Abstract
Huanglongbing {HLB [Candidatus Liberibacter asiaticus (C Las)]} has been one of the biggest challenges in citrus (Citrus sp.) production in Florida and wherever it is present. HLB-affected trees show significant shoot and root dieback, fruit drop, and reduction in yield. Currently, there is no cure for HLB, and there is no commercial HLB-resistant germplasm. Nonetheless, intensive nutrient management has been promising for citrus growers. The nutrient requirement of HLB-affected trees seems to be greater than that of healthy citrus trees. By understanding the nutrient uptake potential of rootstocks, fertilizer programs can be customized accordingly to enhance the performance of a rootstock in existing groves. Moreover, a reduction in the application of nutrients is possible by planting rootstocks with a high nutrient absorption capacity. Use of rootstocks with good nutrient uptake efficiency can take some burden off the growers who are intensively managing HLB-affected citrus groves. Therefore, the objective of this study was to evaluate and understand the nutrient uptake potential of the citrus rootstocks. To achieve this objective, a 100% hydroponic greenhouse study was conducted with six rootstocks with a range of tolerance to HLB. Several physiological and molecular tools were applied to evaluate the rootstocks for their nutrient uptake potential. A+Volk × O-19 (HLB-tolerant) rootstock had greater nutrient uptake efficiency, whereas US-896 (HLB-susceptible) had lesser nutrient uptake efficiency. Swingle, one of the most popular pre-HLB rootstocks, had poor zinc uptake and the least expression of ZINC TRANSPORTER, suggesting that zinc applications should be emphasized in Swingle plantings. US-896 rootstock expressed the least level of nutrient transporter genes, such as IRON TRANSPORTER. UFR-4 (a good performer under HLB conditions) had a large root biomass, but the uptake efficiency for nutrients was poor, suggesting that the nutrient uptake potential is a complex process that is not solely dependent on root biomass. This study is unique because it is one of the first citrus studies to report nutrient uptake efficiency and the potential of rootstocks. The information presented can be used to improve performance or select better-performing rootstocks under HLB conditions.
Huanglongbing (HLB) is presumably caused by the phloem limited bacteria, Candidatus Liberibacter asiaticus (CLas), and is one of the most devastating diseases of citrus (Gottwald, 2010). A distinctive chlorotic mottle on fully expanded leaves is one of the identifying symptoms of HLB (Gottwald et al., 2007). Other symptoms such as stunting and gradual die-back of shoots, lopsided fruit, increased fruit drop, and yield reduction become more evident with the increase in the severity of the disease (Bassanezi et al., 2011; Graham et al., 2013; Tang et al., 2019). Johnson et al. (2014) reported that during the early CLas infection phase in Swingle rootstock, CLas accumulation can be readily seen in the root, possibly because the roots are the sink that receives bulk phloem flow, and CLas mobilizes in the phloem. Depending on the HLB severity, citrus trees can suffer up to 50% fine root loss during later stages of infection (Johnson and Graham, 2015). As a result of a diminishing root system, nutrient uptake can be reduced (Shahzad et al., 2020). Trees affected by HLB do not efficiently transport mineral nutrients from the root to the shoot system (Pustika et al., 2008). These HLB-affected trees have been reported to have lower leaf nutrient concentrations than healthy plants (Huber and Haneklaus, 2007). In several studies, it has been shown that there are lower concentrations of phosphorus (P), iron (Fe), zinc (Zn), calcium (Ca), magnesium (Mg), manganese (Mn), and sulphur (S) in the HLB-affected plants than in healthy trees (Masaoka et al., 2011; Shahzad et al., 2020), suggesting that additional nutrients might be required to compensate for the loss of fine roots.
Currently, there is no cure for HLB. It is well known that HLB-affected citrus trees receiving complete and balanced nutrition perform better over time (Morgan et al., 2016; Shahzad et al., 2020). Field surveys have shown that HLB-affected trees can remain productive for longer durations when provided with nutrients [such as Mn, Zn, Fe, boron (B), Ca, Mg] at higher levels than those recommended for healthy citrus (Vashisth and Grosser, 2018; Xia et al., 2011; Zambon et al., 2019).
The nutrient uptake potential (from root to shoot) of a rootstock is highly linked to its genotype. Rootstocks can influence the leaf nutrient concentration in the scion (Tagliavini et al., 1992); however, the differences can be caused by differences in the nutrient uptake potential of a rootstock or nutrient transport potential. It is important to understand the difference between nutrient uptake and nutrient transport potential. Nutrient uptake potential, as the name suggests, is the capacity of the roots to take up the nutrients from the growing media, whereas nutrient transport potential refers to the mobilization of the nutrients from the roots to leaves (Wutscher, 1973). Nonetheless because most of the nutrients are taken up by the roots with water (hydraulic conductivity) and mobilized via transpiration pull in xylem (Syvertsen and Graham, 1985), nutrient uptake potential and transport potential are related, but they cannot be used interchangeably (Wutscher, 1973). The difference in the nutrient uptake potential of different rootstocks could also lead to the differences in the nutrient concentration in fruit (Fazio et al., 2015). In a study of apple, differences in the nutrient uptake and absorption of several mineral nutrients (including K and Ca) were found to be regulated by genetic components that were strongly inherited from the breeding populations of Geneva rootstocks (Fazio et al., 2013). Because of the success of intensive/enhanced nutrient management programs that mitigate the effects of HLB (Xia et al., 2011), the need to identify rootstocks with good nutrient uptake potential has become important to ensure the effectiveness of intensive fertilizer programs. Use of rootstocks with good nutrient uptake potential can reduce the need to intensively manage the HLB-affected citrus groves. Knowledge about the nutrient uptake potential of rootstocks can further help to optimize the fertilizer program (Jiménez et al., 2004). Additionally, in the last decade, the costs of fertilizer and its application have become more significant to citrus production, with a total production cost of up to 25% (Singerman and Useche, 2016). The reduction in the application of nutrients is possible by using the rootstocks with a high nutrient absorption capacity (Keller et al., 2001). If the newly developed rootstocks are HLB-tolerant and have good nutrient uptake efficiency, then the costs of fertilizer and its application may decrease. Therefore, the objective of this study was to evaluate the nutrient uptake potential of six citrus rootstocks that with a range of tolerance to HLB.
Materials and Methods
Plant materials and experiment setup.
Rootstocks used in this experiment were selected based on rootstock performance under HLB pressure and commercial preferences of the citrus growers in Florida. The six rootstocks used in the experiment were as follows: Swingle citrumelo (C. paradisi × Poncirus trifoliata); UFR-4 [(C. reticulata × C. paradisi) + C. grandis] × [C. reticulata + Poncirus trifoliata], which is an allotetraploid; UFR-17 [C. reticulata/C. paradisi + C. grandis] × [C. aurantium + C. sinensis/Poncirus trifoliata]; US- 896 [C. reticulata × P. trifoliata ‘Rubidoux’]; 46x20–04, which is a diploid ‘Hirado Buntan Pink’ pummelo (C. grandis) × Cleopatra mandarin (C. reticulata Blanco); and A+Volk × Orange 19-11-8 rootstock (which is referred to as A+Volk × O-19 from here onward), which is an allotetraploid hybrid of a cybrid tetraploid Volkamer lemon (C. volkameriana) containing cytoplasm of Amblycarpa crossed with UFR-4. Swingle is a commercial standard and HLB-susceptible rootstock. US-896 is another HLB-susceptible rootstock (Bowman et al., 2016). A+Volk × O-19 is vigorous rootstock that is considered HLB-tolerant (Satpute, 2017). UFR-4 and UFR-17 are recently commercialized rootstocks in Florida that result in trees with a good canopy (Kunwar et al., 2021). The rootstocks were grown from seed for ≈5 months in containers with peat-based medium under standard greenhouse care. Five-month-old rootstock seedlings were budded with ‘Midsweet’ sweet orange and grown for 18 months in 5-L circulars pots with a peat-based medium before their use in these experiments. Throughout the experiment (including plant preparation time), all the plants were kept in a temperature-controlled greenhouse with natural light; the temperature and relative humidity of the greenhouse fluctuated between 22 and 25 °C and 60% to 80%, respectively. The plants were fertilized regularly with a mix of tap water and water-soluble 20N–20P–20K plus micronutrients fertilizer.
The experiment was conducted in a greenhouse at the Citrus Research and Education Center in Lake Alfred, FL. To validate the findings, the experiment was replicated twice in Spring and Summer 2018. Fertilization was withheld from each rootstock for 2 months before experiment initiation to create a nutrient-deficient condition at the onset of the experiment. At the end of 2 months, all the plants were transferred to a 100% hydroponic system (deionized water) with continuously running air pumps. Black 8-L buckets with lids were used for the hydroponic setup. The lids of the buckets were cut to fit the tree trunk, and the trees were suspended in the buckets with help of a trellis. Trees were allowed 1 week to adjust to the hydroponic system before beginning the experiment. The experiment was set in a 100% hydroponic system as a completely randomized design (n = 6) with six rootstocks being the only factor. After 1 week, at the beginning of the experiment (i.e., day 0), Hoagland solution was supplied as the sole source of fertilizer. Hoagland solution was prepared by mixing the reagents as follows: group A (macronutrients), 5 mm Ca(NO3)2⋅4H2O, 5 mm KNO3, 2 mm MgSO4⋅7H2O, and 1 mm KH2PO4; group B (micronutrients), 46 μm H3BO3, 9 μm MnCl2⋅4H2O, 0.8 μm ZnSO4⋅7H2O, 0.4 μm CuSO4⋅7H2O, 0.02 μm (NH4)6MoO4⋅4H2O, 0.02 µm FeSO4⋅7H2O, and 0.02 μm EDTA-Na2. Solutions for groups A and B were diluted to 1:100 and 1:1000 (volume:volume), respectively, in the deionized water. The experiment was conducted for 30 d under 100% hydroponic growing conditions because a long duration can negatively impact the growth and performance of citrus trees (Shahzad et al., 2020). It should be noted that from the time of rootstock seeding sowing until the end of hydroponic experiment, all rootstocks seedlings and grafted plants received the same growing conditions.
Plant growth analysis.
Plant height (measured from the graft union to tip of the plant) and leaf numbers were recorded at the beginning and end of the experiment and are expressed as the percentage change between days 0 and 30. Plant biomass was determined by dividing each plant into the leaf, stem, and root at the end of experiment (day 30). Plant biomass was not measured on day 0 because biomass measurement would have required destruction of the trees.
Growing media and mineral nutrient analysis.
The pH of the hydroponic solution was measured at days 0 and 30 using a pH meter (Orion Star A215; Thermo Fisher Scientific, Beverly, MA) according to the manufacturer’s instructions.
Twenty mL of hydroponic nutrient solution was sampled on days 0, 15, and 30 for nutrient analysis. On days 15 and 30, the water level was replenished to the same level as that at the start of each experiment to accurately measure the nutrient uptake by each plant over the course of 30 d. Growing media sampling was performed on days 15 and 30 so that the nutrient uptake patterns of each rootstock could be studied in two situations: phase 1 (evaluating nutrient uptake from days 0 to 15), which involved nutrient uptake on sudden availability of nutrients after a long period of nutrient starvation; and phase 2 (comparing nutrient uptake from days 15 to 30), which involved nutrient uptake when plants were exposed to nutrient-sufficient conditions. For leaf, stem, and root nutrient analyses, these tissues were sampled at the end of the experiment (day 30). All the harvested tissues were washed with deionized water and a mildly acidic detergent to remove any nutrient residue from the tissue. After washing, leaves were oven-dried at 65 °C for 72 h, and the stem and roots were oven-dried at 65 °C for 120 h. The dried leaves, stem, and root samples were ground using an analytical mill. The sampled growing media solution and ground plant tissues (10 g) were sent to Waters Agricultural Laboratories, Inc. (Camilla, GA) for standard macronutrient and micronutrient analyses.
Nutrient uptake efficiency.
Nutrient uptake efficiency was calculated according to Shahzad et al. (2020). Briefly, the total nutrient uptake was calculated as the difference in the concentration of the nutrients in the hydroponic solution from day 0 to day 30; then, it was divided by the total root biomass of the respective rootstock:
Nutrient uptake efficiency = [Mineral nutrient concentration (mg⋅L−1) in hydroponic solution on day 0 − Mineral nutrient concentration (mg⋅L−1) in hydroponic solution on day 30)] / fresh root biomass (g).
RNA extraction and gene expression analysis.
Fibrous root samples were collected from the plants on days 0 and 30 to extract RNA to analyze the expression of nutrient transporter genes in each rootstock. RNA extraction, cDNA synthesis, and gene expression analyses were performed as described by Ghimire et al. (2020). Briefly, total RNA was extracted from 100 mg of the root tissue with RNeasy Plant Mini Kit (Qiagen, Valencia, CA). When the quality and quantity of RNA were validated using spectrophotometer (Epoch 2 Microplate; BioTek Instruments, Winooski, VT) and denaturing formaldehyde 1.2% agarose gels (Rio, 2015), 1 μg RNA was used for cDNA synthesis with DNase I (Promega, Madison, WI), oligo (dT)15 primer, dNTP mix, and reverse-transcriptase (ImProm-II; Promega) according to the manufacturers’ protocols. Supplemental Table S1 lists the genes used for the expression analysis. The expressions of the listed genes were determined using a real-time quantitative polymerase chain reaction (PCR) system (7500 Fast Real-Time PCR System; Applied Biosystems, Foster City, CA). The quantitative PCR reactions and dissociation-curve analysis, which confirmed that no nonspecific products were formed, were performed as described by Shahzad et al. (2020). Using the quantification cycle (Ct), the levels of relative expressions of genes of interest were expressed as the normalized relative quantity calculated with two reference genes, glyceraldehyde-3-phosphate dehydrogenase C2 (GAPC2) and the gene for DIM1 homologue/YLS8 (Mafra et al., 2012). For the statistical analysis, log2 normalized relative quantity was used to account for heterogeneity of variance of the normalized relative quantity data.
Statistical analysis.
A one-way analysis of variance was used to compare rootstocks using the general linear model in R-studio (R version 3.4.1; R-core team, Vienna, Austria). Tukey’s honestly significant difference was used to perform post hoc comparisons; a significant difference was considered when P ≤ 0.05 according to the analysis of variance test.
Results and Discussion
Plant growth.
There was no significant increase in the height or leaf number among the ‘Midsweet’ orange trees on the six rootstocks (data not shown) on transition from the nutrient-deficient (day 0) to nutrient-sufficient conditions (day 30). The alterations of the root-to-shoot growth ratio is a general adaptive response of plants to the changes in nutrient availability (López-Bucio et al., 2003). Under nutrient deficient conditions, root system architecture is known to transition to a shallower root system with denser lateral roots and root hairs (Péret et al., 2011), which is possibly more efficient for nutrient uptake. When the root system is sufficient to supply all the nutrients required for the aboveground parts, plants may shift back their resources to the developing shoot and reproductive structures (Eissenstat et al., 2000; Taiz et al., 2015).
At the end of the experiment (day 30), of all six rootstocks, A+Volk × O-19 had average dry leaf and root biomass values of 14.75 g (P < 0.0001) and 24.33 g (P < 0.0001), respectively, which were greater than those of all the other rootstocks (Table 1). US-896 had the lowest leaf and root biomass values (9.75 g and 10.75 g, respectively). There were no statistically significant differences in stem dry biomass among rootstocks. None of the rootstocks was statistically different from each other in terms of biomass (root, stem, and leaf), except for A+Volk × O-19; it is possible that the parentage of this rootstock with ‘Volkamer lemon’ may have contributed to the higher growth rate compared to that of the other rootstocks (Wutscher and Bistline, 1988).
Mean (n = 6) leaf, stem, and root biomass (g) values of ‘Midsweet’ orange trees grown on six citrus rootstocks hydroponically with Hoagland solution for 30 d, followed by the SE. Significant differences (P < 0.05) were calculated using Tukey’s honestly significant difference (HSD) test. Means in the column followed by different letters indicate significant differences in the biomass of the respective tissue among the rootstocks.
The pH of the growing media.
An increase in the pH of the nutrient solution from day 0 (pH 6.0) to the end of the experiment was observed; however, the extent of the change in the pH was dependent on the rootstock. At day 30, the pH of the growing media was lowest (P = 0.016) in rootstocks UFR-17 and US-896 (pH ≈6.4) and highest in rootstock A+Volk × O-19 (pH 6.8); the other rootstocks had pH values between this range (Fig. 1).
The pH of the hydroponic solution (growing media) of ‘Midsweet’ orange trees grown on six rootstocks on day 30, with error bars indicating the SE. Significant differences were considered when P < 0.05 using Tukey’s honestly significant difference (HSD) test. Different letters indicate significant differences in the pH of the hydroponic solution among the rootstocks
Citation: HortScience 58, 1; 10.21273/HORTSCI16753-22
The pH of the hydroponic solution (growing media) of ‘Midsweet’ orange trees grown on six rootstocks on day 30, with error bars indicating the SE. Significant differences were considered when P < 0.05 using Tukey’s honestly significant difference (HSD) test. Different letters indicate significant differences in the pH of the hydroponic solution among the rootstocks
Citation: HortScience 58, 1; 10.21273/HORTSCI16753-22
The pH of the hydroponic solution (growing media) of ‘Midsweet’ orange trees grown on six rootstocks on day 30, with error bars indicating the SE. Significant differences were considered when P < 0.05 using Tukey’s honestly significant difference (HSD) test. Different letters indicate significant differences in the pH of the hydroponic solution among the rootstocks
Citation: HortScience 58, 1; 10.21273/HORTSCI16753-22
Tissue nutrient analysis.
The nutrient analyses were performed separately for the leaf, stem, and roots (Tables 2, 3, and 4, respectively). The nutrient analysis of leaves revealed that the highest S (P = 0.03) content was in the leaves of UFR-17; however, UFR-4 had the lowest concentration of S in the leaves. Additionally, the leaf analysis results also showed the highest B (P = 0.02) concentration in 46×20-04-6 and the lowest B concentration in UFR-4. No differences were observed for other nutrients in leaves among the six rootstocks. A similar pattern was observed for the stem nutrient analysis; UFR-4 had the lowest accumulations of secondary macronutrients (Ca, Mg, and S) in the stem, whereas all other rootstocks had similar accumulations. When root nutrient concentrations of the six rootstocks were compared, only N concentrations were different. UFR-4 showed the lowest accumulation of root N, whereas 46×20-04-6 showed the highest N accumulation. Among the six rootstocks compared in this study, UFR-4 consistently had the lowest nutrient accumulation.
Mean (n = 6) nutrient concentration (mg⋅kg−1) in the leaf tissues of ‘Midsweet’ orange trees grown on six citrus rootstocks hydroponically with Hoagland solution for 30 d followed by the SE. Significant differences (P < 0.05) were calculated at using Tukey’s honestly significant difference (HSD) test. Means in the column followed by different letters indicate significant differences in the concentration of respective nutrient in the leaf among the rootstocks.
Mean (n = 6) nutrient concentration (mg⋅kg−1) in the stem of ‘Midsweet’ orange trees grown on six citrus rootstocks hydroponically with Hoagland solution for 30 d followed by the SE. Significant differences (P < 0.05) were calculated using Tukey’s honestly significant difference (HSD) test. Means in the column followed by different letters indicate significant differences in the concentration of respective nutrient in the leaf among the rootstocks.
Mean (n = 6) nutrient concentration (mg⋅kg−1) in the root of ‘Midsweet’ orange trees grown on six citrus rootstocks hydroponically with Hoagland solution for 30 d followed by SE. Significant differences (P < 0.05) were calculated using Tukey’s honestly significant difference (HSD) test. Means in the column followed by different letters indicate significant differences in the concentration of respective nutrient in the leaf among the rootstocks.
Nutrient uptake from the growing media.
For all the rootstocks, the nutrient uptake was evaluated separately in two phases: uptake during days 0 to 15 (Fig. 2A and Supplemental Table S2) and uptake during days 16 to 30 (Fig. 2B and Supplemental Table S2). During phase 1, all rootstocks except US-896 had significant uptake of Mn. Additionally, UFR-4 had significant uptake of N during phase 1, whereas the other rootstocks did not show any significant N uptake. During phase 2, A+Volk × O-19 demonstrated significant uptake of all the nutrients except Mn, whereas 46×20-04-6 and Swingle had significant uptake of all the nutrients except Zn, Mn, and Cu. UFR-17 and US-896 showed the least nutrient uptake. Overall, US-896, UFR-17, and UFR-4 had the lowest nutrient uptake. During the trial period, the trees on UFR-17 only showed increases in four nutrients (N, Ca, Mn, and Fe), whereas US-896 showed a significant uptake of only two nutrients (Mn and P). UFR-4 showed no significant uptake of K, S, B, and Cu; furthermore, the leaves of UFR-4 had the lowest amount of B, S, and Cu (Cu was not statistically different).
Heatmap showing the nutrients significantly taken up by ‘Midsweet’ orange trees grown on six citrus rootstocks from the hydroponic solution on (A) day 15 compared with day 0 Hoagland solution and (B) day 30 compared with day 15. Gray cells represent the nutrients that were not significantly taken up after 15 or 30 d, whereas the black color represents the nutrients that were significantly taken up by the later time point.
Citation: HortScience 58, 1; 10.21273/HORTSCI16753-22
Heatmap showing the nutrients significantly taken up by ‘Midsweet’ orange trees grown on six citrus rootstocks from the hydroponic solution on (A) day 15 compared with day 0 Hoagland solution and (B) day 30 compared with day 15. Gray cells represent the nutrients that were not significantly taken up after 15 or 30 d, whereas the black color represents the nutrients that were significantly taken up by the later time point.
Citation: HortScience 58, 1; 10.21273/HORTSCI16753-22
Heatmap showing the nutrients significantly taken up by ‘Midsweet’ orange trees grown on six citrus rootstocks from the hydroponic solution on (A) day 15 compared with day 0 Hoagland solution and (B) day 30 compared with day 15. Gray cells represent the nutrients that were not significantly taken up after 15 or 30 d, whereas the black color represents the nutrients that were significantly taken up by the later time point.
Citation: HortScience 58, 1; 10.21273/HORTSCI16753-22
To normalize differences in the root biomass of rootstocks, nutrient uptake efficiency (nutrient uptake per gram of root) was calculated. Rootstocks showed different efficiencies in the uptake of P, K, and S (Fig. 3). For S and K, UFR-4 had the least uptake efficiency, whereas A+Volk × O-19 had the greatest uptake efficiency for S (P = 0.01) and K (P = 0.028); furthermore, uptake by A+Volk × O-19 was four-fold greater than that of UFR-4. Similarly, A+Volk × O-19 and UFR-17 had the greatest uptake efficiency for P (P = 0.003), which was eight-fold greater than that of 46×20-04-6 and three-fold greater than that of US-896. The transpiration pull via leaves is another contributing factor in regulating the nutrient uptake in plants (Taiz et al., 2015); therefore, plants with more leaves can have higher nutrient uptake. To address this possibility, the nutrient uptake efficiency among different rootstocks was also calculated considering the differences in leaf biomass (i.e., the nutrient uptake efficiency per gram of leaf). The results of the nutrient uptake efficiency per gram of leaf were similar to the nutrient uptake efficiency per gram of root tissue. A+Volk × O-19 had significantly greater uptake efficiency for N, P, K, Ca, and Mg, as compared with any other rootstock; however, the other rootstocks were not statistically different from each other (data not shown). UFR-17 did not have a greater leaf or root biomass than other rootstocks; however, it did have higher phosphorus uptake efficiency.
Nutrient uptake efficiency per gram of root of ‘Midsweet’ orange trees grown on six rootstocks in the hydroponic system with Hoagland solution for 30 d, with error bars indicating the SE. Significant differences were considered when P < 0.05 using Tukey’s honestly significant difference (HSD) test. Different letters indicate significant differences in the nutrient uptake efficiency per unit of root biomass of the respective nutrient among the rootstocks.
Citation: HortScience 58, 1; 10.21273/HORTSCI16753-22
Nutrient uptake efficiency per gram of root of ‘Midsweet’ orange trees grown on six rootstocks in the hydroponic system with Hoagland solution for 30 d, with error bars indicating the SE. Significant differences were considered when P < 0.05 using Tukey’s honestly significant difference (HSD) test. Different letters indicate significant differences in the nutrient uptake efficiency per unit of root biomass of the respective nutrient among the rootstocks.
Citation: HortScience 58, 1; 10.21273/HORTSCI16753-22
Nutrient uptake efficiency per gram of root of ‘Midsweet’ orange trees grown on six rootstocks in the hydroponic system with Hoagland solution for 30 d, with error bars indicating the SE. Significant differences were considered when P < 0.05 using Tukey’s honestly significant difference (HSD) test. Different letters indicate significant differences in the nutrient uptake efficiency per unit of root biomass of the respective nutrient among the rootstocks.
Citation: HortScience 58, 1; 10.21273/HORTSCI16753-22
A+Volk × O-19 demonstrated the most nutrient uptake and was ranked the highest among all six rootstocks. A larger root system can increase the absorption capacity of mineral nutrients by increasing the contact area with the growing media (Atkinson and Wilson, 1980). However, A+Volk × O-19 had the highest root biomass in the experiment and the highest uptake efficiency for nutrients P, K, and S. UFR-4 had the second largest root biomass of all six rootstocks and had the least nutrient uptake efficiency for these same nutrients. The results of the nutrient analysis showed that UFR-4 had the least concentrations of S and B in leaves, Mg, Ca, and S in the stem, and N in the roots compared with all other rootstocks, suggesting that UFR-4 roots have lower efficiency to uptake nutrients despite large root biomass. Another factor that can contribute to the low nutrient content in tissues apart from low nutrient uptake efficiency could be the partitioning of absorbed nutrients into large biomass or small biomass (for example, in the leaves, stem, and fruit). Moreover, 46×20-04-6 had the greatest concentration of B in the leaves, Mg in the stem, and N in the roots, whereas UFR-17 had the greatest concentration of S in both the stem and leaf tissues. Although in both 46×20-04-6 and UFR-17, the nutrient uptake was not the greatest; however, the nutrient partitioning among few sink tissues (small biomass) could have resulted in a higher accumulation of nutrients. A+Volk × O-19, despite having higher nutrient uptake efficiency and root biomass, had low concentrations of nutrients in tissues, possibly because of more partitioning of nutrients into a large number of sink tissues, thus diluting the nutrient concentrations in the tissues. These results suggested that each rootstock has different nutrient uptake and accumulation characteristics; furthermore, these results indicate the need to increase fertilization with the growth and development of the plants to balance the nutrient concentration in tissues (Obreza and Morgan, 2008). Our results demonstrate that A+Volk × O-19 has higher nutrient uptake efficiency, which is not a factor of biomass; this was possibly caused by the molecular regulation of nutrient uptake and genetic makeup of the rootstocks (Jiménez et al., 2007; Kennedy et al., 1980; Kucukyumuk and Erdal, 2011; Tsipouridis and Thomidis, 2005). Moreover, root morphology studies of A+Volk × O-19 and UFR-4, which are both tetraploids, may reveal the role of root morphology in the nutrient uptake potential of rootstocks.
The results of recent studies suggest that improvements in the performance of HLB-affected trees can be achieved through the use of intensive fertilizer programs, especially those with fortified micronutrients (Morgan et al., 2016; Vashisth and Grosser, 2018). Zambon et al. (2019) reported that HLB-affected plants had improvements in biological functions and HLB tolerance under Mn oversupply in the growing media. In the present study, all the rootstocks except US-896 had taken up Mn during the first 15 d of supply (phase 1); after 30 d, only P and Mn were taken up by US-896. It should be noted that Mn was the first nutrient to be readily taken up by the nutrient-starved plants. A+Volk × O-19 was the only rootstock that was able to take up all the analyzed nutrients within 30 d. Hence, it can be speculated that the below-average performance of US 896 (Castle, 2010) and above-average performance of A+Volk × O-19 (Satpute, 2017) under HLB conditions can be attributed to the nutrient uptake potential and efficiency of rootstocks. In other words, a nutrient uptake-robust rootstock (such as A+Volk × O-19) has high potential to perform better under HLB conditions and remain tolerant. Nonetheless, further validation of these results under HLB-affected conditions is needed because the presence of CLas can alter the physiological process of nutrient uptake.
Differential gene expression.
Of the eight genes studied in this experiment, none was differentially expressed in rootstocks on day 0 (nutrient-deficient condition). However, by day 30, IRON TRANSPORTER (IRT2) (P = 0.001) and ZINC TRANSPORTER (ZIP5) (P = 0.003) were significantly differentially expressed among the rootstocks. Regarding IRT2, US-896 had the lowest expression (Log2 FC = −4.81) and UFR-17 had the highest expression (Log2 FC = 0.33) (Fig. 4B). Regarding ZIP5, Swingle had the lowest expression (Log2 FC = 0.28) and A+Volk × O-19 had the highest expression (Log2 FC = 5.79) (Fig. 4A and B).
Expression analysis of Iron Transporter (IRT2) (A) and Zinc Transporter (ZIP5) (B) in the root tissue of ‘Midsweet’ orange trees grown on six rootstocks on day 0 and day 30, respectively, with error bars indicating the SE. Significant differences (P < 0.05) were calculated within different timepoints using Tukey’s honestly significant difference (HSD) test. Different letters indicate significant differences in the Log2 FC values of the gene on the respective day among the rootstocks.
Citation: HortScience 58, 1; 10.21273/HORTSCI16753-22
Expression analysis of Iron Transporter (IRT2) (A) and Zinc Transporter (ZIP5) (B) in the root tissue of ‘Midsweet’ orange trees grown on six rootstocks on day 0 and day 30, respectively, with error bars indicating the SE. Significant differences (P < 0.05) were calculated within different timepoints using Tukey’s honestly significant difference (HSD) test. Different letters indicate significant differences in the Log2 FC values of the gene on the respective day among the rootstocks.
Citation: HortScience 58, 1; 10.21273/HORTSCI16753-22
Expression analysis of Iron Transporter (IRT2) (A) and Zinc Transporter (ZIP5) (B) in the root tissue of ‘Midsweet’ orange trees grown on six rootstocks on day 0 and day 30, respectively, with error bars indicating the SE. Significant differences (P < 0.05) were calculated within different timepoints using Tukey’s honestly significant difference (HSD) test. Different letters indicate significant differences in the Log2 FC values of the gene on the respective day among the rootstocks.
Citation: HortScience 58, 1; 10.21273/HORTSCI16753-22
Nutrient transporter genes have a critical role in nutrient uptake; therefore, differential expression of these genes can affect the nutrient uptake efficiency of different rootstocks. This also explains why different rootstocks grown under the same conditions can have different nutrient uptake efficiencies. ZIP5 is a transporter gene expressed under Zn or Mn deficiency (Milner et al., 2013; Wintz et al., 2003) that is reported to be one of the most highly expressed genes coding for proteins associated with transport processes in response to CLas infection in citrus rootstocks (Albrecht and Bowman, 2012). A low level of ZIP5 expression was observed in Swingle compared with all other rootstocks on day 30. In Swingle, as a result of the slow uptake of Zn, more Zn was available in the growing media; therefore, ZIP5 is expressed less because of the higher availability of Zn on day 30 in Swingle compared with other rootstocks. Leaves of HLB-affected plants are reported to have lesser concentrations of Zn, Mn, and a few other micronutrients (Masaoka et al., 2011; Shahzad et al., 2020), thus highlighting the importance of Mn and Zn as crucial nutrients needed by HLB-affected plants. It is known that Mn and Zn have a central role in plant defense against damage from reactive oxygen species and are possibly involved in the inhibition of apoptosis (Cakmak, 2000; Dučić and Polle, 2005). Additionally, Zn deficiency interferes with the membrane-bound NADPH oxidase activity inhibiting the detoxification of cellular H2O2, ultimately leading to growth stunting and chlorosis or necrosis (Cakmak, 2000). Furthermore, Mn acts as a cofactor of superoxide dismutase and can scavenge superoxide ion (O2−) and H2O2 (Dučić and Polle, 2005). Our results showing the low expression of ZIP5 in Swingle and well-reported susceptibility of Swingle toward HLB (Ramadugu et al., 2016) could be related to its tendency to take up Zn slowly, thus leading to increased Zn deficiency in the already Zn-deficient leaves (a consequence of rapid Zn metabolism under HLB conditions). The lower expression of IRT2 in US-896 as compared with that of other rootstocks indicated that US-896 has poor capacity to rapidly sequester Fe (Vert et al., 2009) and can be affected during Fe-toxic or Fe-deficient conditions. Vert et al. (2009) concluded that the upregulation of IRT2 is a proactive mechanism of homeostasis, and that when Fe is suddenly available in the growing media immediately after Fe deficiency, strong expression of transporter genes involved with iron uptake could rapidly take up and increase cytosolic iron that are toxic to the cell. The increased expression of IRT2 helps to maintain Fe homeostasis under such conditions and minimizes the chances of Fe toxicity.
Conclusion
The differences in the nutrient uptake potential and efficiency of roots can be attributed to the differences in genetic makeup of each of the rootstock. A+Volk × O-19 rootstock showed the highest nutrient uptake efficiency, whereas US-896 showed the lowest. UFR-4 had a large root biomass, but the uptake efficiency for nutrients was poor, suggesting that the nutrient uptake potential is a complex process that is not solely dependent on root biomass. Root morphology can also be a factor affecting the nutrient uptake potential and efficiency of rootstocks. The obtained rootstock-induced nutrient concentration results may reveal that limitations to the effectiveness of fertilizer applications may curb nutrient deficiency issues under HLB conditions. Rootstocks that are not able to take up nutrients efficiently, such as US-896, should be either avoided or given special attention while designing nutritional programs. It is very important to choose the best rootstock based on site, preferred scion, tree size, and vigor. The rootstock–scion combination that can best meet the overall demands for nutrients will allow the efficient use of fertilizers and enhance orchard management overtime. This study, the first of its kind involving citrus, lays the foundation for studying nutrient uptake differences among different rootstocks. Field validation is still required to strengthen the results because this was a potted hydroponic experiment. Nevertheless, we demonstrated that the rootstocks should be selected after considering the soil nutrient status and equally emphasizing the nutrient uptake potential and absorption efficiency of roots. This information can be useful for the research community because, when evaluating a rootstock for crops requiring high nutrients, it is critical to determine the nutrient uptake potential of the rootstocks.
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Gene-specific primer sequences for quantitative real-time PCR analysis.
Mean (n = 6) nutrient concentration (mg⋅L−1) in growing media of ‘Midsweet’ orange trees grown on six citrus rootstocks hydroponically with Hoagland solution on days 0, 15, and 30. Differences in nutrient concentration in growing media suggest the uptake of nutrients by the trees. Significant differences (P < 0.05) were calculated using Student’s t test for days 0 and 15 (indicated by lowercase letters) or days 15 and 30 (indicated by uppercase letters).
