RAPID AND EFFICIENT DELIVERY OF EFFECTIVE COMPOUNDS INTO CITRUS PHLOEM FOR TREATMENT OF HLB BACTERIA

Citrus huanglongbing (HLB), also known as citrus greening, is the most economically devastating disease of citrus worldwide. It has been estimated that nearly 100 million trees in 40 countries have been affected by HLB. In Florida, Citrus HLB, first detected in August 2005, is now present in all commercial citrus producing counties, and is threatening the survival of the 9 billion dollar citrus industry. The spread and cause of HLB are associated with a phloem-feeding insect (Asian citrus psyllids) and a fastidious bacterium (Candidatus Liberibacter), respectively. The bacterium propagates within the phloem of host plants, resulting in die-back, yellow shoots, mottling of leaves, and off-tasting, malformed fruit. When the psyllid is present, the pathogen spreads rapidly in citrus groves, resulting in more than 95 percent incidence in as short as three years after the first infection. As the disease severity increases, yield is significantly reduced, and thereby the lifespan and profitable productivity of citrus trees is dramatically shortened. To date, there are no adequate control practices for preventing further spread of HLB, and all known citrus species, varieties or combinations of scion and rootstock are susceptible to HLB. Control and management measures are currently limited to reducing inoculum sources by utilizing disease-free propagating stock and destroying symptomatic trees, as well as reducing transmission of the bacterium by control of the insect vector. Sustainability of this approach is in question and concerns over extended reliance on high levels of pesticide use include the possibility of insects developing resistance and environmental and human health impacts.

Experimental design Taking into account the complexity of the transdermal/translaminar delivery process, this research will focus on: i) Designing nanoemulsions by evaluating the ideal adjuvant mixtures of solvents, oils, lipophilic and hydrophilic surfactants based on the physicochemical properties of the compounds; ii) Evaluating penetrants to promote uptake, translocation and diffusion of molecules into phloem based on leaf and bark surface characteristics; iii) Developing an efficient transdermal/translaminar delivery process and ideal formulations of compounds for foliar-spray or basal bark applications.Designing nanoemulsions (NE) by optimizing the ideal adjuvant mixtures for the screened compounds: The uniform experimental design will be conducted for optimizing the adjuvant mixtures for nanoemulsion preparation, which produce the desired droplet contact area on the leaf surface and keep the compounds in a soluble form. The adjuvants will include oils (soy oil, mineral oil, α-tocopherol, cremorphor, p-cymeme, and carvacrol), lipophilic surfactants (Lipoid® S75, Span® 80 and Span® 85), hydrophilic surfactants (Tween® 20, Tween® 80 and Pluronic® F68) and water-miscible solvents (ethanol, acetone, methyl ethyl ketone, methyl acetate and ethyl acetate). The ideal adjuvant mixtures of nanoemulsion formation will be evaluated based on the droplet and particle size, morphology and structure and the load and release rate of compounds when compared to the other nanoparticle systems, i.e. poly (lactide-co-glycolide) (PLGA) and Nano-green (A commercial products from nano-green company). The lipophilic compound will be prepared in oil droplets dispersed in aqueous medium (oil-in-water nanoemulsion formulation) to increase their solubility.Screening ideal penetrants (PT): The epicuticular wax present on the leaf or bark surface has two main forms, crystalline and amorphous. The crystalline wax is undoubtedly an obstacle for spray retention and an intimate contact between antimicrobial compound droplets and leaf surface. Several penetrants, such as DMSO, azone, pyrrolidones, oleic acid, terpenes, terpenoids, oxazolidinones, nonionic organosilicone surfactant products (i.e. Silwet L-77 and Pentrabark®) and Urea will be evaluated based on the time lag, maximum rate of penetration, and total penetrated amount into detached leaves of citrus as described by Baur et al.(1992). The ideal penetrants can increase the permeability of the cuticle and the plasma membrane and have no phytotoxicity to citrus. The ideal penetrants combined with compounds will be applied to citrus grown in the greenhouse. An experimental unit will consist of five leaves on a single clamp, and each treatment will be replicated three times. Each experiment will be conducted three times, and the data will be pooled for analysis and presentation.Developing an efficient transdermal/translaminar delivery process and ideal formulations of compounds for foliar-spray or basal bark application. The combined results of the above experiments determining the ideal adjuvant mixtures and penetrants will be used to prepare formulations of nanomulsions to deliver antimicrobial compounds into HLB-affected citrus by foliar-spray and basal bark applications in the greenhouse and in the field. A split-block design with multiple factors will be conducted with different formulations of antimicrobial compounds as the main blocks; the application rates and times as the factorial plots. Each main block will have five treatments, and five replications for each treatment. In the greenhouse, the HLB-affected citrus seedlings will be treated by foliar-spraying. In the field, six-year old citrus trees infected with Las bacteria will be treated using foliar-spraying or basal bark applications. The activity of the formulations against Las bacterium will be quantified by qPCR.MeasurementsPreparation and characteristics of nanoemulsions of antimicrobial compounds: Nanoemulsions will be prepared in three steps by spontaneous emulsification, ultrasonic or magnetic stirring methods. First, the homogeneous organic solution (S1) composed of oil and lipophilic surfactant in water-miscible solvent will be prepared. The homogeneous aqueous phase (S2) will be formed by water and hydrophilic surfactant. Second, the organic phase (S1) will be injected into the aqueous phase (S2) under magnetic stirring or ultrasonic: the o/w or w/o nanoemulsions will be formed instantaneously by diffusion of the organic solvent into the external aqueous phase. Third, the totality of the water-miscible solvent will be removed by evaporation during 45 min under reduced pressure. Nanoemulsions of oil will be dispersed in an aqueous solution of water and hydrophilic surfactant. The droplets size distribution, one of the most important physical characteristics of nanoemulsions, will be measured by a diffusion method using a light-scattering particle size analyzer Coulter LS 230 (Beckman Coulter, Coultronics France). Morphology and structure of the nanoemulsions will be studied using the transmission electron microscopy (TEM) TOPCON 002B operating at 200 kV and of a 0.18 nm capable point-to-point resolution. The in vitro drug release or load behavior of the nanoemulsion will be determined using a modified ultrafiltration technique. The study will be performed on nanoemulsions containing 10% w/w theoretical drug loading and prepared in HEPES buffer, pH 9.3, as the aqueous phase.Determination of the penetration efficiency of the penetrants: Astomatous cuticular membranes (CM) will be isolated enzymatically from the upper surfaces of fully expanded citrus leaves which will be obtained from trees growing in the greenhouse as described by Schönherr and Riederer (1986). The excised tissue will be incubated in a mixture of 0.2% (w/v) cellulase, 4% (w/v) pectinase, and 1 mM NaN3 (to prevent fungal and bacterial growth) in sodium citrate buffer (50 mM, pH 4.0) prepared in distilled water. Enzyme solution will be changed several times over a 2-week period. The CM released from the tissue will be rinsed repeatedly in distilled water, air-dried, and stored at room temperature. Dewaxed cuticular membranes (DCM) will be prepared by batch extraction of the soluble cuticular lipids with 10 exchanges of 1:1 of chloroform and methanol at 45 ?C over 3 days. Permeability, solute mobility and sorptive properties of isolated CM will be determined using unilateral desorption from the outer surface as compared to non-isolated cuticles. Details of the method have been described by Bukovac and Petracek (1993) and Kirsch et al. (1997). Simulation of foliar uptake will be employed to quantitatively measure rates of cuticular penetration across CM as described by Schönherr and Baur (1996). The method will allow us to study the time course of cuticular penetration for each CM separately.Las bacterial titers quantified by qPCR: Leaf samples will be taken from three positions around the canopy of the treated trees for qPCR assay. Each leaf will be rinsed three times with sterile water. Midribs will be separated from the leaf samples and cut into pieces of 1.0 to 2.0 mm. DNA will be extracted from 0.1 g of tissue (fresh weight) of leaf midribs using Qiagen's DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Quantitative real-time PCR (qPCR) will be performed with the primers and probes (HLBas/HLBr/HLBp) for Las bacterium (Li et al., 2006) using the ABI PRISM 7500 sequence detection system. Data will be analyzed using the ABI 7500 Fast Real-Time PCR System with SDS software. Numbers of Las bacteria will be calculated with the standard linear regressions (y=13.82-0.2866×Ct) of the log concentration of the target DNA copies (y) versus the mean Ct values, which were be developed by Li (Li et al., 2008).