Accurate photosynthetic parameter estimation at low stomatal conductance: effects of cuticular conductance and instrumental noise.

Accurate estimation of photosynthetic parameters is essential for understanding plant physiological limitations and responses to environmental factors from the leaf to the global scale. Gas exchange is a useful tool to measure responses of net CO2 assimilation (A) to internal CO2 concentration (Ci), a necessary step in estimating photosynthetic parameters including the maximum rate of carboxylation (Vcmax) and the electron transport rate (Jmax). However, species and environmental conditions of low stomatal conductance (gsw) reduce the signal-to-noise ratio of gas exchange, challenging estimations of Ci. Previous works showed that not considering cuticular conductance to water (gcw) can lead to significant errors in estimating Ci, because it has a different effect on total conductance to CO2 (gtc) than does gsw. Here we present a systematic assessment of the need for incorporating gcw into Ci estimates. In this study we modeled the effect of gcw and of instrumental noise and quantified these effects on photosynthetic parameters in the cases of four species with varying gsw and gcw, measured using steady-state and constant ramping techniques, like the rapid A/Ci response method. We show that not accounting for gcw quantitatively influences Ci and the resulting Vcmax and Jmax, particularly when gcw exceeds 7% of the total conductance to water. The influence of gcw was not limited to low gsw species, highlighting the importance of species-specific knowledge before assessing A/Ci curves. Furthermore, at low gsw instrumental noise can affect Ci estimation, but the effect of instrumental noise can be minimized using constant-ramping rather than steady-state techniques. By incorporating these considerations, more precise measurements and interpretations of photosynthetic parameters can be obtained in a broader range of species and environmental conditions.

Systemic Uptake of Oxytetracycline and Streptomycin in Huanglongbing-Affected Citrus Groves after Foliar Application and Trunk Injection.

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.

Phloem transport limitation in Huanglongbing-affected sweet orange is dependent on phloem-limited bacteria and callose.

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.

Phenotypic and Genetic Variation in Morphophysiological Traits in Huanglongbing-Affected Mandarin Hybrid Populations.

Huanglongbing (HLB) caused by 'Candidatus Liberibacter asiaticus' (CLas) is the most costly disease for the global citrus industry. Currently, no effective tools have been found to control HLB. Most commercial citrus varieties are susceptible to HLB, though some citrus hybrid cultivars have reduced sensitivity to the disease. Citrus breeding populations contain a large diversity of germplasm, with thousands of unique genotypes exhibiting a broad range of phenotypes. Understanding phenotypic variation and genetic inheritance in HLB-affected mandarin hybrid populations are crucial for breeding tolerant citrus varieties. In this study, we assessed 448 diverse mandarin hybrids coming from 30 crosses, and 45 additional accessions. For HLB tolerance, we measured HLB severity visual score and CLas titers by qPCR. We also measured seven morphophysiological traits indirectly related to HLB tolerance with leaf area index (LAI), leaf area (LA), leaf mass per area (LMA), photosystem II parameters (Fv/Fo, Fv/Fm), and photochemical performance index (PIabs). By estimating the genetic variation in five half-sib families, we estimated the heritability of phenotypic traits and found a significant genetic effect on HLB visual score and photosynthesis parameters, which indicates opportunities for the genetic improvement of HLB tolerance. In addition, although it is easy to identify infected trees based on HLB symptomatic leaves, visually phenotyping whole trees can be difficult and inconsistent due to the interpersonal subjectivity of characterization. We investigated their relationships and found that LAI was highly correlated with HLB score, with correlation coefficients of r = 0.70 and r = 0.77 for the whole population and five half-sib families, respectively. Photochemical parameters showed significant correlation with HLB severity and responded differentially with the side of the canopy. Our study suggests that LAI and photochemical parameters could be used as a rapid and cost-effective method to evaluate HLB tolerance and inheritance in citrus breeding programs.

Effect of Adjuvants on Oxytetracycline Uptake upon Foliar Application in Citrus.

Recently in Florida, foliar treatments using products with the antibiotics oxytetracyclineand streptomycin have been approved for the treatment of citrus Huanglongbing (HLB), which iscaused by the putative bacterial pathogen 'Candidatus Liberibacter asiaticus'. Herein, we assessedthe levels of oxytetracycline and 'Ca. L. asiaticus' titers in citrus trees upon foliar applications withand without a variety of commercial penetrant adjuvants and upon trunk injection. The level ofoxytetracycline 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 upby citrus leaves after foliar sprays of oxytetracycline in water. Addition of various adjuvants to theoxytetracycline solution showed minimal effects on its uptake by citrus leaves. The level ofoxytetracycline in leaves from trunk-injected trees was higher than those treated with all foliarapplications. The titer of 'Ca. L. asiaticus' in the midrib of leaves from trees receiving oxytetracyclineby foliar application was not affected after four days and thirty days of application, whereas thetiter 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 lipidizedcuticle. Perforation of citrus leaf cuticle with a laser significantly increased the uptake ofoxytetracycline, 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 evenafter the use of adjuvants. Our conclusion could help in setting useful recommendations for theapplication of oxytetracycline in citrus to improve tree health, minimize the amount of appliedantibiotic, reduce environmental exposure, and limit off-target effects.

Implications of Heat Treatment and Systemic Delivery of Foliar-Applied Oxytetracycline on Citrus Physiological Management and Therapy Delivery.

Huanglongbing is an economically devastating disease of citrus in Florida and around the world. This study was undertaken to assess two grower-used therapies, heat treatment, and foliar anti-bacterial application. Specifically, there was an industry claim that heat treatment improved subsequent systemic uptake of foliar-applied anti-bacterial compounds. We hypothesized that new vegetative growth induced by heat treatment could lead to increased foliar delivery because of a greater number of new leaves in which cuticles would be more permeable. The study included two factors (1) heat treatment (with or without) and (2) pruning, in which all new leaves, all mature leaves, or no leaves were removed. A commercial formulation of oxytetracycline (OTC) was applied to plants with a non-ionic penetrant surfactant, but one branch on each tree was covered to assess direct versus systemic delivery. The study was repeated twice, destructively assessing whole-plant leaf area and dry weights, as well as OTC content in directly applied and covered leaves. Heat treatment and defoliation treatments reduced growth, but did not affect systemic delivery of OTC. OTC was detected in nearly all covered leaf samples in both repetitions, though at lower concentrations than in directly applied leaves. We conclude that neither heat treatment nor leaf age strongly affect systemic OTC delivery. Implications of this study for leaf age effects on foliar delivery and for phloem delivery of foreign compounds through foliar application are discussed.