How is the HLB-associated bacterium detected in citrus trees and Asian citrus psyllids?
Article written by Greg McCollum, Brianna McGuire, Elizabeth Grafton-Cardwell, Peggy G. Lemaux, & Lukasz Stelinski.
Revised July 24, 2018.
How can detection of CLas infections impact the development of HLB epidemics?
Candidatus Liberibacter asiaticus (CLas) is considered to be the bacterial pathogen that causes the incurable disease Huanglongbing (HLB) in citrus. CLas is transmitted to citrus trees when the insect vector, Asian citrus psyllid (ACP), feeds on plant tissue or when infected plant tissue is grafted onto a citrus tree. No matter how CLas arrives, when ACP invades citrus-growing regions, CLas bacterial infection and HLB soon follow. Maintaining ACP populations as low as possible by aggressive insecticide applications has been clearly shown to slow the spread of HLB. However, ACP can never be 100% eliminated by insecticides. So, if there are CLas-infected trees and even small populations of ACP, the incidence of HLB will increase over time. If trees infected with CLas can be identified and removed as early as possible, the chance of slowing an HLB epidemic improves significantly.
What is the problem?
Detection of CLas in citrus and ACP is necessary for regulatory action (tree removal) and to guide psyllid and disease management strategies. In general, confirming a pathogen’s presence in a host is usually accomplished by isolating the pathogen from tissue suspected to be infected, growing the pathogen in a sterile environment to obtain a pure culture, followed by several means of identification (examining the appearance of cultured cells, determining a pathogen’s DNA sequence, etc.). Unfortunately, culturing CLas outside of either citrus or ACP has not yet been possible, making confirmation of infection especially challenging. At present, the only means to confirm CLas infection is by detection of CLas DNA in extracts prepared from infected citrus tissue or psyllids.
What is the technique?
For citrus extracts and psyllids, the most sensitive and specific technology for detection of CLas DNA is the polymerase chain reaction (PCR) test. DNA is the molecule that contains the genetic information for all life, found in the cells of all organisms. PCR is a technique used to rapidly copy a very small section of DNA sequence so that it can be detected. PCR is somewhat similar to a copy machine. For example, if a single piece of paper is placed in a copy machine and the copy button is pressed 36 times, the end result is 37 copies.
For PCR, starting with a single copy of the DNA target sequence and running the PCR for 36 cycles results each copy making copies of itself, resulting in about 7 billion copies of the original target sequence.
A version of PCR, known as quantitative PCR (qPCR), is the regulatory standard for diagnosing CLas. In qPCR, each copy of target DNA sequence generated gives off a fluorescent signal, and that signal is measured at the end of each PCR cycle. The results of qPCR assays are expressed as the number of PCR cycles required to produce a fluorescent signal of a specific intensity, which is an indication of the starting number of copies of the target DNA. This value is known as the crossing threshold (Ct). The regulatory threshold for a positive plant sample is a Ct of 36, which means that it takes 36 or fewer cycles for sufficient fluorescence to be detected. A plant sample with a Ct value lower than 36 is defined as positive for CLas. If the Ct value is > 37, then the sample is viewed as either noise in the system, a very weak positive, or a negative. The test’s ability to detect a true positive becomes less reliable if there are fewer than three CLas cells in a plant sample.
Plant samples are prepared for PCR by chopping up and freeze drying the petioles, the slender stalks that join leaves to stems, of many leaves on a tree that is suspected to have the disease. These freeze-dried petioles are then ground, and DNA is extracted. qPCR is performed on these DNA extracts. By California law, a tree with a positive qPCR reading must be destroyed.
Psyllid DNA is also tested for CLas presence with qPCR. Up to 25 live psyllids collected from a tree are preserved in alcohol, taken to the laboratory, and crushed to create one unified sample. In California, positive psyllids do not result in tree removal, because it is not necessarily known where the psyllids picked up the bacterium. However, psyllid testing is very important as an early warning system to find the region where the bacterium is present so leaf testing can be intensified.
Who is working on the Project?
Greg McCollum, Research Plant Physiologist, USDA, ARS, Fort Pierce, FL and his research team are working to determine how soon CLas infections can be detected with qPCR following ACP feeding on citrus as well as how CLas moves in citrus. They are also collaborating with researchers at UC Riverside and UC Davis to validate technologies to improve early detection of CLas infections.
What are the challenges and opportunities?
Although qPCR is a very sensitive and specific method for detection of CLas DNA, qPCR is severely limited by sampling and testing capacity. It is only possible to evaluate 100 mg of tissue (about equal to a grain of rice) per sample. Because CLas is typically not evenly distributed in the citrus canopy but found in individual leaves or isolated to a branch, finding an infected leaf is effectively random, unless HLB symptoms are visible. If there are 10,000 leaves on a tree and the CLas infection is limited to a single branch on the tree, and one leaf is collected, the chance of finding CLas is only 1 in 10,000. Though the odds of finding CLas increases as HLB symptoms progress beyond one branch, it is critical to find the infection while limited to one branch, before ACP spreads further. Other early detection technologies (EDTs) measure a whole-tree immune response by CLas-infected trees or rapid distribution of bacterial byproducts throughout the tree rather than the quantity of CLas in a sample of leaves. There are numerous EDTs being studied - please see other EDT snapshots on this website for more details. These approaches could be used to identify quiescent infections prior to qPCR diagnostics, which would result in more efficient and effective use of qPCR resources and would enable regulators to remove trees before psyllids further spread HLB.
Funding source: This project is funded by the USDA, ARS, USDA, APHIS, Citrus Research Board, and California Citrus Nursery Advisory Board.