Adult psyllids were monitored for 6 h on nursery citrus trees treated with two kaolin formulations at 3 and 5% w/v. The two kaolin formulations have a repellent effect on D. citri, causing an overall reduction of 40% of psyllids settled on treated seedlings compared with untreated control. Moreover, both formulations disrupt D. citri probing behavior, with a significant reduction in the proportion of psyllids that reach the phloem compared with untreated nursery citrus trees. In general, there were no differences between the kaolin formulations and among the concentrations tested in both experiments . Then, both formulations could be used in an integrated D. citri management program. These findings reinforce the recommendation of kaolin application on young citrus planting as a useful strategy for HLB management, mainly on the edge of the farms. One of the most crucial steps to find a long-term cure for Huanglongbing is to culture the causative agent which is known as Candidatus Liberibacter asiaticus. In order to culture this bacterium in vitro, it is critical to know the physiochemical conditions in citrus phloem and psyllid organs where CLas present.Micro-electrodes are needle-shaped sensors with tip size of less than 20 µm. The use of micro-electrodes allows minimally-invasive measurement of the micro-scale gradients within native biological system. In this study, oxygen and pH micro-electrodes were used to determine the local oxygen concentration and pH inside citrus phloem and psyllid including intestine and hemolymph. Measurements are done both in healthy and unhealthy psyllids and citrus plants. The results are compared together and with the previous available measurements done with chemical extract from those targets. Results of this study will benefit further design the suitable strategies to culture Candidatus Liberibacter asiaticus in vitro. Many phytophagous hemipteran insects feed by penetration of a stylet bundle into plant tissues to feed on phloem or xylem tissues. Among these are agriculturally important insect vectors of plant diseases which include : white flies, aphids, mealybugs, scales,mobile vertical farm leaf hoppers and psyllids, including the Asian citrus psyllid -the vector of the devastating citrus greening disease.
A common trait of plant vascular feeding hemipterans is ability to form a ‘stylet sheath’ structure that encapsulates the stylet bundle while they penetrate into the plant tissues to feed. The exact function of stylet sheaths in hemipteran feeding are not known; however, trait conservation across diverse phytophagous hemipterans implies a biological importance. It is speculated that sheaths may function to ‘cloak’ the insect feeding process to evade host plant defenses; it also may provide structural stability to the stylets during penetration. We have previously developed a method to isolate pure sheath structures and have used these sheaths to determine gross composition. The primary component of the sheaths is a polymeric glucosyl polysaccharide consisting primarily of “starch-like” alpha-1,4-glucosyl bonds but also a small portion of cellulose-like beta-1,4-glucosyl bonds. This was determined both by mass-spectrometry analysis and demonstration of degradation with amyloglucosidase and cellulase enzymes that are specific for starch and cellulose, respectively. Another major structural component was determined to be proteinaceous as demonstrated by the detection of proteins within purified sheaths and the ability to degrade polymerized sheaths using various proteases. We have further demonstrated that inhibitors of sheath formation can be topically applied to citrus leaves and that this results in inhibition of psyllid feeding on the citrus. This work is presented as a broadly applicable new concept in pest-insect control based on the use of molecules that block the insect’s ability to establish a successful feeding site on the host plant. Huanglongbing , caused by the phloemlimited bacterium Candidatus Liberibacter asiaticus , poses one of the largest threats to citrus production. In ten years, it has spread to most citrus trees in Florida. Citrus production during the 2015/16 crop year is down 54% from the pre-HLB era. After initial leaf infection by Diaphorina citri , Las moves to, colonizes, and damages the entire root system, however Las and HLB symptoms remain sectored in the canopy. Canopy sectoring is thought to occur because of limited lateral connections of phloem sieve tubes. To investigate if lateral movement of Las between sieve tubes is distance or tissue specific, root systems of grafted and seedling trees were split to three different heights: below crown, above crown, and above graft union .
The trees were then graft inoculated at a consistent height above one side of the split root system. Infection of both halves of the root system was monitored weekly. Our results indicate the graft union and crown did not play a unique role in lateral movement. No difference was observed in infection between grafted and seedling trees. Greater variability in the time between Las detection between the inoculated and opposite side in trees with the highest trunk split suggests that vertical distance is an important factor in lateral movement of the bacterium. CLIBASIA_03135 is a highly induced gene in CLas mRNA samples obtained from infected citrus plants. The expression of this gene is undetectable in samples obtained from CLas infected psyllids. The encoded protein could play an important role in helping the bacteria to thrive in the harsh conditions of the plant phloem. To evaluate the biochemical characteristics and its biological significance, we have cloned the gene and purified the encoded protein . Coimmunoprecipitation assays allowed us to identify the GroEL chaperone as the main interacting protein. The specific interaction between LotP and GroEL was confirmed by using a two-hybrid system in Escherichia coli. LotP is a dimer in solution with a native molecular weight of 44 KDa and has ATPase activity in vitro. It displays remarkable structural homology, but no sequence conservation with the amino-terminal region of the Bacillus subtilis LON protease. LotP was systematically annotated as an ATP dependent LON protease, however, the B. subtilis LON protease is an ATP dependent aminopeptidase composed of 6 units each of which are 90 KDa. The results obtained in our analysis allowed us to define that LotP belongs to a different family of proteins involved in protein refolding. LotP is the first member of this large family yet to be biochemically characterized. Our results suggest these proteins modulate the activity of stress-response proteins by direct physical contact depending on the stress conditions present in the environment, hereby opening a possible way to combat HLB disease. The biological agent associated with Huanglongbing in citrus is a phloem-restricted Gram-negative alpha-proteobacteria that belongs to the genus ‘Candidatus Liberibacter’. Among three species of Candidatus Liberibacter, Ca. L. asiaticus has recently invaded several citrus growing regions of the Americas, causing major economic losses. Early HLB detection is necessary in implementing HLB disease management strategies to mitigate the disease.
The major source material for HLB qPCR diagnostic test is symptomatic leaves, but a combination of uneven distribution of the bacteria and the slow development of leaf symptoms which can resemble those induced by various biotic/abiotic stresses, reduces the probability of HLB early detection. We have previously shown that CLas is more evenly distributed in the root system of an HLB-positive tree than in the leaves, suggesting that root tissue may be an alternative source for qPCR diagnosis. We selected about 100 young citrus trees located in an orchard with a low level of infection that is adjacent to an orchard with heavily infected trees from TX and FL, respectively. Leaf and fibrous root samples for HLB qPCR test were collected monthly from these trees since January, 2016. For the current study,vertical farming racks we developed efficient DNA extraction and a new qPCR primers and probe system that target CLas 16s rDNA for root samples. The HLB qPCR data showed that the number of HLB-positive trees confirmed with leaf tissue maintained the steady state during the course of the experiment while the number of HLB-positive trees detected by root tissue has started increasing drastically since May, 2016. The qPCR data showed that about 65% of TX and 80% of FL HLB positive trees were tested positive only with root samples while about 8% of TX and 6% of FL HLB-positive trees were tested positive only with leaf tissue. About 27% and 14% of HLB-positive trees from TX and FL, respectively, were tested positive both with root and leaf tissue. The survey results indicated that the HLB qPCR detected CLas among root tissue substantially more often than among leaf tissue. In response to a pathogen attack, multiple defense mechanisms are triggered in the host plants, including basal defense and gene-for-gene resistance. In particular, Las infection, causes extensive changes in gene expression for several major biological processes including stress responses, signal transduction, transport, cell organization and carbohydrate metabolism. Las deploys effectors that target mitochondria and chloroplasts, which are responsible for the synthesis of adenosine triphosphate and have a critical role in in the creation of reactive oxygen species for both pattern-triggered and effector-triggered immunity signaling in the plant. In this work we investigated HLB disease in relation to ROS and ATP regulation through the analysis of genes correlated to the production and detoxification of H2O2, since H2O2 is one of the major and most stable ROS regulating basic acclamatory, defense and developmental processes in plants. We found that Las infection increased the level of ATP and H2O2 in citrus leaves suppressed the H2O2 detoxification system, resulting in an overall increase in the level of H2O2, which becomes toxic for the plant and initiates damage to the tissues. Trees severely infected by huanglongbing present increased fruit drop, and the harvested fruit are usually smaller and compromise juice quality by being sour, bitter and having off flavor.
It was shown that fruit drop is a result of secondary infection at the calyx zone by the fungus Lasiodiplodia theobromae. The objective of this study was to identify whether those fruit that are ready to abscise have a different quality than fruit that do not have a developed abscission zone. ‘Hamlin’ and ‘Valencia’ orange trees, both healthy and HLB-affected, confirmed by qPCR analysis for Candidatus Liberibacter asiaticus , from two harvests and one harvest were shaken, the dropped fruit collected, and the retained fruit harvested. The harvested fruit were washed, juiced using FMC commercial extractor, pasteurized and frozen for later chemical and sensory analyses. The juice was tasted by 55 untrained panelists in multiple paired-comparison tests where samples were presented as pairs of either “drop/retain”, “drop/drop” or “retain/retain”. Juice from healthy and HLB-affected trees were presented in separate pairs. Panelists could not differentiate juice from healthy fruit retained on the tree or dropped for all three harvests. However, for juice made with HLB-affected fruit, panelists could differentiate juice from fruit that had a developed abscission zone and dropped on the ground after shaking in comparison with fruit that was retained on the trees. The test was statistically significant for all three replications of Hamlin harvested in December, and in two of three replications of Hamlin harvested in January and Valencia harvested in April. A trained descriptive panel also found differences between juice made with fruit having different levels of abscission, with juice from HLB-infected dropped fruit having the most negative off flavor attributes, including sourness, bitterness, astringency, off flavor. Sensory data confirm chemical data and both sets of data were correlated, bringing further insight into off flavor induced by HLB in orange juice.The implications of different control strategies is critical when developing viable disease management plans. Cost, efficacy, disease/pest prevalence, and perceived consequences/benefits, among many other factors, play a role in deciding what control methods to utilize. Additionally, these control methods tend to change over time, reacting or adapting to the current situation. As ACP and HLB continue to spread, the need to assess and implement economically sustainable control options is paramount. We can investigate and compare the production benefits against the fiscal costs of various control strategies through mathematical modeling. We extend a spatially-explicit, stochastic, individual-based compartmental model to incorporate different combinations of control measures such as survey, insecticides/pest control, tree removal, and tree replacement. For surveying, we can implement different survey patterns and probabilities of disease detection. Insecticide spraying can be routine as well as reactive . Further, infected tree removal or localized culling can be invoked to reduce the local inoculum, and replanting can be initiated. Under different combinations of these control measures, we can calculate the costs, model the spread of HLB, and determine the yield/production benefit.