Enhancing PCR capacity in detection of “Candidatus Liberibacter asiaticus” utilizing whole genome sequence information

Authors: BAO, M - South China Agricultural University; ZHENG, Z - South China Agricultural University; SUN, X - Florida Department Of Agriculture And Consumer Services; Chen, Jianchi; DENG, X - South China Agricultural University

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/3/2019
Publication Date: 8/6/2019
Citation: Bao, M., Zheng, Z., Sun, X., Chen, J., Deng, X. 2019. Enhancing PCR capacity in detection of “Candidatus Liberibacter asiaticus” utilizing whole genome sequence information. Plant Disease. 104:527-532. https://doi.org/10.1094/PDIS-05-19-0931-RE.
DOI: https://doi.org/10.1094/PDIS-05-19-0931-RE

Interpretive Summary: Citrus Huanglongbing (HLB, yellow shoot disease) is a high destructive disease in citrus production worldwide. HLB is associated with a bacterium called “Candidatus Liberibacter asiaticus” (CLas). Detection of CLas relies on PCR. A PCR that accurately confirms or excludes CLas infection in citrus tree/Asian citrus psyllid samples is critical for HLB management. HLBas-PCR is currently the standard procedure for CLas detection. However, HLBas-PCR could not accurately detect and confirm CLas at very low concentration. This research was to address such a problem. With the help of recently available CLas whole genome sequence data, a sequence error in HLBas-PCR was identified and corrected. By introducing another PCR system, RNR-PCR, derived from a five-copy gene, ambiguity between low CLas concentration and no CLas was resolved. Furthermore, a new PCR system, 4CP-PCR, based on a four-copy gene, was developed. Techniques developed from this study will help to enhance current HLB management by improving CLas detection sensitivity and accuracy.

Technical Abstract: “Candidatus Liberibacter asiaticus” (CLas) is an unculturable a-proteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease). PCR procedures that accurately confirm or exclude CLas infection in citrus tree / Asian citrus psyllid samples are critical for HLB management. When CLas was described in 1994, a 23-bp signature oligonucleotide sequence (OI1) in the three-copy 16S rRNA gene (rrs) was acquired through Sanger sequencing. OI1 contained single nucleotide polymorphisms (SNPs) separating CLas from non-CLas species. The SNPs were used to design primer HLBas, a key component in the USDA TaqMan PCR (HLBas-PCR), for CLas detection. Recent development in next generation sequencing (NGS) technology has led to the publication of 15 CLas whole genome sequences (WGSs). NGS provided higher sequence quality than that of Sanger sequencing. Based on WGS data, this study re-evaluated the sequence integrity of OI1/HLBas and identified/confirmed a missing nucleotide G in both OI1 and HLBas. At low Ct (<30), HLBas-PCR remained reliable in CLas detection. At high Ct (>35), CLas-PCR could not differentiate samples between low CLas titer and no CLas. The addition of RNR-PCR derived from the five-copy nrdB helped to resolve the problem. Towards the same direction of utilizing recently available genomic information, a 4CP-PCR system, based on a four-copy genomic locus, was developed. Evaluation with 175 HLB samples showed that 4CP-PCR was more sensitive than HLBas-PCR and shared similar sensitivity with RNR-PCR.