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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Citrus and Other Subtropical Products Research » Research » Publications at this Location » Publication #331444
Title: Early detection of CLas infections in citrus

Author
item McCollum, Thomas
item Keremane, Manjunath
item MADHURABABU, KUNTA - Texas A&M University
item LEVESQUE, CYNTHIA - Citrus Research Board
item Hall, David

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 7/5/2016
Publication Date: N/A
Citation: N/A

Interpretive Summary: In the last decade, Florida citrus production has decreased approximately fifty percent and is the lowest it has been in the last fifty years. This decrease in production is mostly the result of Huanglongbing disease (also known as citrus greening). Spread by the Asian citrus psyllid, a tiny insect, citrus greening infects trees, leading to deformed and bitter fruit. Eventually, citrus greening kills the tree. One of the few ways to fight citrus greening is by removing the trees infected with Candidatus Liberibacter asiaticus, causal agent of Huanglongbing. To be effective, infections must be confirmed as soon as possible following infection. By default, to be considered “early”, Candidatus Liberibacter infection must be confirmed prior to visible Huanglongbing symptoms when the bacterial population is extremely low. In this study, we determined that using polymerase chain reaction amplification of Candidatus Liberibacter deoxyribonucleic acid single bacterial cells can be detected. In addition we found that infection could be detected following only two days of exposure to Asian citrus psyllids. These results document that polymerase chain reaction is a sensitive and reliable means for early detection of Candidatus Liberibacter in citrus. Using polymerase chain reaction for detection of Candidatus Liberibacter infection currently provides the earliest diagnostic method capable of detecting the pathogen thereby prompting rapid intervention of Huanglongbing disease.

Technical Abstract: “Early” detection of CLas infection is essential to minimize the risk of Huanglongbing (HLB) epidemics in areas where the pathogen has been recently introduced. Any delay in confirmation of CLas infection results in delays of regulatory and management actions, and increased spread of the pathogen even in areas with aggressive insecticidal ACP control. By default, to be considered “early”, CLas infection must be confirmed prior to visible HLB symptoms. Currently, qPCR with CLas 16S rDNA primers is used to identify suspect infections in large scale surveys of citrus and ACP. We report here on results of experiments that were conducted to verify the sensitivity and consistency of qPCR-based CLas detection. Our results demonstrate that primers used to amplify an 88bp fragment of CLas 16S rDNA target provide essentially 100% amplification efficiency (Log CN = 11.5 – 1/3.32Ct) indicating an appropriate endpoint (CN = 1) of Ct 38.3. These results were verified by analyses conducted among 4 laboratories. Using qPCR we found that CLas infections could be detected in citrus trees following exposure to CLas-infected ACP for 2 days. Our results support the sensitivity and robustness of qPCR for detection of CLas and demonstrate that qPCR is very effective for “early detection” of CLas. However, although pPCR is extremely sensitive and selective for CLas detection, identification of appropriate diagnostic samples in non-symptomatic trees remains challenging.