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.

Spectral light distribution affects photosynthesis, leaf reflective indices, antioxidant activity and growth of Vanillaplanifolia.

Vanilla planifolia is an obligate sciophyte (shade plant) with crassulacean acid metabolism (CAM) photosynthesis. Plants were grown for 12 months under black, blue, green, or red photoselective shade netting (PSN) to alter the spectral light distribution impacting the plants. Light wavelengths were measured in each treatment and plants were assessed for photosynthetic characteristics, leaf chlorophyll index (LCI), maximum quantum yield of photosystem II, leaf reflectance indices, leaf area, growth, antioxidant enzymes, lipid peroxidation, reactive oxygen species (ROS), and osmolyte content. Plants grown under red PSN had a higher quantity of red and far-red light and had greater nocturnal net CO2 assimilation (NocA), leaf area and leaf dry weight than plants in the other treatments. Plants grown under blue PSN had a higher quantity of blue light, resulting in a higher LCI and maximum quantum yield than plants in the other treatments. Plants grown under the red and blue PSN had increased leaf spectral reflectance indices compared to plants in the other treatments, which resulted in the highest levels of antioxidant scavenging enzymes, ascorbic acid (AsA), proline, and glycine betaine, and the lowest levels of H2O2. These findings demonstrate that increasing light in the red and far-red or blue portions of the spectrum by using PSN alters the photosynthetic and/or antioxidant responses of V. planifolia and increasing red and far-red light by using red PSN can also accelerate plant growth, possibly due to higher photosynthesis.

Root system size and root hair length are key phenes for nitrate acquisition and biomass production across natural variation in Arabidopsis.

The role of root phenes in nitrogen (N) acquisition and biomass production was evaluated in 10 contrasting natural accessions of Arabidopsis thaliana L. Seedlings were grown on vertical agar plates with two different nitrate supplies. The low N treatment increased the root to shoot biomass ratio and promoted the proliferation of lateral roots and root hairs. The cost of a larger root system did not impact shoot biomass. Greater biomass production could be achieved through increased root length or through specific root hair characteristics. A greater number of root hairs may provide a low-resistance pathway under elevated N conditions, while root hair length may enhance root zone exploration under low N conditions. The variability of N uptake and the expression levels of genes encoding nitrate transporters were measured. A positive correlation was found between root system size and high-affinity nitrate uptake, emphasizing the benefits of an exploratory root organ in N acquisition. The expression levels of NRT1.2/NPF4.6, NRT2.2, and NRT1.5/NPF7.3 negatively correlated with some root morphological traits. Such basic knowledge in Arabidopsis demonstrates the importance of root phenes to improve N acquisition and paves the way to design eudicot ideotypes.

Physiological Responses and Gene Expression Patterns in Open-Pollinated Seedlings of a Pummelo-Mandarin Hybrid Rootstock Exposed to Salt Stress and Huanglongbing.

Huanglongbing (HLB), caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CaLas), is the primary biotic stress causing significant economic damage to the global citrus industry. Among the abiotic stresses, salinity affects citrus production worldwide, especially in arid and coastal regions. In this study, we evaluated open-pollinated seedlings of the S10 (a diploid rootstock produced from a cross between two siblings of the Hirado Buntan Pink pummelo (Citrus maxima (Burm.) Merr.) with the Shekwasha mandarin (Citrus reticulata Blanco)) for their ability to tolerate HLB and salinity stresses. In a greenhouse study, 'Valencia' sweet orange (either HLB-positive or negative) was grafted onto six clonally propagated lines generated from the screened seedlings in the greenhouse and the trees were irrigated with 150 mM NaCl after eight months of successful grafting and detection of CaLas in the leaf petioles. Cleopatra mandarin was used as a salt-tolerant and HLB-sensitive rootstock control. CaLas infection was monitored using a quantitative polymerase chain reaction before and after NaCl treatments. Following three months of NaCl treatment, 'Valencia' leaves on the S10 rootstock seedlings recorded lower levels of chlorophyll content compared to Cleopatra under similar conditions. Malondialdehyde content was higher in HLB-infected 'Valencia' grafted onto Cleopatra than in the S10 lines. Several plant defense-related genes were significantly upregulated in the S10 lines. Antioxidant and Na+ co-transporter genes were differentially regulated in these lines. Based on our results, selected S10 lines have potential as salt-tolerant rootstocks of 'Valencia' sweet orange under endemic HLB conditions. However, it is necessary to propagate selected lines through tissue culture or cuttings because of the high percentage of zygotic seedlings derived from S10.

The Role of the Xylem in Oxytetracycline Translocation within Citrus Trees.

Antibiotics have been successfully used to control plant diseases for more than fifty years. Recently, oxytetracycline and streptomycin have been approved for the treatment of Huanglongbing, which is threatening the citrus industry in many regions. Because the efficiency of antibiotics in planta is highly affected by their movement and distribution, understanding the mechanism of antibiotics' uptake and distribution could lead to a better control of plant pathogens. Herein, we investigated the movement of oxytetracycline within citrus plants. Oxytetracycline was applied by root drenching to both girdled and non-girdled citrus seedlings. In addition, oxytetracycline was applied by trunk injection to girdled and non-girdled citrus trees. After the exposure time (24 h), citrus seedlings were dissected and the levels of oxytetracycline in the different tissues were measured using an oxytetracycline ELISA kit. Upon root application (laboratory experiment), oxytetracycline was detected in the inner part of the stem (xylem-associated tissue), cortex (phloem-associated tissue), and leaves above and below the girdled area. Likewise, oxytetracycline was also detected in leaves of trunk-injected field trees (girdled and non-girdled) three days post treatment. Interestingly, cortex girdling did not affect the distribution and translocation of oxytetracycline, indicating that the xylem is the main path for oxytetracycline translocation. Taken together, our results indicate that oxytetracycline translocation mainly occurs via xylem vessels, and that movement into the phloem occurs subsequent to xylem translocation. Our findings also clearly demonstrated that upon trunk injection, only trace levels of oxytetracycline reached the roots, minimizing its therapeutic value there. Thus, our recommendation is to time tree injections to coincide with the flushing periods when the bacteria are moving into new shoots to maximize the efficiency of oxytetracycline.

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.

A plant pathogenic bacterium exploits the tricarboxylic acid cycle metabolic pathway of its insect vector.

Huanglongbing in citrus is caused by a phloem-limited, uncultivable, gram-negative α-proteobacterium, Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by the phloem-sucking insect, Diaphorina citri (Hemiptera: Liviidae), in a persistent, circulative, and propagative manner. In this study, we investigated the metabolomic and respiration rates changes in D. citri upon infection with CLas using gas chromatography-mass spectrometry (GC-MS) and gas exchange analysis. The level of glycine, L-serine, L-threonine, and gamma-amino butyric acid were higher in CLas-infected D. citri, while L-proline, L-aspartic acid, and L-pyroglutamic acid were lower in CLas-infected D. citri compared with the control. Citric acid was increased in CLas-infected D. citri, whereas malic and succinic acids were reduced. Interestingly, most of the reduced metabolites such as malate, succinate, aspartate, and L-proline are required for the growth of CLas. The increase in citric acid, serine, and glycine indicated that CLas induced glycolysis and the tricarboxylic acid cycle (TCA) in its vector. In agreement with the GC-MS results, the gene expression results also indicated that glycolysis and TCA were induced in CLas-infected D. citri and this was accompanied with an increases in respiration rate. Phosphoric acid and most of the sugar alcohols were higher in CLas-infected D. citri, indicating a response to the biotic stress or cell damage. Only slight increases in the levels of few sugars were observed in CLas-infected D. citri, which indicated that sugars are tightly regulated by D. citri. Our results indicated that CLas induces nutrient and energetic stress in its host insect. This study may provide some insights into the mechanism of colonization of CLas in its vector.