Huanglongbing (HLB), caused by Candidatus `Liberibacter asiaticus' (CLas), is a phloem-limited disease that disrupts citrus production in affected areas. In HLB-affected plants, phloem sieve plate pores accumulate callose, and leaf carbohydrate export is reduced. However, whether HLB causes a reduction in carbohydrate phloem translocation speed and the quantitative relationships among callose, CLas population and phloem translocation are still unknown. In this work, a procedure was developed to concurrently measure sugar transport, callose deposition and relative pathogen population at different locations throughout the stem. Increasing quantities of CLas genetic material were positively correlated with quantity and density of callose deposits and negatively correlated with phloem translocation speed. Callose deposit quantity was position and rootstock dependent and was negatively correlated with phloem translocation speed, suggesting a localized relationship. Remarkably, callose accumulation and phloem translocation disruption in the scion were dependent on rootstock genotype. Regression results suggested that the interaction of Ct values and number of phloem callose depositions, but not their size or density, explained the effects on translocation speed. Sucrose, starch and sink ¹⁴C label allocation data support the interpretation of a transport pathway limitation by CLas infection. This work shows that the interaction of local accumulation of callose and CLas affects phloem transport. Furthermore, the extent of this accumulation is attenuated by the rootstock and provides important information about the disease mechanism of phloem-inhabiting bacteria. Together, these results constitute the first example of a demonstrated transport limitation of phloem function by a microbial infection.

Huanglongbing (HLB), which is caused by the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus,’ is an economically important disease of citrus in many regions of the world. Due to the significant damage caused by the HLB disease in recent years, the use of antibiotics was recommended for the therapy of this destructive disease. Products with active ingredients oxytetracycline and streptomycin have been approved for the control of the HLB via foliar application. However, previous work raised questions about the efficacy of foliar delivery of antibiotics in the field. In this study, we examined the effects of a variety of adjuvants on the uptake of oxytetracycline and streptomycin using the foliar application. We also compared the efficiency of foliar application of oxytetracycline and streptomycin with trunk injection. The ‘Ca. L. asiaticus’ titers in citrus plants were measured using quantitative PCR, and the levels of antibiotics were determined using the ELISA assay. Our results include extremely low levels of oxytetracycline and streptomycin in leaves that were covered during foliar application, indicating that neither streptomycin nor oxytetracycline was successfully systemically delivered by foliar application even after being mixed with adjuvants. Likewise, the ‘Ca. L. asiaticus’ titer0 was not affected by any of the foliar applications. High levels of streptomycin were detected in leaves that were exposed to direct foliar application, indicating that streptomycin was adsorbed or bound to citrus leaves. On the other hand, the trunk injection of oxytetracycline resulted in high levels of this antibiotic in leaves and significantly reduced the level of ‘Ca. L. asiaticus’ titer in citrus trees. Unfortunately, the trunk injection of streptomycin resulted in low levels of streptomycin in citrus leaves and did not affect the ‘Ca. L. asiaticus’ titer, indicating that streptomycin was either bound in the xylem of citrus trees or it was not applied in sufficient quantity required for the inhibition of ‘Ca. L. asiaticus.’ Taken together, our current results demonstrated that foliar application of oxytetracycline and streptomycin did not effectively deliver antibiotics in citrus despite using adjuvants. Our results also suggested that oxytetracycline could be more effective against the HLB pathogen than streptomycin, which is possibly due to differences between the two in systemic movement in citrus trees.

Recently in Florida, foliar treatments using products with the antibiotics oxytetracycline and streptomycin have been approved for the treatment of citrus Huanglongbing (HLB), which is caused by the putative bacterial pathogen ‘Candidatus Liberibacter asiaticus’. Herein, we assessed the levels of oxytetracycline and ‘Ca. L. asiaticus’ titers in citrus trees upon foliar applications with and without a variety of commercial penetrant adjuvants and upon trunk injection. The level of oxytetracycline in citrus leaves was measured using an oxytetracycline ELISA kit and ‘Ca. L. asiaticus’ titer was measured using quantitative PCR. Low levels of oxytetracycline were taken up by citrus leaves after foliar sprays of oxytetracycline in water. Addition of various adjuvants to the oxytetracycline solution showed minimal effects on its uptake by citrus leaves. The level of oxytetracycline in leaves from trunk-injected trees was higher than those treated with all foliar applications. The titer of ‘Ca. L. asiaticus’ in the midrib of leaves from trees receiving oxytetracycline by foliar application was not affected after four days and thirty days of application, whereas the titer was significantly reduced in oxytetracycline-injected trees thirty days after treatment. Investigation of citrus leaves using microscopy showed that they are covered by a thick lipidized cuticle. Perforation of citrus leaf cuticle with a laser significantly increased the uptake of oxytetracycline, decreasing the titer of ‘Ca. L. asiaticus’ in citrus leaves upon foliar application. Taken together, our findings indicate that trunk injection is more efficient than foliar spray even after the use of adjuvants. Our conclusion could help in setting useful recommendations for the application of oxytetracycline in citrus to improve tree health, minimize the amount of applied antibiotic, reduce environmental exposure, and limit off-target effects.

Our study aimed to evaluate the physiological responses following salinity treatment of three putatively salt-tolerant Citrus rootstocks recently developed by the University of Florida’s Citrus breeding program. Four-month-old seedlings from each of the three rootstocks (HS1, HS17, and HC15) were irrigated with 0, 60, 80, and 100 mm NaCl solution. The seedlings were evaluated together with the salt-tolerant Cleopatra mandarin as a positive control, Volkamer lemon as a moderately salt-tolerant rootstock, and the salt-sensitive Carrizo rootstock as a negative control. Our results demonstrated that chlorophyll content, net CO₂ assimilation rate (A), transpiration rate (E), and stomatal conductance (g_sw) significantly decreased in response to salinity. Na⁺ and Cl⁻ levels were higher in leaf tissues than in the roots. Relatively little damage to the cellular membrane was recorded in HC15 and Cleopatra rootstocks under the 100 mm NaCl treatment, along with high accumulation of total phenolic content (TPC), while HS17 had the highest proline levels. Our results indicate that HC15 and HS17 rootstocks exhibited salt tolerance capacity via different strategies under salt stress and could be suitable replacements to the commercially available, salt-tolerant Cleopatra rootstock.