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Evaluation of Real‐time PCR Primer Sets for the Diagnosis of Huanlongbing (HLB) in Citrus Root Tissue
Author(s) -
Avila Courtney J.,
Park JongWon,
Kunta Madhurababu
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.663.20
Subject(s) - diaphorina citri , biology , vector (molecular biology) , horticulture , botany , hemiptera , biochemistry , gene , recombinant dna
Through the Step 2 program undergraduate students are placed into internships that will help foster their knowledge and readiness for careers in the field of agriculture and research studies. Collaboration between South Texas College and the diagnostics lab at the Texas A&M Kingsville Citrus Center has allowed student studies within the scope of plant and soil sciences. Huanglongbing, also known as citrus greening, is one of the most destructive diseases in citrus of which causal agents are unculturable phloem‐limited Gram‐negative Alphaproteobacteria that belongs to the genus ‘Candidatus Liberibacter’. So far, three species of Candidatus Liberibacter, CL asiaticus (CLas), americanus (CLam) and africanus (CLaf), are known to be causing HLB. While CLas is the most widely spread, CLaf and CLam are geographically limited to Africa and South America, respectively. These HLB causing bacteria are transmitted by citrus psyllids, Diaphorina citri in Asia and America and Trioza erytreae in Africa. As there is no known resistant citrus cultivar available for commercial cultivation, the early HLB detection is a critical step to deploy an efficient disease control strategy that is heavily dependent on chemical method to control insect vector population. Currently, symptomatic citrus leaves are the source material for the detection of HLB‐causing bacteria in citrus by real‐time PCR. However, the uneven distribution of HLB‐causing bacteria in a tree canopy can lead to a misdiagnosis. Previous work conducted in our lab indicated that HLB‐positive plant exhibits more uniform distribution of HLB‐causing bacteria in the root system, suggesting that root tissue can be used for HLB diagnosis. However, currently available real‐time PCR primers based on CLas 16s rDNA cannot be used for root HLB test due to non‐target amplification. In this study, we designed new sets of primers and probes based on 16s rDNA of all three HLB‐causing bacteria that can be used for HLB diagnosis with root tissue. BLASTn analysis against prokaryotic ribosomal RNA database after exclusion of 16s rDNAs of HLB causing bacteria revealed that the newly designed forward and reverse primers had ~61% and ~90% sequence identity, respectively, to the prokaryotic 16s rDNA database. The current study evaluated these three new real‐time PCR primer sets, each of which targets CLas, CLam and CLaf, using DNA templates prepared with root DNA extracts. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .