Application of gamma-aminobutyric acid increased the level of phytohormones in Citrus sinensis.

MAIN CONCLUSION: In the current study, we showed that exogenous GABA supplementation increases the endogenous GABA level, several amino acids, and phytohormones in citrus plants, suggesting that GABA works in harmony with phytohormones. Gamma-aminobutyric acid (GABA) plays a key role in cytosolic regulation of pH, controlling of carbon and nitrogen metabolism, and protection against biotic and abiotic stresses. Although it is well-known that GABA is implicated in plant defense and it could act as a signaling molecule, its effect on phytohormones is not completely understood. In this study, we investigated the effect of exogenous GABA on citrus phytohormones using gas chromatography-mass spectrometry. A significant increase in endogenous GABA was observed in GABA-treated plants. The highest increase in GABA was recorded in plants treated with 10 mM 7 days post-treatment. In addition, we observed a moderate increase in several amino acids including glycine, L-alanine, L-proline, L-asparagine, and L-glutamine. The levels of benzoic acid, cinnamic acid, salicylic acid, trans-jasmonic acid, indole acetic acid, indole propionic acid, and abscisic acid were significantly increased in GABA-treated plants compared to the control. The gene expression showed that GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) were induced in GABA-treated plants, indicating a conversion of GABA to succinate. In addition, the gene expression of the regulatory enzymes of the TCA cycle (malate dehydrogenase and succinic dehydrogenase) was upregulated in GABA-treated plants, indicating an induction of respiration. In agreement with the chemical analysis, the gene expression results showed that most of the genes implicated in the biosynthesis of phytohormones were also upregulated in GABA-treated plants. Our results indicated that GABA works in harmony with phytohormones and suggested that regulation of phytohormones by exogenous GABA could play a key role in reducing plant stress.

A Plant Bacterial Pathogen Manipulates Its Insect Vector's Energy Metabolism.

Insect-transmitted plant-pathogenic bacteria may alter their vectors' fitness, survival, behavior, and metabolism. Because these pathogens interact with their vectors on the cellular and organismal levels, potential changes at the biochemical level might occur. "Candidatus Liberibacter asiaticus" (CLas) is transmitted in a persistent, circulative, and propagative manner. The genome of CLas revealed the presence of an ATP translocase that mediates the uptake of ATP and other nucleotides from medium to achieve its biological processes, such as growth and multiplication. Here, we showed that the levels of ATP and many other nucleotides were significantly higher in CLas-infected than healthy psyllids. Gene expression analysis showed upregulation for ATP synthase subunits, while ATPase enzyme activity showed a decrease in ATPase activity. These results indicated that CLas stimulated Diaphorina citri to produce more ATP and many other energetic nucleotides, while it may inhibit their consumption by the insect. As a result of ATP accumulation, the adenylated energy charge (AEC) increased and the AMP/ATP and ADP/ATP ratios decreased in CLas-infected D. citri psyllids. Survival analysis confirmed a shorter life span for CLas-infected D. citri psyllids. In addition, electropenetrography showed a significant reduction in total nonprobing time, salivation time, and time from the last E2 (phloem ingestion) to the end of recording, indicating that CLas-infected psyllids were at a higher hunger level and they tended to forage more often. This increased feeding activity reflects the CLas-induced energetic stress. In conclusion, CLas alters the energy metabolism of its psyllid vector, D. citri, in order to secure its need for energetic nucleotides.IMPORTANCE Insect transmission of plant-pathogenic bacteria involves propagation and circulation of the bacteria within their vectors. The transmission process is complex and requires specific interactions at the molecular and biochemical levels. The growth of the plant-pathogenic bacteria in the hemolymph of their vectors indicated that the hemolymph contains all the necessary nutrients for their growth. In addition to nutrients, "Candidatus Liberibacter asiaticus" (CLas) can take up energetic nucleotides, such as ATP, from its vector, Diaphorina citri, using ATP translocase. In this study, we found that the CLas pathogen manipulates the energy metabolism of its insect vector. The accumulation of ATP in CLas-infected D. citri psyllids indicated that CLas induces ATP production to fulfill its need for this energetic compound. As a result of ATP accumulation, a shorter life span and altered feeding behavior were observed. These findings increase our knowledge of insect transmission of the persistent-circulative-propagative type of plant pathogens vectored by insects.

RNA interference of acetylcholinesterase in the Asian citrus psyllid, Diaphorina citri, increases its susceptibility to carbamate and organophosphate insecticides.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Lividae) transmits the Candidatus Liberibacter asiaticus, which causes citrus greening disease or Huanglongbing, (HLB). To date, there is no efficient cure for HLB disease and the control of D. citri using insecticides became the most important tools for the management of HLB. However, the extensive use of insecticides could increase D. citri resistance to these insecticides. The objective of this study was to investigate the effect of RNA interference of acetylcholinesterase (AChE) on the mortality and susceptibility of D. citri to the four major insecticides used in Florida. In this study, we used a consensus sequence derived from the two AChE genes and cholinesterase 2-like (ChE-2-like) gene to target all of the three genes. Treatment with dsRNA-AChE increased the mortality percentages of both nymphs and adults of D. citri. The mortality percentage increased with the increase in the concentration of applied dsRNA-AChE, and the highest mortality (> 60%) was observed at the highest applied concentration (125ng/µl). Treatments of nymphs or adults with dsRNA-AChE down-regulated the expression of the three targeted genes of D. citri. Silencing of AChE and ChE in D. citri nymphs increased the susceptibility of emerged adults to chlorpyrifos and carbaryl, which act as AChE inhibitors. However, treatment with dsRNA-AChE did not increase the susceptibility of emerged adults to imidacloprid, which acts as an agonist of nicotinic acetylcholine receptors. In the same manner, treatment of adults with dsRNA-AChE increased their susceptibility to chlorpyrifos and carbaryl, but did not affect their susceptibility to imidacloprid. The ANOVA did not show any significant increase in susceptibility of D. citri adults to fenpropathrin after treatment with dsRNA-AChE, either as nymphs or as adults. However, simple linear regression showed that treatment with dsRNA-AChE increased D. citri susceptibility to fenpropathrin, which indicated that AChE could be involved in the metabolism of fenpropathrin. Our results indicated that silencing of AChE and ChE genes in D. citri to increase its susceptibility to insecticides could be a promising tool for the control of this important vector.

Impact of different temperatures on survival and energy metabolism in the Asian citrus psyllid, Diaphorina citri Kuwayama.

Temperature influences the life history and metabolic parameters of insects. Asian citrus psyllid (ACP), Diaphorina citri is a tropical and subtropical pest. ACP invaded new regions around the world and threatened the citrus industry as a vector for Huanglongbing (HLB) disease. ACP is widely distributed and can survive high (up to 45 °C) and low temperatures (as low as -6 °C). The precise mechanism of temperature tolerance in ACP is poorly understood. We investigated adult survival, cellular energy balance, gene expression, and nucleotide and sugar-nucleotide changes under the effect of different temperature regimes (0 °C to 45 °C with 5 °C intervals). The optimum temperatures for survival were 20 and 25 °C. Low temperatures of 0 °C and 5 °C caused 50% mortality after 2 and 4 days respectively, while one day at high temperature (40 °C and 45 °C) caused more than 95% mortality. The lowest quantity of ATP (3.69 ± 1.6 ng/insect) and the maximum ATPase enzyme activities (57.43 ± 7.6 µU/insect) were observed at 25 °C. Correlation between ATP quantities and ATPase activity was negative. Gene expression of hsp 70, V-type proton ATPase catalytic subunit A and ATP synthase α subunit matched these results. Twenty-four nucleotides and sugar-nucleotides were quantified using HPLC in ACP adults maintained at low, high, and optimum temperatures. The nucleotide profiles were different among treatments. The ratios between AMP:ATP and ADP:ATP were significantly decreased and positively correlated to adults survival, whereas the adenylate energy charge was increased in response to low and high temperatures. Exploring energy metabolic regulation in relation with adult survival might help in understanding the physiological basis of how ACP tolerates newly invaded regions.

Development of delayed bitterness and effect of harvest date in stored juice from two complex citrus hybrids.

BACKGROUND: Mandarins and mandarin hybrids have excellent flavor and color attributes, making them good candidates for consumption as fresh fruit. When processed into juice, however, they are less palatable, as they develop delayed bitterness when stored for a period of time. In this study the kinetics of delayed bitterness in two citrus mandarin hybrid siblings, 'Ambersweet' and USDA 1-105-106, was explored by sensory and instrumental analyses. In addition to the bitter limonoids, other quality factors (i.e. sugars, acids, pH, soluble solids content (SSC), titratable acidity (TA) and the ratio SSC/TA) were also measured. RESULTS: The two citrus hybrid siblings had different chemical profiles, which were perceived by taste panels. USDA 1-105-106 developed delayed bitterness when the juice was stored for more than 4 h, similar to juice from 'Navel' oranges, but 'Ambersweet' did not. Bitterness in 'Ambersweet' was more affected by harvest maturity, as juice from earlier harvest had lower SSC but higher TA and bitter limonoids. CONCLUSION: Since juice of USDA 1-105-106 shows delayed bitterness when stored for more than 4 h, this cultivar is not suitable for juice processing. Our finding that siblings can differ in chemical and sensory properties emphasize the importance of post-processing storage studies before releasing cultivars for juice.

Viability of 'Candidatus Liberibacter asiaticus' prolonged by addition of citrus juice to culture medium.

Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium 'Candidatus Liberibacter asiaticus'. Infection with 'Ca. L. asiaticus' is incurable; therefore, knowledge regarding 'Ca. L. asiaticus' biology and pathogenesis is essential to develop a treatment. However, 'Ca. L. asiaticus' cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of 'Ca. L. asiaticus' in vitro, 'Ca. L. asiaticus' inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima 'Mato Buntan') was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King's B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air-liquid interface of juice cultures contained 'Ca. L. asiaticus' cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining 'Ca. L. asiaticus' viability in vitro, which will contribute to future development of a culture medium for 'Ca. L. asiaticus'.

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.

Citrate Mediated Europium-Based Detection of Oxytetracycline in Citrus Tissues.

Oxytetracycline (OTC) and streptomycin have been used for the control of several plant diseases and were recently permitted for the control of citrus greening disease, Huanglongbing. Consequently, sensitive and reliable methods are highly needed for the detection of OTC in citrus tissues. Herein, we studied the replacement of cetyltrimethylammonium chloride (CTAC) by citrate (Cit) as a sensitizing agent for the analysis of OTC in citrus tissues using the recently established europium (Eu) method. In addition, we determined the optimal conditions for the formation of the Eu-OTC-Cit ternary complex in tris buffer. Our results showed that the plant matrix significantly decreased the fluorescence intensity of the Eu-OTC-Cit complex even after the replacement of CTAC. Our investigations showed that phenols such as gallic acid degrade slowly at high pH and their degradation was enhanced in the presence of the (Eu+3) cation. To reduce the plant matrix interference, the sample extract was cleaned using solid-phase extraction (SPE). The OTC recoveries from spiked healthy and Candidatus Liberibacter asiaticus (CLas)-infected trees were 91.4 ± 7.8% and 82.4 ± 3.9%, respectively. We also used the citrate method to determine the level of OTC in trunk-injected trees. The level of OTC as measured using the Eu-OTC-Cit complex (117.5 ± 20.3 µg g-1 fresh weight "FWT") was similar to that measured using Eu-OTC-CTAC complex (97.5 ± 14 µg g-1 FWT). In addition, we were able to visualize the OTC in citrus leaf extract, under ultraviolet light (400 nm), after it was cleaned with the SPE. Our study showed that the citrate can be successfully used to replace the harmful CTAC surfactant, which could also react with phenols.

Detection of Oxytetracycline in Citrus Phloem and Xylem Saps Using Europium-Based Method.

Oxytetracycline (OTC) has been used for the control of several plant diseases and was recently approved for the control of Huanglongbing, the citrus greening disease. Huanglongbing is caused by the phloem limited 'Candidatus Liberibacter asiaticus'. Determination of OTC in the xylem and phloem of citrus plants is of great interest as they are the main routes of translocation in citrus. In addition, the determination of the level of OTC in the phloem sap is necessary for the control of the 'Ca. L. asiaticus' pathogen, which resides in the phloem. Herein, we demonstrated that the level of OTC in the citrus phloem and xylem saps obtained using the centrifugation method can be successfully measured using the europium (Eu) method directly or with cleanup by solid-phase extraction (SPE). Recovery of OTC from spiked sap samples purified by solid-phase extraction (SPE) was higher than 90%, while recovery from saps without SPE cleanup were nearly 100%. The 'Ca. L. asiaticus'-infected leaf and phloem sap samples showed higher inhibition of the fluorescence intensity of the OTC standard compared to non-infected control leaf and phloem samples. In agreement with this finding, the levels of phenols and flavonoids in 'Ca. L. asiaticus'-infected leaves were higher than those controls and were shown to interfere with the Eu method. Therefore, the SPE cleanup step only improved OTC recovery from leaf samples containing the interfering compounds. The Eu method was then used to determine OTC levels in the phloem and xylem sap of OTC-treated plants, and the results were similar whether measured directly or after SPE. Visualization under ultraviolet light (400 nm) showed the presence of OTC in citrus xylem and phloem saps with and without the use of SPE.

Development of Europium-Sensitized Fluorescence-Based Method for Sensitive Detection of Oxytetracycline in Citrus Tissues.

Antimicrobial compounds have been successfully used to control many plant and animal diseases. Recently, oxytetracycline (OTC) and streptomycin have been approved for the treatment of Huanglongbing in citrus. Since the application of OTC is under strict regulations, several methods have been developed to determine and monitor its levels in the environment including high-performance liquid chromatography, ELISA, colorimetric, and fluorometric assays. In this study, we developed a fluorometric method for the determination of OTC in plant tissues based on its complexation with europium. Our preliminary trials showed that phenols and flavonoids interfere with the europium assay by reacting with the sensitizing reagent, cetyltrimethylammonium chloride. Consequently, we used the 60 mg hydrophilic-lipophilic balanced (HLB) cartridges to purify the OTC from the plant matrix. The recovery of OTC from spiked leaf samples was 75 ± 7.6%. Using the 500 mg HLB, we were able to detect 0.3 ppm OTC in the final sample extract, which corresponds to 3 µg g-1 fresh weight (FWT). The developed method was successfully used to measure the level of OTC in leaves obtained from trunk-injected trees. The results obtained by the europium method were similar to those obtained using the ELISA assay. We also tested the cross-reactivity of OTC metabolites with the europium method. The 4-epi-OTC showed a high cross-reactivity (50.0 ± 3.6%) with europium assay, whereas α-apo-OTC and ß-apo-OTC showed small cross-reactivity. We showed that the europium-sensitized fluorescence-based method can be successfully used to assess OTC in citrus plant tissues after a cleanup step. Our results showed that this method was sensitive, reproducible, and can be used to analyze many samples simultaneously.

Evaluation of Oxytetracycline Metabolites Cross-Reactivity with Oxytetracycline Enzyme-Linked Immunosorbent Assay (ELISA).

Antibiotics have been successfully used for the control of several plant diseases for many years. Recently, streptomycin and oxytetracycline have been approved for the treatment of Huanglongbing (HLB) in Florida. The enzyme-linked immunosorbent assay (ELISA) is the most commonly used assay for the detection of these antibiotics because it is quick, simple, and can be used to analyze many samples at the same time. However, ELISA can react with the metabolites of the parent compound and its structurally related compounds. In this study, we investigated the cross-reactivity of the oxytetracycline ACCEL ELISA kitTM with three of oxytetracycline metabolites (4-epi-oxytetracycline, α-apo-oxytetracycline, and ß-apo-oxytetracycline). The α-apo-oxytetracycline and ß-apo-oxytetracycline metabolite did not show any cross-reactivity in the linear range (1.5-50 ng mL-1) of the assay. Whereas 4-epi-oxytetracycline showed high cross-reactivity, and its response was similar to oxytetracycline. Our results indicated that the oxytetracycline ELISA kits estimate the level of oxytetracycline as well as its main metabolite, 4-epi-oxytetracycline.

The use of deuterium-labeled gamma-aminobutyric (D6-GABA) to study uptake, translocation, and metabolism of exogenous GABA in plants.

BACKGROUND: Exogenous application of gamma-aminobutyric acid (GABA) could relieve stress symptoms caused by abiotic stresses including anoxia, heat and cold shock, drought, and salt. However, studying translocation and metabolism of exogenous GABA is challenged by the presence of endogenous GABA. RESULTS: Herein, we used D6-GABA in order to investigate the uptake, translocation, and the metabolism of exogenous GABA in Mexican lime (Citrus aurantifolia) seedlings using gas chromatography-mass spectrometry (GC-MS). The GC-MS analysis showed that D6-GABA could be easily distinguished from the non-labeled GABA after methyl chloroformate (MCF) derivatization. The D6-GABA was detected in the cortex (phloem), inner stem (xylem), and leaves after root drench. Girdling did not affect the translocation of D6-GABA, indicating that it is mainly translocated via the xylem. In addition, D4-labled succinic acid was detected in D6-GABA-treated plants, indicating that exogenous GABA was metabolized to succinic acid. The half-life of D6-GABA in citrus was about 1.3 h, indicating a quick conversion to succinic acid. CONCLUSION: The use of D6-GABA offers a valuable tool to study the translocation and metabolism of GABA in plants. D6-GABA and its metabolite (D4-succinic acid) can be easily distinguished from the endogenous GABA and succinic acid using GC-MS.

Phenolics, flavonoids and antioxidant capacities in Citrus species with different degree of tolerance to Huanglongbing.

Huanglongbing (HLB) is a highly destructive disease to the citrus industry in Florida caused by the bacterium, Candidatus Liberibacter asiaticus(CLas) and is transmitted by Diaphorina citri. It is hypothesized that plants with high phenolic contents show higher tolerance to certain plant pathogens. In this regard, different citrus varieties and plants of genera related to Citrus were analyzed for their total phenolic and flavonoids contents, and their antioxidant capacities. In addition, the secondary metabolites in the leaves of seven citrus species were analyzed using high performance liquid chromatography-mass spectrometry (HPLC-MS). Colorimetric assays showed that curry leaf contained the highest total phenolic content and free radical scavenging activity (DPPH). Curry leaf also contained high concentrations of an unusual class of carbazole alkaloids. Tolerant Citrus species contained high levels of phenolics and flavonoids and showed high antioxidant capacities. Our results suggest that high phenolic and flavonoid leaf contents correlate with increased citrus tolerance to CLas bacterium. The results also suggest that the high level of carbazole alkaloids, known for their strong antimicrobial properties in curry leaf, could make it immune to the CLas bacteria. Understanding the mechanisms underpinning citrus tolerance to HLB will contribute to the development of commercially tolerant citrus cultivars.

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.

Knock-down of δ-aminolevulinic acid dehydratase via virus-induced gene silencing alters the microRNA biogenesis and causes stress-related reactions in citrus plants.

The δ-aminolevulinic acid (δ-ALA) is an intermediate in the biosynthetic pathway of tetrapyrroles. Tetrapyrroles play vital roles in many biological processes such as photosynthesis, respiration, and light-sensing. ALA-dehydratase (ALAD) combines two molecules of δ-ALA to form porphobilinogen. In citrus, the silencing of ALAD caused discrete yellow spots and necrosis in leaves and stems. Additionally, it caused rapid death in developing new shoots. Herein, we hypothesize that the accumulation of δ-ALA results in severe stress and reduced meristem development. For that reason, we investigated the dynamic changes in the expression profiles of 23 microRNA (miRNA) identified through small RNA sequencing, from CTV-tALAD plants in comparison with healthy C. macrophylla and C. macrophylla infiltrated with CTV-wt. Furthermore, we reported the effect of ALAD silencing on the total phenolics, H2O2, and reactive oxygen species (ROS) levels, to examine the possibilities of miRNAs involving the regulation of these pathways. Our results showed that the total phenolics content, H2O2, and O2- levels were increased in CTV-tALAD plants. Moreover, 63 conserved miRNA members belonging to 23 different miRNA families were differentially expressed in CTV-tALAD plants compared to controls. The identified miRNAs are implicated in auxin biosynthesis and signaling, axillary shoot meristem formation and leaf morphology, starch metabolism, and oxidative stress. Collectively, our findings suggested that ALAD silencing initiates stress on citrus plants. As a result, CTV-tALAD plants exhibit reduced metabolic rate, growth, and development in order to cope with the stress that resulted from the accumulation of δ-ALA. This cascade of events led to leaf, stem, and meristem necrosis and failure of new shoot development.

Effect of fruit maturity on volatiles and sensory descriptors of four mandarin hybrids.

Mandarins (or tangerines) are mainly consumed as fresh fruits due to the ease of peeling and desirable flavor. Sweetness, acidity, and flavor of mandarin are the most important criteria for consumer preference. The objective of this study was to evaluate the effects of harvest date on sensory and chemical components of four mandarin cultivars (Murcott, 411, Temple, and 'LB8-9' Sugar Belle®). Volatiles were extracted from the headspace of juice samples with solid phase microextraction (SPME) and analyzed using gas chromatography-mass spectrometry (GC-MS). The optimum harvest window for eating quality of 411 was late January to mid-February (soluble solids content [SSC]/titratable acidity [TA]: 11.3 to 14.0), Sugar Belle® fruits were best tasted when harvested from mid- to end of January (SSC/TA: 14.1 to 16.1), and February was the best month for harvesting Murcott (SSC/TA: 13.10 to 18.0) and Temple (SSC/TA:10.3 to 12.50). Sensory perception of sweetness, ripeness, and juiciness increased as SSC/TA increased while sourness and bitterness decreased. Pumpkin flavor, an indicator of overripe fruit, was mainly noticed late in the season. Tangerine flavor tended to decrease, whereas fruity-noncitrus flavor tended to increase with fruit maturity. Monoterpenes were the most abundant volatiles and tended to decrease with fruit maturity, whereas alcohols, esters, and aldehydes increase. Aldehydes, esters, and alcohols were positively correlated with sweetness, ripeness, juiciness, and fruity characteristics, and negatively with sourness and bitterness. On the other hand, monoterpenes were positively correlated with bitterness and tangerine flavor, and negatively correlated with sweetness and fruity-noncitrus flavor. The highest number of esters was found in Temple, whereas Murcott and 411 were high in aldehydes.

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.

Metabolic Profiling of Hybrids Generated from Pummelo and Citrus latipes in Relation to Their Attraction to Diaphorina citri, the Vector of Huanglongbing.

The citrus industry at present is severely affected by huanglongbing disease (HLB). HLB is caused by the supposed bacterial pathogen "Candidatus Liberibacter asiaticus" and is transmitted by the insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. Developing new citrus hybrids to improve HLB management is much needed. In this study, we investigated the metabolomic profiles of three new hybrids produced from the cross of C2-5-12 Pummelo (Citrus maxima (L.) Osbeck) × pollen from Citrus latipes. The hybrids were selected based on leaf morphology and seedling vigor. The selected hybrids exhibited compact and upright tree architecture as seen in C. latipes. Hybrids were verified by simple sequence repeat markers, and were subjected to metabolomic analysis using gas chromatography-mass spectrometry. The volatile organic compounds (VOCs) and polar metabolites profiling also showed that the new hybrids were different from their parents. Interestingly, the levels of stored VOCs in hybrid II were higher than those observed in its parents and other hybrids. The level of most VOCs released by hybrid II was also higher than that released from its parents. Additionally, the preference assay showed that hybrid II was more attractive to D. citri than its parents and other hybrids. The leaf morphology, compact and upright architecture of hybrid II, and its attraction to D. citri suggest that it could be used as a windbreak and trap tree for D. citri (double duty), once its tolerance to HLB disease is confirmed. Our results showed that metabolomic analysis could be successfully used to understand the biochemical mechanisms controlling the interaction of D. citri with its host plants.

Exogenous GABA is quickly metabolized to succinic acid and fed into the plant TCA cycle.

Exogenous application of γ-aminobutyric acid (GABA) could improve plant tolerance to environmental stresses such as hypoxia, heat, cold, drought, and salt stress. However, the mechanism by which GABA relieves stress is poorly understood. Here, we studied the uptake and metabolism of exogenous GABA in citrus leaves. Leaves were incubated in GABA solutions and the levels of endogenous GABA, succinic acid, and fumaric acid were investigated. Interestingly, the levels of endogenous GABA, succinic acid, and fumaric acid were quickly increased in GABA-treated leaves. This result indicated that GABA was taken up by the leaf, metabolized to succinic acid, and fed into the tricarboxylic acid cycle (TCA). This finding suggested that exogenous GABA could enhance plant resistance to biotic and abiotic stresses by generating more energy through the activation of the GABA shunt pathway and TCA cycle.