Since the advent of Huanglongbing [HLB ( Candidatus Liberibacter asiaticus)] in Florida, several preliminary reports have emerged about the positive effects of mineral nutrition on the performance of HLB-affected citrus ( Citrus sp.) trees. HLB-affected trees are known to undergo significant feeder root loss. Therefore, studies have focused on foliar nutrient application instead of soil-applied nutrients speculating that the HLB-affected trees root systems may not be competent in nutrient uptake. Some studies also suggest that HLB-affected trees benefit from micronutrients at higher than the recommended rates; however, the results are often inconclusive and inconsistent. To address this, the goal of the present study was to evaluate the nutrient uptake efficiency and the quantitative and qualitative differences in nutrient uptake of HLB-affected trees. HLB-affected and healthy sweet orange ( Citrus sinensis ) trees were grown in a 100% hydroponic system with Hoagland solution for 8 weeks. The trees were deprived of any fertilization for 6 months before the transfer of trees to the hydroponic solution. Altogether, the four treatments studied in the hydroponic system were healthy trees fertilized (HLY-F) and not fertilized (HLY-NF), and HLB-affected trees fertilized (HLB-F) and not fertilized (HLB-NF). HLY-F and HLY-NF trees were found to have similar levels of leaf nutrients except for N, which was found to be low in nonfertilized trees (HLY and HLB). Both HLB-F and HLB-NF trees had lower levels of Ca, Mg, and S compared with HLY trees. In addition, HLB-NF trees had significantly lower levels of micronutrients Mn, Zn, and Fe, compared with HLY-NF trees. The hydroponic solution analysis showed that HLB-F and HLY-F trees had similar uptake of all the nutrients. Considering that HLB-affected trees have a lower root-to-shoot ratio than healthy trees, nutrient uptake efficiency per kilogram of root tissue was significantly higher in HLB trees compared with HLY trees. Under nutrient-deficient conditions (day 0) only nine genes were differentially expressed in HLB roots compared with HLY roots. On the other hand, when fertilizer was supplied for ≈1 week, ≈2300 genes were differentially expressed in HLB-F roots compared with HLY-F roots. A large number of differentially expressed genes in HLB-F were related to ion transport, root growth and development, anatomic changes, cell death, and apoptosis compared with HLY-F trees. Overall, anatomic and transcriptomic analyses revealed that HLB-affected roots undergo remarkable changes on transitioning from no nutrients to a nutrient solution, possibly facilitating a high uptake of nutrients. Our results suggest the roots of HLB-affected trees are highly efficient in nutrient uptake; however, a small root mass is a major limitation in nutrient uptake. Certain micronutrients and secondary macronutrients are also metabolized (possibly involved in tree defense or oxidative stress response) at a higher rate in HLB-affected trees than healthy trees. Therefore, a constant supply of fertilizer at a slightly higher rate than what is recommended for micronutrients and secondary macronutrients would be beneficial for managing HLB-affected trees.

For field-grown ‘Valencia’ sweet orange ( Citrus sinensis ) affected by Huanglongbing [HLB ( Candidatus Liberibacter asiaticus ( C Las)], trees that displayed more severe HLB symptoms (severe trees) had 74% fruit drop before harvest; however, the drop rate for less symptomatic trees (mild trees) was 45%. For mature fruit (3 weeks before harvest) still attached to the branches, 60% of them from severe trees were “loose fruit” [fruit detachment force (FT) < 6 kgf]. In contrast, only 13% of the attached fruit from the mild trees were loose. Overall, fresh weight and size of loose fruit were lower than “tight fruit” (FT > 6 kgf). Irrespective of the symptom levels of trees, the concentrations of glucose, fructose, and inositol in juice of loose fruit were the same or larger than those of tight fruit, suggesting that the shortage of carbohydrates is not the dominant cause of HLB-associated preharvest fruit drop. Expression levels of the cell wall modification genes encoding cellulase (endo-1,4-β-glucanase), polygalacturonase, and pectate lyase were greater in the calyx abscission zones of loose fruit compared to tight fruit, indicating that cell separation was occurring in the former at the time of collection. No differences in the expression levels of genes encoding the ethylene biosynthesis enzymes, including 1-aminocyclopropane-1-carboxylic acid (ACC) synthase ( ACS ) and ACC oxidase ( ACO ), and an ethylene-responsive transcription factor 1 ( ERF1 ) were observed in tissues of loose and tight fruit. Interestingly, ACS , ACO , and EFR1 expressions were lower in calyx abscission zones and in leaves of the severe trees compared with those of mild trees, suggesting an ostensible, HLB-dependent reduction in ethylene biosynthesis and/or signaling close to harvest time. However, the role of ethylene in HLB-associated preharvest fruit drop remains to be determined. The results leave open the possibility of early ethylene production and action before the initiation of fruit abscission.

Devastating citrus disease Huanglongbing (HLB) is without existing cures. Herein, we present results demonstrating the possible mechanisms (hypoxia stress) behind HLB-triggered shoot dieback by comparing the transcriptomes, hormone profiles, and key enzyme activities in buds of severely and mildly symptomatic ‘Hamlin’ sweet orange (Citrus sinensis). Within six months (October – May) in field conditions, severe trees had 23% bud dieback, greater than mild trees (11%), with a concomitant reduction in canopy density. In February, differentially expressed genes (DEGs) associated with responses to osmotic stress, low oxygen levels, and cell death were upregulated, with those for photosynthesis and cell cycle downregulated in severe versus mild trees. For severe trees, not only were the key markers for hypoxia, including anaerobic fermentation, reactive oxygen species (ROS) production, and lipid oxidation, transcriptionally upregulated, but also alcohol dehydrogenase activity was significantly greater compared to mild trees, indicating a link between bud dieback and hypoxia. Tricarboxylic acid cycle revival, given the upregulation of glutamate dehydrogenase and alanine aminotransferase DEGs, suggests that ROS may also be generated during hypoxia-reoxygenation. Greater (hormonal) ratios of abscisic acid to cytokinins and jasmonates and upregulated DEGs encoding NADPH oxidases in severe versus mild trees indicate additional ROS production under limited oxygen availability due to stomata closure. Altogether, our results provided evidence that as HLB progresses, excessive ROS produced in response to hypoxia and during hypoxia-reoxygenation likely intensify the oxidative stress in buds leading to cell death, contributing to marked bud and shoot dieback and decline of the severely symptomatic sweet orange trees.

To determine how the dormancy-breaking agent hydrogen cyanamide (HC) advances budbreak in peach ( Prunus persica ), this study compared the transcriptome of buds of low-chill ‘TropicBeauty’ peach trees treated with 1% (v/v) HC and that of nontreated trees at 3 and 7 days after treatment (DAT), respectively, using an RNA sequencing analysis. The peak of total budbreak occurred 6 weeks earlier in the HC-treated trees (at 32 DAT) than the nontreated trees (at 74 DAT). There were 1312 and 1095 differentially expressed genes (DEGs) at 3 and 7 DAT, respectively. At 3 DAT, DEGs related to oxidative stress, including the response to hypoxia, lipid oxidation, and reactive oxygen species (ROS) metabolic process, were upregulated in HC-treated buds. Additionally, DEGs encoding enzymes for ROS scavenging and the pentose phosphate pathway were upregulated at 3 DAT but they were not differently expressed at 7 DAT, indicating a temporary demand for defense mechanisms against HC-triggered oxidative stress. Upregulation of DEGs for cell division and development at 7 DAT, which were downregulated at 3 DAT, suggests that cell activity was initially suppressed but was enhanced within 7 DAT. At 7 DAT, DEGs related to cell wall degradation and modification were upregulated, which was possibly responsible for the burst of buds. The results of this study strongly suggest that HC induces transient oxidative stress shortly after application, leading to the release of bud dormancy and, subsequently, causing an increase in cell activity and cell wall loosening, thereby accelerating budbreak in peach.

Citrus disease Huanglongbing (HLB) causes sparse (thinner) canopies due to reduced leaf and shoot biomass. Herein, we present results demonstrating the possible mechanisms behind compromised leaf growth of HLB-affected ‘Valencia’ sweet orange trees by comparing morphological, transcriptome, and phytohormone profiles at different leaf development phases (1. buds at the start of the experiment; 2. buds on day 5; . 3. leaf emergence; 4. leaf expansion; and 5. leaf maturation) to healthy trees. Over a period of 3 months (in greenhouse conditions), HLB-affected trees had ≈40% reduction in growth traits such as tree height, number of shoots per tree, shoot length, internode length, and leaf size compared to healthy trees. In addition, buds from HLB-affected trees lagged by ≈1 week in sprouting as well as leaf growth. Throughout the leaf development, high accumulation of defense hormones, salicylic acid (SA) and abscisic acid (ABA), and low levels of growth-promoting hormone (auxin) were found in HLB-affected trees compared to healthy trees. Concomitantly, HLB-affected trees had upregulated differentially expressed genes (DEGs) encoding SA, ABA, and ethylene-related proteins in comparison to healthy trees. The total number of cells per leaf was lower in HLB-affected trees compared to healthy trees, which suggests that reduced cell division may coincide with low levels of growth-promoting hormones leading to small leaf size. Both bud dieback and leaf drop were higher in HLB-affected trees than in healthy trees, with concomitant upregulated DEGs encoding senescence-related proteins in HLB-affected trees that possibly resulted in accelerated aging and cell death. Taken together, it can be concluded that HLB-affected trees had a higher tradeoff of resources on defense over growth, leading to sparse canopies and a high tree mortality rate with HLB progression.

Previous research has shown that Huanglongbing {HLB [causal agent Candidatus Liberibacter asiaticus ( C Las)]}-affected sweet orange ( Citrus sinensis ) trees have a reduced root-to-shoot ratio, potentially due to the high rate of root death. The diminished root system cannot support the existing aboveground canopy and a cycle of imbalance begins. As a result, the tree enters into a continuous carbohydrate stress cycle and, eventually, the tree declines. Therefore, the goal of this study was to evaluate pruning as a strategy to adjust the root-to-shoot ratio to improve growth and productivity of HLB-affected trees. In Jan. 2015, a 3-year trial was initiated on a 14-year-old grove of ‘Hamlin’ sweet orange on Swingle citrumelo ( Citrus paradisi × Poncirus trifoliate ) rootstock that was symptomatic of HLB and produced less than 180 lb of fruit per tree. The four pruning treatments were as follows: 1) 0% pruning (no canopy removal), 2) 25% pruning (canopy removed), 3) 50% pruning (canopy removed), and 4) 80% pruning (canopy removed). In a split-plot design, two sources of fertilizer were evaluated in combination with the pruning: 1) conventional fertilizer [CNV (dry granular)] applied at 200 lb/acre nitrogen (N) in five split applications per year, and 2) controlled-release fertilizer (CRF) applied at 150 lb/acre N, split in three applications per year. Within each pruning treatment, half of the trees received CNV and the other half received CRF. The fertilizer treatments were applied in each of the 3 years; however, pruning was performed only once in the beginning of the experiment. The trees that were pruned produced new vegetative growth that looked healthy with no visual HLB symptoms (initially); however, the trees remained positive for CLas throughout the study as determined by quantitative real-time polymerase chain reaction. The 80% pruned trees grew vigorously over the course of 3 years but remained significantly smaller in canopy than control trees (0% pruning) for both CRF and CNV treatments. The 25% and 50% pruned tree canopies grew back and were similar in canopy size as 0% pruning (control) treatment by the end of year 2. At the end of the study, the use of CRF on 25% pruned trees resulted in a significantly higher leaf area index as compared with trees receiving CNV. A significant positive linear correlation was observed between canopy volume and root density; the root density decreased with intensive pruning. A significant positive correlation was also observed between canopy volume and yield, and a negative correlation between canopy volume and fruit drop. There were no significant increases in yield resulting from any pruning or fertilization treatments compared with controls (0% pruning). However, with the use of CRF, the amount of N and frequency of application were reduced. Overall, our results indicate that pruning did not improve the productivity of HLB-affected trees over the course of 3 years. Therefore, severe pruning is not a viable option to rejuvenate the HLB-affected trees.