Detection of Citrus Huanglongbing by Fourier Transform Infrared–Attenuated Total Reflection Spectroscopy SAMANTHA A. HAWKINS,* BOSOON PARK, GAVIN H. POOLE, TIMOTHY GOTTWALD, WILLIAM R. WINDHAM, and KURT C. LAWRENCE US Department of Agriculture- Agricultural Research Service Russell Research Center, 950 College Station Rd, Athens, Georgia 30605 (S.H., B.P., B.W., K.L.); and US Department of Agriculture- Agricultural Research Service, US Horticultural Research Laboratory, Fort Pierce, Florida 34945 (G.P., T.G.) Citrus Huanglongbing (HLB, also known as citrus greening disease) was discovered in Florida in 2005 and is spreading rapidly amongst the citrus growing regions of the state. Detection via visual symptoms of the disease is not a long-term viable option. New techniques are being developed to test for the disease in its earlier presymptomatic stages. Fourier transform infrared–attenuated total reflection (FT-IR-ATR) spectroscopy is a candidate for rapid, inexpensive, early detection of the disease. The mid-infrared region of the spectrum reveals dramatic changes that take place in the infected leaves when compared to healthy non-infected leaves. The carbohydrates that give rise to peaks in the 900–1180 cm À1 range are reliable in distinguishing leaves from infected plants versus non-infected plants. A model based on chemometrics was developed using the spectra from 179 plants of known disease status. This model then correctly predicted the status of .95% of the plants tested. Index Headings: Citrus greening disease; Fourier transform infrared spectroscopy; Attenuated total reflection; FT-IR-ATR; Chemometrics; HLB. INTRODUCTION For several decades the occurrence of citrus greening disease, also known as Huanglongbing (HLB) or Yellow Dragon disease, has been of great concern in the citrus growing regions of the southern United States as well as in other major citrus regions of the world. 1–7 The citrus psyllid (Diaphorina citri) acts as a vector spreading the disease from infected trees to nearby and distant uninfected trees. 1–3,7 These psyllids were detected in Florida in 1998, 1 and HLB was confirmed to be in Florida citrus in 2005. 8 Since that time it has spread to 32 counties across the state, with the psyllid being found in two additional counties. 9 Unfortunately, once the tree is infected the disease does not always appear immediately. It may take multiple months to years for the infection to express visual signs depending on tree age and horticultural health. During this latent period the tree can, however, be contagious and infect numerous other trees. Thus, it is imperative that reliable techniques are developed for early, presymptomatic detection of the disease. Currently, the technique used most frequently is polymerase chain reaction (PCR) DNA analysis. 4,5,7–13 The PCR detection method is very accurate and is able to detect the disease before the disease becomes symptomatic but not before the bacteria proliferate in the portion of the plant assayed. Unfortunately, this method is also somewhat time-consuming and is relatively expensive. Several studies have been reported using other techniques, including spectroscopic methods, to attempt to quickly identify the citrus greening disease’s presence in plants. 14–17 Some of the reported methods are subjective or are not reproducible. Ideally, a technique needs to be developed that is highly reliable, quick, and inexpensive. Fourier transform infrared–attenuated total reflection (FT-IR-ATR) spectroscopy may be such a technique. In this study leaves from plants that were identified as infected and uninfected with the citrus greening disease using both conventional and real- time PCR methods were collected and studied by the ATR technique. The results of this study, which are presented below, are potentially very promising in providing a new method for the rapid detection of citrus greening. EXPERIMENTAL DETAILS Samples of leaves were collected in Florida from orange and grapefruit trees. There were several symptom types (subsets) identified during collection. These subsets include greenhouse quarantined negative (negative control), visual negative, and samples that showed visible symptoms of the HLB infection, including foliar blotchy mottle, yellow dragon (entire yellow shoots), foliar green islands, and various stages of chlorosis ranging from mild to severe. Several leaves were collected from each plant or area of a plant and combined as a single sample. HLB infected plants generally produce smaller leaves, which necessitates the use of several leaves per sample. The mid-rib veins of the leaves were separated for testing by PCR. The DNA was isolated using a modified sodium dodecyl sulfate/potassium acetate extraction method. 18 These samples were then normalized to contain 50 ng of DNA per lL of water. Analysis of 2 lL sample was performed in duplicate via a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City , CA) using Invitrogen Express Premix (Carlsbad, CA), along with primers and probe (labeled with NED- MGB TM ) developed by Li. 19 The thermal cycling protocol was 95 8C for 20 s, then forty cycles of 95 8C for 3 s and 60 8C for 30 s. PCR is a technique that amplifies and detects the amount of predetermined DNA strands. 20 Briefly, a probe to a specific piece of DNA, in this case a probe to a small section of the DNA sequence for HLB, is added to the DNA extracted from the sample. Through the cycling process, the DNA section to which the probe attaches is amplified. Under optimum conditions, and with perfect efficiency, the amount of DNA of interest is doubled with each cycle. With real-time PCR, the amplicon is ‘‘monitored’’ with each cycle. This can be done by tracking how much dye is turned on by the amplification process. In the technique used in this paper, a dye is attached to the DNA probe, along with a quencher. Once the probe attaches to the DNA of interest, the dye and quencher are cleaved from the probe (and each other), and therefore the dye fluoresces. 21 The amount of fluorescence is measured at the end Received 29 July 2009; accepted 26 October 2009. * Author to whom correspondence should be sent. E-mail: samantha. hawkins@ars.usda.gov. 100 Volume 64, Number 1, 2010 APPLIED SPECTROSCOPY 0003-7028/10/6401-0100$2.00/0 Ó 2010 Society for Applied Spectroscopy