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ARS Home » Pacific West Area » Parlier, California » San Joaquin Valley Agricultural Sciences Center » Crop Diseases, Pests and Genetics Research » Research » Publications at this Location » Publication #359702

Research Project: Identification of Novel Management Strategies for Key Pests and Pathogens of Grapevine with Emphasis on the Xylella Fastidiosa Pathosystem

Location: Crop Diseases, Pests and Genetics Research

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

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

Submitted to: International Research Conference on Huanglongbing
Publication Type: Abstract Only
Publication Acceptance Date: 2/18/2019
Publication Date: 3/6/2019
Citation: Bao, M., Zheng, Z., Sun, X., Chen, J., Deng, X. 2019. Enhancing PCR capacity in early detection of “Candidatus Liberibacter asiaticus” utilizing whole genome sequence information. International Research Conference on Huanglongbing.

Interpretive Summary:

Technical Abstract: “Candidatus Liberibacter asiaticus” (CLas), an unculturable a-proteobacterium, is associated with citrus Huanglongbing (HLB, yellow shoot disease). PCR procedures that effectively confirm or exclude CLas infection in asymptomatic or high Ct (>35) samples are important for HLB management. CLas was defined mainly by a 23-bp signature oligonucleotide sequence (OI1) in the 16S rRNA gene (rrs) acquired through Sanger sequencing in 1994. OI1 contained single nucleotide polymorphisms (SNPs) separating CLas from non-CLas species. The SNP region was used in primer HLBas, a key component of the current USDA TaqMan PCR (HLBas/HLBprobe/HLBr) system for CLas detection. By analyzing the 11 whole genome sequences of CLas currently available in GenBank database, a missing nucleotide G in OI1/HLBas was found. This study evaluated the effect of the missed-G in CLas detection. A corrected primer CLas-4G was developed to replace HLBas in the TaqMan PCR system. The CLas-4G PCR system reduced Ct by 0.76 (n=175) comparing to that of the HLBas PCR system. The second study developed a gene-copy-ratio PCR (gcrPCR) system (used in both TaqMan probe and CYBR green formats). Primer set RNR1f/RNR1r, derived from the 5-copy nrdB gene encoding ribonucleotide reductase (RNR), was referenced to primer set CLas-4G/HLBr, derived from the 3-copy rrs gene. The Ct ratio (Rrnd:rrs) of the gcrPCR effectively differentiated the result of CLas positive (Rrnd:rrs <1) from that of CLas negative (Rrnd:rrs >1) at high Ct situation. And, thirdly, a new primer set 4CPf/4CPr from a 4-copy genomic locus of CLas was developed. PCR with 4CPf/4CPr had a Ct reduction of at least 0.6 (n=100) from those of the rrs-based primer sets. However, the genetic nature of the 4CPf/4CPr locus remained unknown.