Since Huanglongbing (HLB) was first found in Florida, growers have recognized the importance of controlling the Asian citrus psyllid (ACP) that vectors the disease.   ACP management programs have been developed and applied over large acreages, yet in many instances, the incidence of the disease continues to increase.   When asked, the growers invariably report that they are controlling ACP and it is obvious that the control programs are reducing the level of ACP in the groves.  However there is an outstanding question as to whether the levels of ACP are being reduced enough to limit the spread of HLB.  In most cases, growers have not implemented ACP scouting programs so the level of infestation is based on rough grove surveys and feelings.  Even if scouting programs are in place, there are no established data-based thresholds to trigger additional applications of pesticides resulting in a “chasing” approach where applications are made after an infestation is found.  Southern Gardens has had an ACP and HLB scouting program in place for many years.  By comparing the annual ACP levels to resulting HLB infection succeeding years, it may be possible to elucidate thresholds and then design ACP control programs that effectively limit the spread of HLB.  Data will be presented for three years of ACP scouting and four years of HLB scouting in an attempt to show the level of control that must be achieved in order to limit the spread of HLB.  The bottom line is that control thresholds are very low and the level of ACP control that must be achieved in order to limit spread HLB is much lower than most growers realize.

Invasive plant diseases can have devastating consequences on the local plant populations, in both agricultural and natural landscapes. Knowledge of the spatial patterns of pathogen spread can be used to guide more time- and cost-effective disease management strategies. Based on disease dispersal principles and consideration of host pattern, an improved plant disease epidemiological model was developed and tested for plant disease mapping. The model is able to characterize the disease dispersal gradient and predict infection risk, with indication of uncertainty, through heterogeneous environments without reference to the source of infection. As a result, sampling methods can be informed by the predicted prevalence map of the disease. In order to better describe the shapes of the dispersal gradients, three different dispersal functions (Exponential, Modified power law, and Cauchy distribution) were considered in the model. Two data sets of disease observations of Huanglongbing (HLB) of citrus in different landscapes (Southern Garden and Devils Garden plantation) in Florida were used to evaluate the performance of the improved method for disease mapping. The results showed that the improved model provided estimates of greater precision for unsampled hosts. With all different dispersal models compared, the exponential dispersal gradient gave the most satisfactory performance. All the determined information can help decision makers understand the spatial aspects of disease processes, and formulate decisions about disease control accordingly.

Florida growers have reported that enhanced nutritional programs (ENPs) maintain productivity of HLB-infected trees.  However, efficacy and sustainability of the nutritional approach for HLB disease management remains uncertain.  Complementary studies of multiple ENPs and their individual components compared to the standard nutritional program (SNP) on nursery and field trees were initiated in 2010.  Two independent nursery trials were initiated with final data collection of the second trial currently underway.  The field site was chosen for its mix of healthy, presymptomatic, and HLB symptomatic trees to determine if observed differences resulted from effects on healthy or infected trees.  We have found no evidence of reduced phloem plugging in ENP treated nursery trees.  Candidatus Liberibacter asiaticus (Las) populations are similar for ENPs and the SNP.  Minor differences in Las movement have been observed.  Las invaded new flush tissue faster in ENP treated trees than SNP trees.  Phosphite treatments have caused Las to favor early invasion of root tissue compared to other treatments.  Preliminary observations of the second nursery trial suggest that foliar symptoms are more apparent on the standard nutrient program compared to ENPs; however, root and canopy decline are unaffected.  Fruit yield and HLB symptoms in field trees treated with ENPs have not differed significantly from the standard nutritional program after two years.  Third year yield data will be presented.

The current standard to diagnose Huanglongbing (HLB) for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and apply quantitative real-time PCR (qPCR) test to detect 16S rDNA of Candidatus Liberibacter asiaticus (CLas), the putative causal pathogen. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high pectin concentration, low pH and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice, and detection of CLas by qPCR. Homogenization using a sonicator increased DNA extraction by 86%, and stabilized quantification of 16S rDNA in comparison to mortar and pestle extraction, which showed wide variability of Ct values of 16S rDNA. Orange juice is rich in pectin, which has similar physiochemical features to DNA: soluble in water and precipitates in ethanol/isopropanol solutions. Thus, it is difficult to separate the DNA from pectin. However, DNA was successfully extracted by adding pectinase to hydrolyze the pectin. Without going through an elution column, the amplification of plant and microbial DNA in orange juice samples was inhibited by an unknown compound. Thus application of an elution column successfully eliminated the inhibitor. To eliminate errors caused by different methods of sampling, DNA extraction and qPCR procedures, Ct of a cytochrome oxidase (COX) to represent citrus plant DNA was detected as a reference, and a relative unit, ΔCt_{16S rDNA-COX} was introduced to express the relative CLas population.

Huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus (Las), was first detected in Florida in late 2005 and is now widely distributed throughout the commercial citrus-growing regions. In recent seasons, concurrent with freeze and drought episodes, symptomatic HLB-infected trees were much more affected by the extremes of temperature and moisture than trees without HLB. Symptoms exhibited by the stressed trees were excessive leaf loss and premature fruit drop even when HLB-infected trees were managed with good nutritional and irrigation practices recommended to support health of HLB-affected trees. This stress intolerance may be due to a loss of fibrous roots. To assess root status of HLB-infected trees on Swingle citrumelo rootstock (Citrus paradisi × Poncirus trifoliata), blocks of 2,307 three-yr-old Hamlin orange trees and 2,693 four-yr-old Valencia orange trees were surveyed visually and with a real time polymerase chain reaction (PCR) assay to determine Las infection status. The incidence of Las-infected trees (pre-symptomatic: Las+, visually negative, and symptomatic: Las+, visually positive) trees was 89% for the Hamlin block and 88% for the Valencia block. Las+ trees had 30 and 37% lower fibrous root mass density for pre-symptomatic and symptomatic trees, respectively, compared to Las- trees. In a second survey, 10- to 25-yr-old Valencia trees on ‘Swingle’ citrumelo or ‘Carrizo’ citrange (C. sinensis (L.) × P. trifoliata) rootstock were sampled within 3-6 months after identification of visual HLB status as symptomatic (Las+, visually positive) or non-symptomatic (Las-, visually negative) in orchards located in the central ridge, south-central and southwest flatwoods. Pairs of HLB symptomatic and non-symptomatic trees were evaluated for PCR status, fibrous root mass density and Phytophthora nicotianae progagules in the rhizosphere soil. Las+ trees had 27-40% lower fibrous root mass density and in one location higher P. nicotianae per root but Phytophthora populations per cm³ soil were high on both Las+ and Las- trees.  Fibrous root loss from HLB damage interacted with P. nicotianae depending on orchard location and time of year.

Transgenic citrus scion (mostly grapefruit and sweet orange) and rootstock cultivars (Carrizo and experimental complex tetraploids) were transformed with gene(s) encoding antimicrobial peptides or systemic acquired resistance (SAR) proteins. Each transgene was under control of an enhanced CaMV 35S promoter. Several genes were also under control of a phloem specific Arabidopsis SUC2 (AtSUC2) promoter.  A number of clones of each transgenic line (at least 3 replicate plants per clone) were evaluated for resistance to Huanglongbing (HLB, caused by Candidatus Liberibacter asiaticus). 650 clones, from over 180 individual transgenic lines planted in spring 2009 in a heavily HLB infected Martin County grove were tested using qPCR for infection to HLB after 30 months in field. 396 trees tested negative for the HLB bacterium. Approximately 200 clones were observed to be healthy and flushing after 40 months in the field and were again evaluated using qPCR during June 2012. We did not detect the Ca. Liberibacter asiaticus bacterium in a majority of these trees. In a separate trial in St. Lucie County, 300 clones, from over 80 individual transgenic lines planted during 2010 were evaluated in October 2012. Similar trends were observed to that seen in our Martin County site.  345 transgenic clones and controls containing the same transgene(s) were also placed in a greenhouse containing free flying HLB-infected Asian citrus psyllids (ACP) during April 2011. All trees were evaluated for infection after 12 months by qPCR, and 80% of the transgenic trees tested negative for the bacterium.  These results suggest that some of the antimicrobial peptides and SAR-inducing proteins can provide long-term resistance against HLB.