Injections of antibiotics have been the primary focus and several issues have hampered commercial development: cost of treatment, damage inflicted on the tree as a result of multiple injections, phytotoxicity of antibiotic treatment, and concerns over bactericide residue in the fruit. Cost can be high because of the labor intensive process of injecting each tree and/or the cost of the injection apparatus. Since the bacterium resides within the phloem cells, effective control requires the bactericides to first enter the tree and then move systemically throughout the vascular tissues. Damage from the injection process can create scar tissue that impedes normal transport of nutrients throughout the tree, and phytotoxicity is exacerbated because treatments require a high dose of the bactericide applied to a small application site. Our research was conducted to determine if effective treatment application strategies can be developed that reduce phytotoxicity and plant damage and also reduce the long-term cost by reducing labor costs of using bactericides in an HLB IPM strategy. We have shown that three bactericides—oxytetracycline, penicillin,maceta de plastico cuadrada and streptomycin—can be applied in ways that cause the molecules to penetrate plant tissues and move systemically throughout the plant. We have also developed a strategy for therapeutic delivery of the antibiotic in a “time-release” strategy that should reduce the phytotoxicity and cost of multiple applications.
Encouraging results on greenhouse and research plots have led to current commercial grove application trials testing different application strategies and comparing effectiveness. Huanglongbing is caused by a phloem-limited bacterium, Ca. Liberibacter asiaticus in the United States. The bacterium often is present at a low concentration and unevenly distributed in the early stage of infection, making reliable and early diagnosis a serious challenge. Conventional diagnostic techniques, including real-time PCR , have often failed at early detection of HLB. We have demonstrated a promising novel diagnostic assay based on digital PCR for early and reliable detection of HLB. dPCR has revolutionized the detection of rare pathogens and nucleic acid molecules as it partitions samples into tens of thousands of picoliter wells in a single reaction.The number of negative and positive wells can then be fitted into a Poisson distribution to allow absolute and precise quantification of the target molecules in a sample without the use of a standard curve. The large number of independent reactions in a single assay makes it possible to apply statistical tools to estimate a level of precision and confidence interval of the measurement. Using probes targeting the Las 16s rDNA and the integrated prophage repeat sequences, we show that as few as 1–2 copies of the targeted DNA molecules per microliter can be detected, with the prophage probe providing the best sensitivity. The copy number measurement of the targeted DNA molecules can be statistically differentiated from the healthy sample and negative water controls. Furthermore, this assay can quantitate the copy number of the 16S rDNA and the phage repeat DNA simultaneously, permitting the tracking of lysogenic and lytic activities of the Las prophage/phage accurately.
The dPCR‐based assay will not only provide a reliable and early diagnostic tool but also an enabling technology to advance research on HLB therapies. Accurate and timely detection is critical for the eradication of Huanglongbing . Unfortunately, early HLB detection is challenging because infected citrus plants remain asymptomatic, sometimes for years; furthermore, the HLB-associated bacterial pathogen Candidatus Liberibacter spp. exhibits uneven distributions in infected trees with low titers. Very often, CLas cannot be detected by quantitative PCR even in symptomatic branches or leaves. To date, reliable methods for HLB early detection and the ability to process a large amount of samples necessary to track HLB epidemic are urgently needed. In the past several years, we have identified unique secreted proteins of CLas as markers for HLB detection and developed an antibody-based detection method using a simple branch imprint assay. The basic idea is that although CLas has sporadic distribution in the phloem of infected trees, proteins secreted from CLas cells into the phloem can be systematically distributed in the infected trees through the vascular flow. As such, serological detection methods based on CLas-specific secreted proteins will better cope with the large variability in the distribution of CLas cells within infected trees and the various degrees of disease progression, and thereby increasing the chances of HLB detection in a direct and highly specific manner. Our preliminary research suggest that this method holds promise for allowing early detection of newly infected trees and large-scale field surveys. Since its earliest known occurrence in California, the Asian Citrus Psyllid has spread rapidly across the urban landscape of southern California. It is not surprising that much of the observed ACP infestation occurs within the urban areas of Los Angeles. However, to-date little is known about the factors that influence the spread and distribution of ACP at the landscape level.
Our study focuses on understanding the colonization and distribution pattern of ACP across a predominantly urban landscape by using a combination of GIS tools and spatial models. More specifically, we first explore the role of roads in the colonization of ACP during early stages of spread and then develop several regression models to identify the key exogenous and endogenous factors that influence the eventual landscape-level distribution of ACP. Analyses of road-proximity patterns among ACP occurrences captured at different temporal stages of spread for each county showed that the initial occurrences of ACP were strongly and positively influenced by the network of roads for the counties of Los Angeles and San Diego. Logistic regression model highlighted the strong influence of ‘percent impervious surface’ and ‘NDVI’ as the key landscape factors along with ‘rain in warm quarter’ as the most influential bio-climatic factor in determining the occurrence of ACP in the urban landscape. Inclusion of spatial and temporal patterns of ACP occurrence as an auto-covariate further improved the fit of regression models, suggesting the role of fine scale endogenous process like dispersal in determining ACP occurrence. Results from the best-fitting spatiotemporal auto-logistic regression model shall eventually be used to develop habitat suitability map for ACP. Before HLB, the useful lives of the plants were larger than 20 years. Now it was estimated, even with modern managements, an average is 14 years. When plants are managed with HLB, eradicating the plant stand decreases, making low productivity and impeding the maintenance of the area, and making it necessary to eradicate all plants and plant new plants. A good strategy to have new citrus area is to induce vigor to have high productivity rates and earliness, getting “payback” in the first years. Some insecticides, like the neonicotinoids group, have demonstrated physiological action on treated plants – like increased vigor, shoot and root development, better absorption of nutrients and water, increased enzyme activity, resistance, and productivity. To evaluate vigor, healthy nursery trees and standardized Valencia orange single stem produced in greenhouse were planted in pots in randomized blocks and treated with systemic insecticides action indicated to manage the psyllid. The experiment was conducted with six treatments—three different doses of thiamethoxam, one of Imidacloprid, Aldicarb,maceteros reciclados de plastico and Check without insecticide. We evaluated the development of plants every 15 days until 150 days after planting, evidencing that all the treatments had an increase in trunk diameter, with the thiamethoxam treatments differing significantly from Control. Thiamethoxam at a dose of 1 g per plant produced fresh and dry weight of root differing from Check. This treatment also had higher weight of shoots. That way, treatment with thiamethoxam at a dose of 1 g per plant had the best results in all studied characteristics providing better vigor and plant development during the study period. Considerable research has been conducted toward developing and implementing HLB and ACP management strategies. With respect to ACP control, of interest is that reports indicate guava, Psidium guajava, can be repellent to ACP. We conducted research to further assess repellency of guava to ACP. In one set of experiments, guava oil from five Brazilian cultivars was extracted from leaves by hydrodistillation in a Clevenger apparatus and evaluated for psyllid repellency. In a second set of experiments, repellency of guava leaves to ACP was investigated using leaves from two guava cultivars, ‘Pink’ and ‘Thai White’. Repellency was evaluated by releasing ACP adults into a cage with two vials, one containing a young flush of Murraya exoticaand one with M. exotica flush and the test material of interest . The adults were free to move throughout the cage and into the vials. After 24 hours, the number of psyllids in each vial was counted.
The results obtained showed that all guava materials tested had at least some repellency to ACP. Mature guava leaves had a greater repellent effect than young flush. Each of the five oils exhibited repellency. There were no differences between males and females with respect to guava repellency. A report in the literature suggested that sulfur compounds associated with guava may be responsible for ACP repellency. Interestingly, the five guava oils we studied were repellent to ACP, but three did not appear to contain any sulfur compounds. Identification of the constituents responsible could lead to new management tactics. Orchard renewal is a special challenge where HLB is endemic. Young trees are especially susceptible to the disease and continuously attractive to the psyllid vector due to frequent flushing. Therefore, heavy reliance is placed on soil‐applied systemic insecticides to protect young trees. We conducted a one year study to evaluate foliar concentrations of neonicotinoids and cyantraniliprole applications and two multi‐year studies to assess long‐term ACP control and consequent protection from HLB. In the first study, we found detectable levels of cyantraniliprole and significant reduction of ACP nymphs 295 days after a drench application to 2‐month-old trees. In the first long‐term trial, ACP adults averaged 1.4 per tap under heavy pressure on untreated trees over 2.5 years, compared to 0.06–0.11 on trees treated at 3‐month intervals with rotations of neonicotionoid and cyantraniliprole drenches. Incidence of HLB reached 49% on untreated trees after 2 years compared to 6.3% on treated trees which later rose to 98% after 3.5 years. Trunk diameter on treated trees after 3.7 years was 37% larger and yield 6.5 times greater compared to untreated trees. During 15 months after planting the second long‐term trial, shoot infestation averaged 60% and adults 0.9 per tap compared to 7.1% and 0.04 per tap for trees treated with a neonicotinoid – cyantraniliprole rotation. The numbers for repeated imidacloprid application were 12.7% and 0.08 per tap and repeated cyantraniliprole 18.0% and 0.08 per tap. HLB incidence was already 46% in the check compared to 12.5% for the rotation and 29 and 25% for imidacloprid and cyantraniliprole, respectively. Drip injection worked better than drench, once roots had established around emitters spaced at 30 cm. Huanglongbing , the most devastating citrus disease worldwide, is threatening the citrus industry in several countries, mainly Brazil and United States. HLB is a bacterial-caused disease transmitted by the Asian Citrus psyllid . Currently there is no cure for this disease and no identified genetic resistance in the genus Citrus. An emerging technology, RNA interference , which is a natural biological process that selectively down regulates the expression of a specific gene, is being developed as a more environmentally friendly approach to control insects. The feasibility of RNA interference strategies supports a method that would be highly specific ACP populations. One question that needed to be addressed was the effect, if any, on ACP response to plants treated with an RNAi product. To evaluate if ACP sensed the presence of an ACP-specific dsRNA in plants, the experiment examined psyllid response to dsRNA treated plants versus non-treated plants. Four groups of six plant flush each were placed in each corner of a cage, and 100 ACP were released at the center. They were observed for 15 days, and the number of ACP on each flush recorded daily. A significant difference, using t-test analyses, showed more psyllids on the dsRNA treated plants than on untreated controls . Psyllids appear not to be sensitive to dsRNA ingestion, as they may be for traditional chemical insecticides, thus were not repelled. These preliminary results suggest that RNAi-based products may actually increase psyllid feeding from the treated trees, thus increasing RNAi efficacy, by increasing the amount of dsRNA each psyllid would ingest.The Asian citrus psyllid, Diaphorina citri, is an oligophagous herbivore that transmits Candidatus Liberibacter asiaticus, the devastating pathogen causing citrus greening disease.