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Title: Exploring the use of NGS technology for citrus HLB diagnosis and microbiome research

Author
item ZHENG, ZHENG - South China Agricultural University
item WU, FENIGIAN - South China Agricultural University
item XU, M. - South China Agricultural University
item DENG, X. - South China Agricultural University
item STULBERG, M. - Animal And Plant Health Inspection Service (APHIS)
item RASCOE, J. - Animal And Plant Health Inspection Service (APHIS)
item SUN, X. - Florida Department Of Agriculture And Consumer Services
item JEYAPRAKASH, A. - Florida Department Of Agriculture And Consumer Services
item Chen, Jianchi

Submitted to: American Phytopathological Society Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 8/18/2017
Publication Date: 12/1/2017
Citation: Zheng, Z., Wu, F., Xu, M., Deng, X., Stulberg, M., Rascoe, J., Sun, X., Jeyaprakash, A., Chen, J. 2017. Exploring the use of NGS technology for citrus HLB diagnosis and microbiome research. American Phytopathological Society Abstracts. S5:55.

Interpretive Summary:

Technical Abstract: Citrus Huanglongbing (HLB) is currently threatening citrus production around the world. HLB is most prevalently associated with “Candidatus Liberibacter asiaticus” (CLas), an unculturable alfa-proteobacterium. Accurate diagnosis of HLB exclusively depends on PCR detection of CLas, which is determined by the presence of a targeted DNA sequence (standard PCR) or/and a Ct value below the defined threshold (TaqMan real-time PCR). In the case of a low level of PCR product or high Ct value, it is difficult to distinguish low CLas titer from non-specific DNA amplification. In this study, next generation sequencing (NGS) technology was used in HLB diagnoses and microbiome research. Fourteen NGS (10 MiSeq and 4 HiSeq) data sets from whole genome sequencing of CLas samples, which varied in Ct values measured by the primer set HLBas/HLBr, were subject to sequence read count assisted by BLASTn search. The results showed that: 1) Increase of HLBas reads was correlated to decrease of Ct values; 2) at high Ct values (e.g. Ct>35), a sample could have either low titer of CLas, or no CLas; 3) NGS detection sensitivity was related to sequencing throughput (HiSeq of 40M reads > MiSeq 20M reads); 4) detection of the nrdB gene (5 copies) was more sensitive than that on 16S rRNA gene (3 copies); 5) target (e.g. 16S rRNA gene) sequencing was more cost-effective than whole genome sequencing for CLas detection; and 6) other non-CLas bacteria, fungi and DNA viruses in HLB samples also were detected.