Park, Jong-Won

Prophages/phages are important components of the genome of ‘Candidatus Liberibacter asiaticus’ (CLas), an unculturable alphaproteobacterium associated with citrus huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida but different to those reported in California and China. Further studies were performed on prophage type diversity. Analyses on Type 1-specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originating from Florida, and Type 1-B, clustered with prophage P-SGCA5-1 originating in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2-specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. The results from this study provide an initial baseline of information on genomic and population diversity of CLas in Texas.

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.