot be detected consistently in plants until symptoms develop, presumably because of low titer and uneven distribution of the causal bacteria in nonsymptomatic tissues. In the present study, TaqMan based real-time quantitative polymerase chain reaction methodology was developed for detection of 'Ca. L. asiaticus' in D. citri. Over 1,200 samples of psyllid adults and nymphs, collected from various locations in Florida, from visually healthy and HLB symptomatic trees at different times of the year were analyzed to monitor the incidence and spread of HLB. The results showed that spread of 'Ca. L. asiaticus' in an area may be detected one to several years before the development of HLB symptoms in plants. The study suggests that discount garden centers and retail nurseries may have played a significant role in the widespread distribution of psyllids and plants carrying HLB pathogens in Florida.

Novel set of real-time PCR primers for simultaneous detection of Liberibacter species associated with citrus Huanglongbing

“…For this purpose, the β-operon primer set f-rplLAs/r-rplLAs was selected for The rapid expansion of HLB in Brazil and Florida are examples where the confirmation of the disease occurred much later than the infection by the pathogen (Coletta-Filho et al, 2004;Manjunath et al, 2008), which clearly attests that the plant had been infected for a long period of time when disease symptoms appeared. It is rather difficult to diagnose HLB on the basis of disease symptoms, and when it happens, it is too late to prevent it from spreading.…”

“…This test uses a 16S rDNA-based TaqMan probe and a set of three different forward primers for the detection of the three different species of Liberibacter and one common reverse primer. This set has been successfully applied in the detection, identification and quantification of Las in host plants of citrus (Li et al, 2009;Tatineni et al, 2008) and in the psyllid vector (Manjunath et al, 2008).…”

“…Microscopy, serology and DNA-DNA hybridization used to detect the pathogens (Bové, 2006;Villechanoux et al, 1990) are either time consuming or lacking in sensitivity. Methods based on PCR have been widely used to detect HLB-associated Liberibacter, mainly based on the 16S rDNA and the beta operon sequences (Hocquellet et al, 1999;Hung et al, 1999;Jagoueix et al, 1996;Teixeira et al, 2005), and several real-time quantitative PCR assays (qPCR) have been designed to detect and quantify low Liberibacter titers, both in plants (Li et al, 2006;Teixeira et al, 2008) and insects (Manjunath et al, 2008).…”

Base científica para a erradicação de plantas sintomáticas e assintomáticas de Huanglongbing (HLB, Greening) visando o controle efetivo da doença

“…Dados preliminares de coletas de adultos de D. citri, realizadas em pomares infestados em São Paulo, revelaram porcentagens superiores a 60% de insetos infectivos quando originários de populações presentes em pomares com mais que 10% de plantas sintomáticas (Yamamoto et al, dados não publicados). Porcentagens similares de adultos infectivos foram observadas em diferentes locais na Flórida, mesmo quando em períodos de baixas populações do inseto (Manjunath et al, 2008). No entanto, o exemplo mais claro da presença de insetos infectivos, originários de áreas sem controle da doença foi apresentado por Tersi (2008).…”

First report of ‘Candidatus Liberibacter asiaticus’ in Diaphorina communis

“…The purified DNA amplification product from one adult specimen collected in Kamichu was directly sequenced in both directions at the Australian Genome Research Facility Ltd with automated sequencing using an Applied Biosystems 3730xl capillary sequencer (www.agrf.org.au) to confirm the identity of the amplified fragment. Real-time PCR was performed as per Li et al (2006) with an internal control for monitoring the quality of psyllid DNA extraction coding for the wingless gene using the primers and probe (DCF, DCR and DCP) described by Manjunath et al (2008).…”