Early Detection of Citrus Greening (Huanglongbing,HLB)

Zhongguo Xiong, University of Arizona

The disease and the Pathogen

Citrus Greening, also known as Huanglongbing (HLB, 黄龙病 or "yellow dragon disease" in Chinese), is the most destructive of all citrus diseases, dubbed as “the green menace” (Callaway, 2012). It is widely believed to be caused by the bacterium “Candidatus Liberibacter asiaticus” (Las) in all citrus varieties. Two related bacteria, Ca. L. africanus and Ca. L. americanus, are also suspects. These Gram-negative bacteria live exclusively in the phloem of the host plants and spread from tree to tree by a sap-sucking insect: the Asian citrus psyllid, Diaphorina citri, in the US and Asia (Bové, 2006). The disease has already spread to Florida and Texas, and will likely spread to Arizona and California if no vigilance is taken. The psyllid has already been found in Arizona and California, but the disease has not been reported with the exception of an already eradicated, HLB-positive pummelo tree in the Los Angeles County (CDFA News, 2012). 

Trees with HLB decline rapidly and eventually produce green fruit of little value or stop producing completely. Typical symptoms of HLB start with leaf yellowing on an individual limb or in one sector of a tree. Individual leaves display blotchy yellowing or mottling throughout the leaf or mottling one side and green on the other side of a leaf. As the disease progresses, yellow shots on individual trees become evident. Diseased trees bear smaller fruit with bitter juice, and the fruit may stay partially green. Twigs and limbs of the affected trees gradually wither, followed by tree death. The initial symptoms could be observed in the greenhouse under experimental conditions within a few months; however, the symptoms might not be visible two to three years after a tree becomes infected in the field.

Fig. 1. Asian citrus psyllids. Brown adult, yellow nymphs, and white wax produced by the nymphs. Photo credit: Michael E. Rogers, University of Florida

Fig. 2 HLB symptoms. Photo credits: CREC, University of Florida

Economic Significance

Citrus and its value-added products are a major agricultural industry in the US. As of 2013, citrus production is valued at $3.2 billion (packhouse-door equivalent value) (USDA NAAS, 2013), and the economic impact of the citrus industry is many billion dollars more if value-added products are included in the estimate. The majority of acreage of citrus is planted in Florida (61%), followed by California (33%) with remaining acreage in Texas and Arizona. Despite its lower acreage, the citrus production value in California is nearly the same as that of Florida in 2013 (USDA NASS, 2013).

This highly valued industry is severely impacted by HLB. According a University of Florida estimate (Hodges and Spreen, 2012), the economic impact of HLB on Florida fresh citrus juice production was enormous between 2006, the year after the arrival of HLB, to 2011. The total citrus production dropped by 23 percent, cumulative orange juice production has reduced from 6.32 billion gallons in 2006 to 4.62 billion gallons in 2011, and Florida growers lost annual revenue of $1.7 billion. The same study also estimated a loss of more than $4.5 billion in the overall economic impact including value-added revenues and related operations. The cost to the public was also significant. Orange and juice prices have steadily increase; increased pesticide spread in order to slow down the spread of the disease has negatively impacted other IPM programs and increased pesticide residues in fresh fruit and juice. A similar economic impact would be likely if the HLB is introduced and established in Arizona and California, the other major citrus-producing states in the US.

HLB management

There are no cures for HLB at the moment despite intensive research efforts in the last few years. A few promising treatments, including heat therapies and micronutrient supplements, are currently being investigated. They may prolong the productive life of the infected trees, but their effectiveness is still unclear. The best management for this disease is to prevent its introduction and establishment, and immediate eradication if the disease is found right away. For this to be successful, a highly sensitive method must be developed for the detection of the bacterium and/or the disease at the earliest possible time. The major challenge is the detection sensitivity as the infested trees and psyllids often carry only a tiny amount of the bacterium. As the bacterium must infect before any signs or physiological changes occur in the infested trees, direct test for the presence of the bacterium would be the best logical choice for early detection.

HLB detection:

Currently, PCR-based detection methods are available, however they are not sensitive and reliable enough to detect the bacterium early before symptoms occur. A number of volatile chemical and metabolite-based detection methods are also being developed and tested but they are yet to be validated and “approved”. More sensitive and earlier detection of HLB is important in stopping the spread of HLB and helping currently affected citrus growers to manage the disease better. An example of a more sensitive PCR test based on the repeat sequences of a prophage in Las is show below. The phage-repeat based detection (Fig. 3, top) is 10 to 100 times more sensitive that the detection targeting Las 16S ribosomal DNA (Fig. 3, bottom). A new promising technology is digital PCR, which carries out 20,000 independent PCR reactions in one test and allows direct counting of the copy number of the target DNA molecules (Fig. 4).

Fig. 3. Amplification of Las-specific DNA by realtime PCR. DNA extracted from healthy citrus (lanes 1, 3, 5, 7,9, 11) and Las-infected citrus (lanes 2, 4, 6, 8. 10. 12) was diluted and amplified with primers specific for the prophage repeats (top panel) and 16S ribosomal DNA (bottom panel). Dilutions: 1,2, 1:5; 3,4, 1:50; 5,6, 1:500; 7,8, 1:5,000; 9,10, 1:50,000; 10,11, 1:500,000.  (Xiong, Z., unpublished data)

Fig. 4. Scanned images of a section of digital PCR chips. Each reaction on a chip is carried out in a volume of 0.7 nanoliters. The sample at the left contains 100 times more target Las DNA than the sample at the right. Two different fluorophores are used to probe either the Las phage repeat targets or the 16S rDNA target. Red dots represent amplification of phage repeat DNA, blue dots represent amplification of Las 16S rDNA, and green dots represent amplification of both target DNA molecules. (Xiong, Z., unpublished data)

References:

Baker, M. 2012. Digital PCR hits its stride. Nature Methods  9, 541–544

Bové, J. M. 2006. Huanglongbing: A destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology 88, 7-37.

Callaway E. 2008. Bioterror: The green menace. Nature, 452, 148-150

CDFA News. 2012. Citrus disease Huanglongbing detected in Hacienda Heights area of Los Angeles county. http://www.cdfa.ca.gov/egov/Press_Releases/Press_Release.asp?PRnum=12-012

Duan Y. P., Zhou, L., Hall, D. G., and et al. 2009. Complete genome sequence of citrus huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’ Obtained through metagenomics. Molecular Plant Microbe Interactions 22, 1011-1020.

Hodges, A. W., and Spreen, T. H. 2012. Economic Impacts of Citrus Greening (HLB) in Florida, 2006/07–2010/11. University of Florida IFAS extension publication EDIS document FE903. http://edis.ifas.ufl.edu

Li, W. B., Hartung, J. S., and Levy, L. 2006. Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. Journal of Microbiological Methods 66, 104-115

USDA National Agriculural Statistics Service (NASS).  (2013). Citrus Fruits 2013 Summary.  ISSN: 1948-9048

Huggett, J. F, Foy, C. A., Benes, V. et al. 2013. The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments. Clinical Chemistry 6:892–902.