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United States Department of Agriculture

Agricultural Research Service

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Location: Subtropical Plant Pathology Research

2013 Annual Report

1a. Objectives (from AD-416):
1. Directly compare the inoculum potential of fruit and foliage. 2. Examine the effect of packingline treatments in reducing the population of Xcc. 3. Confirm the efficacy of pretreatment with detergent vs. chlorine for decontamination. 4. Ascertain the survival of bacteria in wounds in the peel. 5. Examine survival and contribution of cull piles to inoculum dispersal. 6. Examine the incidence, severity and inoculum production capability of fruit being culled from packingline runs. 7. Establish the effect of flash heating (or cooling) for reducing the population of Xcc in fruit lesions.

1b. Approach (from AD-416):
We will make direct, replicated comparisons of fruit and foliage inoculum production capability. The experiments will be repeated throughout the season on fruit and foliage through harvest and processing to collect these data.We will compare packingline processed and unprocessed fruit for canker lesion activity, and each stage of the process checked independently (rinse/chlorine/detergent/waxing) to ascertain which are the most efficacious at reducing the activity of the lesion. Both bioassay tests and cultures of samples will be taken from each treatment to quantify survival. Treatments will be replicated and analyzed using standard statistical procedures.

3. Progress Report:
This research related to inhouse project objective 1c: Characterize meteorological components affecting the epidemiology of Asiatic citrus canker (ACC), the interaction of the Asian leaf miner with ACC, the Asian citrus psyllid with Huanglongbing (HLB), the interaction of whiteflies with Squash vein yellowing virus (SqVYV), and the interaction of thrips with Groundnut ringspot virus (GRSV). The risk of introduction of Asiatic citrus canker (Xanthomonas citri subsp. Citri (Xcc)) to new, unaffected citrus producing areas both within the U.S. and internationally is a major concern for those citrus producing areas attempting to remain free of citrus canker. Prior research by an international group, which included the proposing investigators, resulted in a publication that served as the justification for USDA’s APHIS to promulgate a new regulation (7 CFR Part 301, FR Doc E915508 “Citrus Canker; Movement of Fruit from Quarantined Areas). This rule eliminated the requirement that fruit lots be inspected at the packinghouse and found to be free of canker symptoms and reduces industry costs by an estimated $15 million annually. It also removed the prohibition imposed on the movement of fruit from a quarantined area to commercial citrus producing states. While relieving restrictions on interstate movement of fresh citrus fruit within the U.S., the new ruling still protects canker-free areas from citrus canker. However, international trading partners remain unwilling to import fresh fruit from canker infected areas and seek further evidence that fruit is not a pathway for Xcc introduction to new areas, and that Xcc does not survive on fruit in sufficient populations to establish new infections. This project is devised to address this barrier to international trade by providing additional objective data to support the hypothesis (in previous work) that indicated lesions on harvested, packingline processed fruit are of no risk to citrus industries in canker-free areas overseas. To explore the potential of various temperature treatments on Xcc survival, the temperature change within and around the fruit while it was in the different bath temperatures. To accomplish this, we used our custom made temperature probe, to record temperatures at the surface of the fruit, 2 mm in (inside the rind), then 1, 2, 3, 4, and 5 cm into the fruit. This data gives an accurate analysis of how quickly and how much heat gets into the fruit during treatment. From the analysis of the 2012 replications, we noticed that at the higher test temperatures 55 and 60ºC), a majority of the heat is absorbed into the fruit within the first 1.5 minutes, however, only the outermost readings were affected. For example, after the fruit is immersed in 60ºC for 5 minutes, the temperature just inside the peel reached an ultimate temperature of 50.4ºC, but by 1.5min into the immersion, the temperature had already reached 44ºC. When looking deeper within the fruit, the heat treatments didn’t appear to affect the core temperatures much at all. Even at 2cm into the fruit, when immersed in 60ºC for 5 min, the temperature only was raised 7.5ºC. In order to reduce the variability in lesions and fruit used for heat and cold treatments we are studying the population dynamics of Xcc in grapefruit on tree (i.e., in situ) utilizing a needle free pulse injection device to infiltrate stomata in the fruit rind with a controlled volume of bacterial suspension by injection with pressurized gas. The device produces a uniform inoculation of Xcc into the fruit as measured by the volume of infiltration and diameter of the infiltrated area. Reproducible lesion size, number and populations of Xcc are obtained after repeated sampling of the fruit lesions through time. The in situ assay of fruit is being used to study Xcc population development in lesions through the season to determine the susceptibility of the rind to infection and the population dynamics as fruit reach full expansion before harvest. Fruit inoculations were initiated at the beginning of June and were repeated every three weeks to evaluate susceptibility of different fruit size classes. Inoculum concentrations higher than105 CFU on small fruit size classes produced individual stomatal lesions but at 107 CFU produced total necrosis and fruit drop. Fruit 3-5 cm in diameter inoculated with 105 CFU developed typical erumpent lesions with an initial population of ~7.2 x 108. Lesions expanded from 1-2 mm in diameter after 2 weeks to 7-9 mm after 12 weeks of fruit development and maintained populations with little or no decline for 10 weeks after inoculation. Fruit inoculated at 7-9 cm diameter developed lesions but these lesions were less erumpent and did not expand to more than 1-2 mm in diameter. Populations in these lesions were in the range of 107. Multiple attempts to injection-infiltrate fruit > 8-9 cm (after the end of July) produced very little inoculum penetration of rind and failed to produce lesions. These fruit inoculation trials will be repeated in 2013 to confirm reproducibility of lesion population dynamics. Citrus canker (Xanthomonas citri subsp. citri [Xcc]) can cause yield loss of susceptible citrus and result in trade restrictions of fresh fruit. Both for regulatory purposes and for experiments studying the epidemiology and spread of the disease, accurate detection and quantification of viable inoculum is critical. To compare bioassay (the gold standard) to a media-based culture method for detection of Xcc, lesions were taken from fruit, leaves and shoots in a commercial orchard in Florida in 2009-10 and 2010-11 and assayed via both methods. There were some misclassifications, but true positives (sensitivity) and true negatives (specificity) were the dominant classes. The apparent false diagnosis, false positives had the highest proportion of miscalculations (4.3-21.4%). This was due to more frequent detection of Xcc at concentrations =102 Xcc/ml when using culture compared to the bioassay. The false negative rate was consistently low (0.9-5.7%), confirming that in only a few cases did culture fail to detect Xcc when Xcc was present. Area under the curve for receiver operator characteristic analysis ranged from 0.79-0.97, confirming that culture was an accurate test. Of the different lesion sources, lesions from shoots tended to have the highest proportion of false positives (13.1-21.4% compared to 4.3-15.7% for lesions from fruit or leaves). There was a comparatively high frequency of lesions on shoots producing =102 Xcc/ml compared to lesions on fruit or leaves. The data demonstrate the value of culture as a reliable and complementary way to detect and quantify. Two additional packingline experiments are now complete. The data analysis are underway, but due to the preponderance of zeroes (no pathogen detected) among some of the samples novel methods for analysis of these zero-inflated data are being developed. Preliminary results suggest that time in storage has a dramatic effect in reducing lesion activity. Later harvested fruit have lower lesion activity. These data confirm previous year’s results showing that the risk posed by fruit lesions as a source of inoculum declines rapidly with weeks in storage and with harvest date.” Xcc compared to bioassay alone or other published methods.

4. Accomplishments

Last Modified: 2/23/2016
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