Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, ‘<i>Candidatus</i> Liberibacter asiaticus’ (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, <i>Diaphorina citri</i> Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy <i>nrd</i>B gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the <i>nrd</i>B target as low as ~2.6 Log<sub>10</sub> copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.
AbstractHuanglongbing (HLB), or Citrus Greening, is one of the most devastating diseases affecting agriculture today. Widespread throughout Citrus growing regions of the world, it has had severe economic consequences in all areas it has invaded. With no treatment available, management strategies focus on suppression and containment. Effective use of these costly control strategies relies on rapid and accurate identification of infected plants. Unfortunately, symptoms of the disease are slow to develop and indistinct from symptoms of other biotic/abiotic stressors. As a result, diagnosticians have focused on detecting the pathogen, Candidatus Liberibacter asiaticus, by DNA-based detection strategies utilizing leaf midribs for sampling. Recent work has shown that fibrous root decline occurs in HLB-affected trees before symptom development among leaves. Moreover, the pathogen, Ca. Liberibacter asiaticus, has been shown to be more evenly distributed within roots than within the canopy. Motivated by these observations, a longitudinal study of young asymptomatic trees was established to observe the spread of disease through time and test the relative effectiveness of leaf- and root-based detection strategies. Detection of the pathogen occurred earlier, more consistently, and more often in root samples than in leaf samples. Moreover, little influence of geography or host variety was found on the probability of detection.
ABSTRACT
We report here the draft genome sequence of “
Candidatus
Liberibacter asiaticus” strain TX2351, collected from Asian citrus psyllids in south Texas, USA. The TX2351 genome has a size of 1,252,043 bp, a G+C content of 36.5%, 1,184 predicted open reading frames, and 52 RNA genes.