Progress 10/01/09 to 09/30/10
Progress Report Objectives (from AD-416) Develop canine detection methods for detection of Huanglungbing (HLB). Dogs will first be trained on infected versus non infected trees. Isolate a unique chemical signature that can be used to train the canines for the detection of HLB positive trees in the fields. Identify volatile compounds that are unique to HLB. Training of canines for the detection of HLB infected citrus trees (via a commercial canine scent training facility). Investigative use of canines technology for detection of HLB and Citrus Canker (CC). Simulate a grove survey and assess results. Investigative use of scent transfer units in determining thresholds of detection. Investigative use of canines technology in the detection of various lesion ages of CC infection, as well as fruit vs. foliar infections and detection on differing cultivars of citrus. Deployment � determine how best to deploy canine technology for disease detection in commercial groves and dooryards. Approach (from AD-416) Identity, quantity, and characteristics of volatile compounds specific to HLB by anlayzing infected trees using gas chromatography (in the lab) to separate the volatile components followed by mass spectrophotometric methods to characterize and identify the individual chemical components. Once the compound or compounds of interest are determined and the olfactory signature of HLB is determined, the canines will be trained as they were with the canker samples. If the data in the labs and the canine training prove positive, we will investigate the use of canines for detection of HLB and CC in a grove survey simulation. Known positive and negative trees will be put in close proximity to each other, and the two assay techniques will be tested against one another. Replicated tests will be conducted to determine the statistical reliability of both canine and estimate the proportion of false negative and false positive detections. This will be done by Receiver Operating Characteristic (ROC) curve analyses. Replicated tests will also be run to determine the maximum effective range of the canine, as well as minimal threshold levels of detection. Replicated studies will be undertaken to investigate the use of canines in the detection of various lesion ages of canker infection, as well as fruit vs. foliar infections, and detection on differing cultivars of citrus. Tests will be conducted to determine optimal deployment strategies for using the canines in real world situations, both in grove settings, packinghouses, and in the detection of dooryard tree contamination. This research relates to inhouse objective 2: Develop/refine rapid, sensitive reliable detection/sampling methods for pathogens. Our initial efforts in using canines to detect canker started in 2000 but were thwarted by the September 11 attack on the US, which permanently diverted the canine we had been training (it was included in the much needed efforts to detect explosives). However, prior to this dog�s reassignment, our initial research with canines for canker detection demonstrated their ability to detect citrus and citrus canker volatiles/aromas/essences, i.e., minute concentrations of volatile/aromatic compounds, deposited on cloth and other materials as well as directly from the air. A second attempt with USDA, APHIS agriculture detector dogs was cut short by decreasing budgets combined by increasing needs of detection of contraband agricultural products at points of entry into the US. However, our third and ongoing attempt has been much more successful. This most recent canine, �Juice�, has demonstrated an ability to detect canker infected citrus fruit with high reliability. The initial success of this project with regards to canker detection, suggests that the same technique may be useful for detection of Huanglongbing, perhaps presymptomatic while the disease is still latent. Such a tool would be very useful and is much needed, as it would allow both detection and early removal of presymptomatic (latently infected) trees, which also may be below the titer required for psyllids transmission. Two dogs have been trained to recognize the scent of citrus canker. Dogs were trained on infected versus non-infected trees and fruit. Training of the canines for the detection of canker infected citrus trees was accomplished via a commercial canine training facility in North Florida. As indicated above, �Juice�, demonstrated an ability to detect canker infected citrus fruit with high reliability (> 99.3%). Juice was capable of discriminating canker infected from non-infected fruit in 30 runs of 5 blind stations each, with only one false positive and no false negatives. Results indicate that canines can detect and differentiate citrus fruit infected with canker from non-infected tissues. The canine was then trained on infected versus non-infected Duncan grapefruit seedlings. Field trials were conduced in early April and again in July at the USHRL farm. Trees (75) were placed in the field in a 3 row by 25 tree/row design. Ten replications were conducted in which the proportion of disease trees ranged from 2-10%. The conditions were sunny but with a 15-20 MPH breeze. Even so, detection highly reliable. Data are presently under analyses but suggest 98% reliability. Short movie footage was also taken to document the dogs search and detection behavior. More field trials are scheduled over the next few months to validate the canine's performance.