Abstract
The spread of Huanglongbing (HLB), a bacterial disease presumed to be caused by Candidatus Liberibacter asiaticus, throughout the state of Florida has coincided with a steady decline in total citrus (Citrus sp.) production. This decline is partially attributable to the high rates of preharvest fruit drop seen in HLB-affected trees. Although mature fruit drop is a natural phenomenon, the drop rates continue to increase as HLB symptom severity worsens. Unfortunately, how HLB causes this increase in fruit drop remains unknown. The current study aimed to determine the fruit characteristics associated with mature fruit drop in sweet orange (Citrus ×sinensis) and to provide an understanding of the possible role of endogenous ethylene, carbohydrates, and water deficit in HLB-associated preharvest fruit drop. Therefore, preharvest fruit drop rates of ‘Hamlin’ and ‘Valencia’ trees exhibiting mild, moderate, or severe HLB symptoms were monitored during the preharvest period (October–December for ‘Hamlin’ and January–May for ‘Valencia’). In addition, a subset of 20 fruit was collected to measure the fruit detachment force (FDF), which is the amount of force necessary to detach the fruit from the stem. After performing FDF measurements, eight additional physical and biochemical variables of tight and loose fruit (categorized by FDF) were measured. The total fruit drop rate during the preharvest period was higher for trees with severe visual HLB symptoms than for mild trees. Similarly, this increase in drop rates was negatively correlated with the canopy density. The fruit from severe trees (with high preharvest drop) showed increases in gene activity related to ethylene and abscisic acid earlier in the preharvest drop season, but not late in the season. No consistent carbohydrate pattern in tight and loose fruit was observed. Fruit likely to drop (those with lower FDF) were also consistently smaller than the fruit likely to be maintained on the tree (those with higher FDF). Therefore, it is proposed that the suppression of fruit growth early in the developmental period (possibly caused by water deficit) determines the fate (to drop or not) of the fruit before they have reached physiological maturity. Thus, strategies to mitigate preharvest fruit drop should be applied earlier in the season, and possibly during early stages of fruit development. By the time actual fruit drop becomes evident, the fruit drop-related signals have already been triggered, and treatments may not effectively reduce drop.
Huanglongbing has spread rapidly throughout Florida since it was discovered in Aug 2005 (Gottwald 2010), and it has resulted in a steep yield decline in total citrus (Citrus sp.) production (Spreen et al. 2014). The US Department of Agriculture (USDA) reported a dramatic increase in preharvest fruit drop in sweet orange (Citrus ×sinensis) from 12% during the 2005–06 season to 26% during the 2018–19 season as HLB became epidemic throughout Florida (US Department of Agriculture, National Agricultural Statistics Service 2019). This HLB-associated preharvest fruit drop takes place 2 to 4 months before harvest, when fruit are physiologically mature (end of stage ΙΙΙ of fruit development), thereby limiting the final yield. For healthy citrus, some amount of preharvest fruit drop is a natural phenomenon; however, this drop has escalated in the presence of HLB (Albrigo and Stover 2015; Tang et al. 2019). The preharvest fruit drop rates of HLB-affected ‘Valencia’ and ‘Hamlin’ sweet orange are significantly higher for trees that exhibit severe visual HLB symptoms than for those that exhibit mild symptoms, thus indicating a strong correlation between preharvest fruit drop and HLB severity (Tang et al. 2019, 2020). Trees with severe HLB symptoms have smaller fruit than sweet orange trees with mild HLB symptoms (Tang et al. 2019, 2020). Moreover, Tang et al. (2020) reported that the dropped fruit from HLB-affected sweet orange trees were smaller than the fruit that remained attached to the tree until harvest (Tang et al. 2020). Decreased fruit size in trees with HLB symptoms compared with that in asymptomatic or healthy trees has been documented by several researchers (Baldwin et al. 2018; Bové 2006; Liao and Burns 2012; Rosales and Burns 2011; Spann and Oswalt 2008). In addition, Singh et al. (2022) reported that the repeated exogenous application of gibberellic acid to HLB-affected trees at stage II of fruit development resulted in larger fruit and less fruit drop compared with untreated trees. These findings suggest that fruit development defects and preharvest fruit drop of HLB-affected trees are connected. Nonetheless, the nature and timing of the signal for preharvest fruit drop in HLB-affected trees are not yet clear.
To develop effective strategies to reduce preharvest fruit drop, it is critical to understand the cause of HLB-associated fruit drop, attributes of the fruit that drop, and the progression of events that lead to drop. Therefore, the first objective of this study was to investigate the natural progression of HLB-associated preharvest fruit drop and determine the fruit characteristics associated with drop. The second objective was to understand the role of endogenous ethylene, carbohydrates, and water stress in HLB-associated preharvest fruit drop.
Materials and Methods
Plant materials
Two cultivars of sweet orange, early maturing Hamlin (17-year-old) and late maturing Valencia (17-year-old), on Swingle citrumelo (C. paradisi MacFaden × Poncirus trifoliata L. Raf.) rootstock in experimental orchards at the Citrus Research and Education Center, Lake Alfred, FL, USA, were selected for this experiment. Because it is unlikely to find healthy Candidatus Liberibacter asiaticus (CLas)-negative sweet orange trees as a control in open uncontrolled conditions, the selected trees (n = 4) were categorized as mild, moderate, and severe based on the visual HLB symptoms and as described by the rating system developed by the Citrus Research and Development Foundation, Inc. (Slinski 2016). The photosynthetic photon flux density (PPFD), which is a measure of the flux density of photons between 400 and 700 nm), is a measure of the intensity of photosynthetically active radiation (PAR). The PPFD was measured using a ceptometer (MQ-301 Line Quantum with 10 sensors and handheld single external quantum sensor; Apogee Instruments, Inc., Logan, UT, USA) and expressed in μmol·m−2·s−1. PPFD was measured beneath the canopy of each tree to act as an index of canopy density and to explore correlations between canopy density and visual HLB symptoms. Greater PPFD was correlated with decreased canopy density (Levy et al. 2023).
Preharvest fruit drop.
The ground below the trees was raked and cleared before the start of the experiment. For all trees, the dropped fruit on the ground were counted and removed twice per week until harvest from 10 Oct 2018 to 4 Dec 2018 for ‘Hamlin’, and from 4 Feb 2019 to 22 May 2019 for ‘Valencia’. The fruit harvested were counted to calculate the cumulative fruit drop for each cultivar. The total fruit drop rate is presented as the percentage of the original crop load that dropped during the preharvest period.
Fruit detachment force.
The fruit detachment force (FDF) the force required to remove the fruit from the stem. A generally accepted concept is as follows: the lower the FDF, the higher the chances of drop, and vice versa (Malladi and Burns 2008; Pozo et al. 2007; Tang et al. 2019). A digital force gauge (Force One; Wagner Instruments, Greenwich, CT, USA) was used to measure FDF, which is expressed in kilograms-force (kgf). For ‘Hamlin’, the FDF analysis was performed at the following two timepoints: 5 weeks (Oct) and 1 week (Nov) before harvest. For ‘Valencia’, FDF was measured at the following four timepoints: 16 weeks (January), 13 weeks (February), 8 weeks (March), and 1 week (May) before harvest. To measure the FDF, 20 branches (length, ∼20 cm), each with a single subtending fruit and mature leaves, were clipped and brought to the laboratory, where measurements were performed. The fruit were categorized into “tight” fruit and “loose” fruit based on a threshold of 6 kgf.
Physical and chemical characteristics of fruit and leaves.
After FDF measurements were performed, eight variables were collected separately for each fruit measured within each tree replicate. The equatorial fruit diameter was measured using a vernier caliper. The fruit juice was extracted to quantify the total soluble solids (TSS) and titratable acidity (TA) using a handheld refractometer (Pocket PAL-BX1 ACID1; Atago USA, Bellevue, WA, USA). The number of healthy and aborted seeds of each fruit were counted (Rosales and Burns 2011). The total numbers of mature healthy leaves and blotchy mottled source leaves (as described by Koch and Avigne 1984) on each sampled branch were counted, and the chlorophyll content was estimated using the MC-100 chlorophyll concentration meter (Apogee Instruments, Inc.) and expressed as the soil–plant analysis development (SPAD) value.
For each combination of tree symptom level (mild, moderate, and severe) and fruit type (tight and loose), the calyx abscission zone (AZ-C) and mature leaves were pooled, immediately frozen in liquid nitrogen, and stored at −80 °C for a gene expression analysis. The fruit juice was also pooled and stored at −20 °C for carbohydrate quantification. Samples from trees with moderate symptoms were not used for gene expression and carbohydrate quantification. This decision was made based on physical data and to allow a manageable analysis.
Carbohydrate quantification in fruit juice.
Pooled juice samples from tight and loose fruit from both mild and severe trees were used for carbohydrate quantification. Samples from both timepoints (October and November) were used for ‘Hamlin’, whereas samples from only February (beginning of the fruit drop period) and May (before harvest) were used for ‘Valencia’. The concentrations of sucrose, glucose, fructose, and inositol were quantified using ion chromatography and expressed in g⋅L−1. After centrifugation of 2 mL of juice at 12,000 gn for 5 minutes, 10 μL of the supernatant was added to 29.90 mL of deionized water. The diluted sample was filtered through a prefilled chromatography column (1-X8 resin; Biorad Laboratories, Hercules, CA, USA). To remove the anion contaminants, 250 μL of the filtered sample was transferred to a 0.45-μM polytetrafluoroethylene filter vial (Restek, Bellafonte, PA, USA) to perform a second filtration. Then, 25 μL of elute was injected into ion chromatography equipped with an anion exchange column (250 mm × 3 mm) with a guard column (50 mm × 3 mm) (CarboPac PA 200; Dionex, Sunnyvale, CA, USA). The isocratic mobile phase consisted of 95% deionized water and 5% NaOH (1 N), with a flow rate of 0.4 mL per minute. Standard curves for sucrose, fructose, glucose, and inositol were run with all the samples and used for quantification.
Relative gene expression.
Leaf and AZ-C tissues stored from mild and severely symptomatic ‘Hamlin’ trees (October and November) and ‘Valencia’ trees (February and May) were used for gene expression analyses. The RNA extractions from leaf and AZ-C tissues were performed using the Invitrogen PureLink RNA Mini Kit (ThermoFisher Scientific, Waltham, MA, USA) and RNeasy Plant Mini Kit (Qiagen, Valecia, CA, USA), respectively, according to the manufacturer’s instructions. The RNA quality and quantity were determined by the Epoch 2 Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). Then, 1 µg of total RNA was treated with RQ1 Rnase-Free Dnase (Promega, Madison, WI, USA) and used for cDNA synthesis using Oligo (dT)15 Primer, dNTP mix, and ImProm-II™ reverse transcriptase (Promega) according to the manufacturer’s protocol.
The relative expressions of genes involved in cell wall remodeling, ethylene biosynthesis and signaling, carbohydrate metabolism and transport, phloem function, auxin and abscisic acid metabolism and signaling, and oxidative stress in the AZ-C and leaves were determined using a quantitative real-time polymerase chain reaction. The gene-specific primers are listed in Supplemental Table 1. Additionally, 10-μL quantitative real-time polymerase chain reactions containing 2 µL of diluted cDNA, 3 μL of forward and reverse primer mix (0.3 μM), and 5 μL of PowerUp™ SYBR™ Green Master Mix (2X) (Applied Biosystems, Foster City, CA, USA) were performed using a 7500 Fast Real-Time PCR System (Applied Biosystems). Each reaction was allowed to run first at 50 °C for 2 min, then at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. The dissociation curve ranging from 60 to 95 °C was analyzed at the end of each quantitative real-time polymerase chain reaction to confirm that nonspecific products were not formed. The relative expressions (fold change) of genes were calculated using the quantification cycle and Pfaffl method (Hellemans et al. 2007; Pfaffl 2001) and normalized relative to the geometric mean of ACT7 (actin 7) and DIM1 (thioredoxin-like protein YLS8) (Mafra et al. 2012).
Leaf water potential.
During the first week of May 2020 (1 week before harvest), 10 fruit per tree were categorized into small and large (five each) based on a fruit diameter threshold of 58 mm or based on mild and severe symptoms of ‘Valencia’ trees. The mature leaf closest to each fruit was clipped to measure the water potential using a Scholander pressure chamber (PMS Instrument Company, Albany, OR, USA). The FDF and fruit size of the same fruit were measured to calculate the correlation of the water potential with the FDF and fruit size.
Statistical analysis
R (R Core Team 2018) was used to conduct the statistical analysis. A one-way analysis of variance (ANOVA) was used to test the differences in the total fruit drop rates among symptom levels (mild, moderate, or severe). The effects of the disease symptom category and its interactions with the characteristics, juice carbohydrate content, and relative gene expression among tight and loose fruit were tested by performing an ANOVA using the lmer() function in the {lme4} package with a nested mixed effects model in which the fruit type variable (tight or loose) was nested within each individual tree, and each individual tree was considered a random effect, whereas the fruit type and disease severity variables were fixed effects. For the post hoc analysis, significant differences were considered when α = 0.05; Tukey’s honest significant difference test was performed to determine the mean separation among groups when significant differences were indicated by the ANOVA. Using Pearson’s correlation method, the correlations between the fruit drop rate and PPFD, water potential and FDF, and water potential and fruit size were tested.
Results
Fruit drop rate and PPFD.
The PPFD of each tree increased with the increasing disease severity for both cultivars (Supplemental Fig. 1), where severely symptomatic trees had the highest PPFD beneath the canopy, and mildly symptomatic trees had the lowest PPFD beneath the canopy. ‘Hamlin’ sweet orange trees that exhibited mild, moderate, and severe HLB symptoms had similar fruit drop rates (5%, 3%, and 6% respectively) in the beginning of the preharvest period (early October) (Fig. 1A). The fruit drop rate of mild and moderate trees increased steadily, without any significant difference between the two types of trees throughout the preharvest period until harvest. However, the severe trees showed an increase in fruit drop during the preharvest period when compared with mild trees. At the end of the survey, severe trees had the highest fruit drop rate (28%), whereas mild and moderate trees had fruit drop rates of 15% and 18%, respectively. A similar pattern of fruit drop rates was found for ‘Valencia’ sweet orange, with mild, moderate, and severe trees having similar fruit drop rates from the beginning of the survey until mid-March (Fig. 1B). By the second week of March, the fruit drop rate of severe trees increased compared with mild and moderate trees, which had steady increases. Before harvest, the total fruit drop rates of mild, moderate, and severe trees were 30%, 34%, and 42%, respectively. The total fruit drop rate was correlated with the PAR intensity of both Hamlin (r = 0.84; P < 0.001) and Valencia (r = 0.67; P < 0.001) sweet orange cultivars (Supplemental Fig. 2). The results indicated that the fruit drop rate increased with the decreasing canopy density.
Cumulative preharvest fruit drop rates of Huanglongbing-affected (A) ‘Hamlin’ and (B) ‘Valencia’ sweet orange trees with the following three visual symptom levels (SLs): mild, moderate, and severe. A one-way analysis of variance (ANOVA) was used to determine whether the difference in the total fruit drop rate of the three SLs was significant. Different letters indicate significant differences among means according to Tukey’s honest significant difference test (α = 0.05).
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05380-24
Fruit detachment force.
For Hamlin, the average FDF was not different between mild, moderate, and severe trees at either timepoint, but the average FDF of fruit from all trees decreased from October to November. However, the average FDF at the time of harvest was higher than 6 kgf for all three HLB symptom levels (Supplemental Fig. 3A). Nonetheless, the number of loose fruit increased from 4% and 12% in October to ∼22% in November for both moderate and severe trees, respectively, whereas the number of loose fruit in mild trees remained the same (Supplemental Fig. 4A). Similarly, for ‘Valencia’, the average FDF decreased in May (Supplemental Fig. 3B). The number of loose fruit also increased in May as compared with January, February, and March. This increase in the percent of loose fruit was greater for moderate (30% increase) and severe trees (30% increase) than for mild trees (20% increase) (Supplemental Fig. 4B).
Physical and chemical characteristics of fruit and leaves.
Among the eight variables of tight and loose fruit from mild, moderate, and severe trees that were studied, fruit size showed a consistent and distinct pattern at all the timepoints for both cultivars (Tables 1–6). Briefly, regarding ‘Hamlin’, loose fruit (lower FDF) were smaller than tight fruit (higher FDF), irrespective of HLB symptoms in October and November (Tables 1 and 2). Regarding ‘Valencia’, a similar pattern of loose fruit being smaller in size than tight fruit was observed in January, February, March, and May (Tables 3–6). There was no significant interaction between the HLB symptom level and fruit type for fruit size. A positive correlation between the FDF and fruit size of ‘Hamlin’ and ‘Valencia’ was observed, suggesting that smaller fruit are more likely to drop than larger fruit (Fig. 2).
Physical and chemical characteristics of tight (fruit detachment force >6 kgf) and loose (fruit detachment force ≤6 kgf) fruit of ‘Hamlin’ sweet orange trees in October (5 weeks before harvest). The trees were categorized as mild, moderate, and severe based on visual Huanglongbing (HLB) symptoms.
Physical and chemical characteristics of tight (fruit detachment force >6 kgf) and loose (fruit detachment force ≤6 kgf) fruit of ‘Hamlin’ sweet orange trees in November (1 week before harvest). The trees were categorized as mild, moderate, and severe based on visual Huanglongbing (HLB) symptoms.
Physical and chemical characteristics of tight (fruit detachment force >6 kgf) and loose (fruit detachment force ≤6 kgf) fruit of ‘Valencia’ sweet orange trees in January (16 weeks before harvest). The trees were categorized as mild, moderate, and severe based on visual Huanglongbing (HLB) symptoms.
Physical and chemical characteristics of tight (fruit detachment force >6 kgf) and loose (fruit detachment force ≤6 kgf) fruit of ‘Valencia’ sweet orange trees in February (13 weeks before harvest). The trees were categorized as mild, moderate, and severe based on visual Huanglongbing (HLB) symptoms.
Physical and chemical characteristics of tight (fruit detachment force >6 kgf) and loose (fruit detachment force ≤6 kgf) fruit of ‘Valencia’ sweet orange trees in March (8 weeks before harvest). The trees were categorized as mild, moderate, and severe based on visual Huanglongbing (HLB) symptoms.
Physical and chemical characteristics of tight (fruit detachment force >6 kgf) and loose (fruit detachment force ≤6 kgf) fruit of ‘Valencia’ sweet orange trees in May (1 week before harvest). The trees were categorized as mild, moderate, and severe based on visual Huanglongbing (HLB) symptoms.
Pearson’s correlation between the fruit detachment force (FDF) and fruit size of Huanglongbing-affected ‘Hamlin’ and ‘Valencia’ sweet orange trees.
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05380-24
Regarding ‘Hamlin’, juice from tight fruit had significantly higher TSS than loose fruit in November, but not in October (Tables 1 and 2), irrespective of the HLB symptoms. However, regarding ‘Valencia’, the juice TSS was not significantly different between tight and loose fruit or among mild, moderate, and severe trees (Tables 3–6), for any of the timepoints except March, when tight fruit juice had more TSS. Similarly, there was no significant difference in the TA from juice based on symptom level, fruit type, or interaction for both cultivars (Tables 1–6), except in March, when fruit from ‘Valencia’ trees with severe HLB symptoms had lower TA than mild trees (Table 5). No significant differences were observed in the number of healthy seeds and aborted seeds of tight or loose fruit or the mild, moderate, and severe trees for both cultivars at any timepoint, with the exception of ‘Valencia’ in January (Tables 1–6). During the January sampling of ‘Valencia’, the tight fruit had a higher number of healthy seeds than loose fruit, irrespective of HLB symptoms (Table 3). The numbers of healthy leaves, blotchy mottled leaves, and leaf chlorophyll contents (SPAD value) were not different between fruit types or HLB symptom levels (Tables 1–6).
Regarding ‘Valencia’, the water potential of the mature leaf closest to the small or large fruit from mild, moderate, and severe trees was measured during first week of May (2 weeks before harvest). No differences in leaf water potential were observed between the three tree symptom levels or fruit size groups (data not shown). Additionally, there was no significant correlation between water potential and FDF or between water potential and fruit size.
Carbohydrates in fruit juice.
Regarding ‘Hamlin’, glucose, fructose, and inositol concentrations in the fruit juice from severe and mild trees (Fig. 3A, 3B) as well as in juice from tight and loose fruit (Fig. 3A, 3B, 3D) were similar. The concentration of sucrose in fruit juice was not different among symptom levels; however, loose fruit had lower sucrose levels than tight fruit at both timepoints (Fig. 3C).
Average glucose, fructose, sucrose, and inositol concentrations in juice of two fruit types (FTs), tight and loose fruit, with fruit detachment forces (FDFs) >6 and ≤6 kgf, respectively, of ‘Hamlin’ (October and November) and ‘Valencia’ (February and May) sweet orange trees with mild and severe Huanglongbing symptom levels (SLs). Data are means ± SE of four biological replicates. An analysis of variance (ANOVA) was used to determine significant differences between two SLs, two FTs, and their interactions at each timepoint. Different letters indicate significant differences according to Tukey’s honest significant difference test. ns = nonsignificant difference.
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05380-24
Similarly, regarding ‘Valencia’, glucose, fructose, and inositol concentrations were not different among two symptom levels or two fruit types at both timepoints (Fig. 3E, 3F, 3H). Loose fruit had lower sucrose levels in juice than tight fruit, irrespective of the symptom level in both February and May (Fig. 3G).
Relative expression of cell wall remodeling genes in AZ-C.
Compared with those in tight fruit, cell wall modification genes were highly expressed in AZ-C of loose fruit (Fig. 4) in both cultivars. In ‘Hamlin’, the fruit type and interaction between fruit type and symptom level affected transcript levels of cellulase (CEL6) (Fig. 4A). CEL6 expression was higher in the AZ-C of loose fruit from severe trees than in the AZ-C of tight fruit from severe trees and in both mild and loose fruit from mild trees in October (Fig. 4A). However, in November, CEL6 expression in the AZ-C of loose fruit was higher than that in tight fruit irrespective of the symptom level (Fig. 4A). The expression of polygalacturonase (PG20) was not significantly different based on symptom level, fruit type, or the interaction of the two at both timepoints (Fig. 4B); however, a trend of numerically higher expression was seen in loose fruit from both mild and severe trees. In October, pectate lyase (PL5) transcripts were higher in fruit AZ-C from severe trees than in fruit AZ-C from mild trees, and loose fruit had a higher expression of PL5 than tight fruit (Fig. 4C); however, there was no interaction effect. Additionally, no effect of tree symptom level was observed on PL5 in November; however, loose fruit had numerically higher expression levels of PL5 than tight fruit at both symptom levels.
The relative expressions of cellulase (CEL6), polygalacturonase (PG20), and pectate lyase (PL5) in the calyx abscission zone (AZ-C) of two fruit types (FTs), tight and loose fruit, with fruit detachment forces (FDFs) >6 and ≤6 kgf, respectively, of ‘Hamlin’ (October and November) and ‘Valencia’ (February and May) sweet orange trees with mild and severe Huanglongbing symptom levels (SLs). Data are means ± SE of four biological replicates. An analysis of variance (ANOVA) was used to determine significant difference between SLs, FTs, and their interactions at each timepoint. Different letters indicate significant differences according to Tukey’s honest significant difference test. ns = nonsignificant difference.
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05380-24
Regarding ‘Valencia’, the AZ-C of loose fruit had higher expression levels of CEL6 and PG20 than the AZ-C of tight fruit, irrespective of symptom level, in February (Fig. 4D, 4E); however, no significant effect was observed in May. The symptom level and fruit type had no significant effects on the expression of PL5 at both timepoints (Fig. 4F).
Relative expression of ethylene biosynthesis and signaling genes in AZ-C.
Regarding ‘Hamlin’, severely symptomatic trees had a significantly higher expression of 1-aminocyclopropane-1-carboxylate oxidase (ACO) in the AZ-C compared with that of mildly symptomatic trees in October (Fig. 5A). The transcript levels of ACO and the gene encoding ethylene responsive factor (ERF1) were higher in the AZ-C of loose fruit than in the AZ-C of tight fruit in October (Fig. 5A, 5B); however, there was no difference in November. There was no difference in the expression of ethylene-insensitive protein (EIN2) based on the symptom level or fruit type at either timepoint (Fig. 5C).
The expression of 1-aminocyclopropane-1-carboxylate oxidase (ACO), ethylene response factor 1 (ERF1), and ethylene insensitive protein 2 (EIN2) in the calyx abscission zone (AZ-C) of two fruit types (FTs), tight and loose fruit, with fruit detachment forces (FDFs) >6 and ≤6 kgf, respectively, of ‘Hamlin’ (October and November) and ‘Valencia’ (February and May) sweet orange trees with mild and severe Huanglongbing symptom levels (SLs). Data are the means ± SE of four biological replicates. An analysis of variance (ANOVA) was used to determine significant differences between the two SLs, two FTs, and their interactions at each timepoint. Different letters indicate significant differences according to Tukey’s honest significant difference test. ns = nonsignificant difference.
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05380-24
Regarding ‘Valencia’, the expressions of ACO and ERF1 in the AZ-C were not different between fruit types or symptom levels at both timepoints (Fig. 5D, 5E). However, loose fruit had higher EIN2 expression compared with tight fruit irrespective of the symptom level in February (Fig. 5F). Moreover, severely symptomatic trees had a higher expression of EIN2 than mild trees in May (Fig. 5F).
Relative expression of stress-related and other genes in AZ-C.
The 9-cis-epoxycarotenoid dioxygenase 2 (NCED2) involved in abscisic acid (ABA) biosynthesis was greater in the AZ-C of loose fruit than in the AZ-C of tight fruit in October for ‘Hamlin’ and in February for ‘Valencia’ (Fig. 6A, 6D). Additionally, severe trees had a higher expression of NCED2 in the AZ-C than mild trees at both timepoints for ‘Valencia’ (Fig. 6D); however, no similar pattern was observed for ‘Hamlin’ (Fig. 6A). For ‘Hamlin’, the AZ-C of severe trees had higher phloem protein (PP2B15) expression levels than mild trees, and tight fruit had higher expression levels than loose fruit in October, but not in November (Fig. 6B). Additionally, the gene encoding antioxidant enzyme ascorbate peroxidase (APX) was highly expressed in severe trees compared with that in mild trees of ‘Hamlin’ in October, but not in November (Fig. 6C). For ‘Valencia’, the symptom level and fruit type had no effect on the expressions of PP2B15 and APX (Fig. 6E, 6F). Other genes studied (listed in Supplemental Table 1) did not differ significantly in their expressions in the AZ-C based on the symptom level or fruit type for both ‘Hamlin’ and ‘Valencia’ (Supplemental Tables 2 and 3).
The expression of 9-cis-epoxycarotenoid dioxygenase 2 (NCED2), phloem protein 2-B15 (PP2B15), and ascorbate peroxidase (APX) in the calyx abscission zone (AZ-C) of two fruit types (FTs), tight and loose fruit, with fruit detachment forces (FDFs) >6 and ≤6 kgf, respectively, of ‘Hamlin’ (October and November) and ‘Valencia’ (February and May) sweet orange trees with mild and severe Huanglongbing symptom levels (SLs). Data are the means ± SE of four biological replicates. An analysis of variance (ANOVA) was used to determine significant differences between the two SLs, two FTs, and their interactions at each timepoint. Different letters indicate significant differences according to Tukey’s honest significant difference test. ns = nonsignificant difference.
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05380-24
Relative gene expression in leaves.
The relative expressions of 20 genes in leaves associated with tight and loose fruit from mildly and severely HLB symptomatic trees were determined. However, there were no significant differences in the expressions of genes related to carbohydrate metabolism, ethylene biosynthesis and signaling, other plant hormones, and oxidative stress in leaves based on fruit type or symptom level (Supplemental Tables 4 and 5).
Discussion
This study adds further evidence to the existing body of evidence indicating that preharvest fruit drop is linked with HLB symptoms at the whole canopy level. The cumulative fruit drop rate during the preharvest period was higher for trees with severe visual HLB symptoms than for mild trees, whereas moderate trees had fruit drop rates between those of mild and severely symptomatic trees with both ‘Hamlin’ and ‘Valencia’ sweet oranges. Similar results indicating an increase in fruit drop with an increase in HLB disease severity have been reported for ‘Valencia’ and ‘Hamlin’ sweet orange (Tang et al. 2019, 2020). These results are supported by USDA reports that have shown a dramatic increase in preharvest fruit drop of sweet orange during the past decade because HLB has become epidemic throughout Florida (US Department of Agriculture, National Agricultural Statistics Service 2019). Moreover, preharvest fruit drop was negatively correlated with canopy density (but positively correlated with PPFD beneath the canopy). Trees affected with HLB show significant leaf drop and shoot dieback, resulting in the thinning of the canopy (Bové 2006) and an increase in PAR intensity below the canopy (Levy et al. 2023). The citrus genotypes with more tolerance to HLB have higher canopy densities and better growth than susceptible genotypes (Miles et al. 2017; Yu et al. 2022). ‘LB8-9’ (Sugar Belle®), an HLB-tolerant mandarin [C. reticulata × (Citrus paradisi × C. reticulata)], is also known to have significantly lower fruit drop rates than the more susceptible ‘Hamlin’ sweet orange (Tang and Vashisth 2020). LB8-9 mandarins also have higher canopy density than HLB-susceptible sweet orange cultivars, despite high rates of “blotchy-mottle” symptoms in the tolerant cultivar (Deng et al. 2019). Similarly, in the present study, no differences in the number of healthy and blotchy-mottled source leaves on 20-cm-long branches or in the leaf chlorophyll were found between HLB symptom level and fruit type (tight or loose) at any timepoint, suggesting that canopy density, more so than individual leaf characteristics (during preharvest fruit drop period), is indicative of fruit retention through harvest. Relevantly, Levy et al. (2023) recently proposed that canopy density is a more accurate predictor of the tree yield potential of HLB-affected trees than CLas titer.
In the present study, the average FDF of each tree decreased closer to harvest regardless of symptom level. This decrease corresponded with an increase in the proportion of loose fruit. Cumulative drop rates of severely symptomatic trees increased more sharply than those of mildly symptomatic trees so that, at the time of harvest, the severely symptomatic trees had dropped a larger proportion of their crop load than the mildly symptomatic trees. This suggests that preharvest fruit drop is heightened by HLB severity. This also suggests that although HLB-affected fruit experience a general decrease in FDF (Chen et al. 2016), further reductions in the average FDF as the disease severity increases is the result of an increase in the number of loose fruit. Furthermore, the correlation between preharvest fruit drop rates and canopy density observed during this study further suggests that practices and sweet orange cultivars that improve or maintain tree health, as observed in canopy density, are crucial to preventing the increased rates of preharvest fruit drop associated with HLB.
During this study, we found a direct positive relationship between fruit size and FDF, with FDF increasing with the increasing fruit diameter. Loose fruit (those more likely to drop) were consistently smaller than the tight fruit of both Hamlin and Valencia sweet orange cultivars at all timepoints (i.e., even several weeks before harvest, when fruit had completed growth and maturation). Previous studies have reported that HLB-affected trees yield smaller fruit than healthy (CLas-negative) trees (Baldwin et al. 2018; Bové 2006), that fruit size has decreased significantly with the establishment of HLB in Florida (Spann and Oswalt 2008), and that fruit size decreases with the increasing disease severity (Tang et al. 2019, 2020). Consistently, symptomatic branches on HLB-affected ‘Valencia’ sweet orange trees yielded smaller fruit than asymptomatic branches (Rosales and Burns 2011). It was recently reported that the dropped fruit from HLB-affected sweet orange trees were smaller than the fruit that remained attached to the tree until harvest (Tang et al. 2020). The present work established the direct connection between fruit size and FDF. Thus, it can be concluded that HLB-associated reductions in fruit size are related to the likelihood of mature fruit drop.
Fruit size of citrus is determined by the increase in cell number attributable to cell division during stage I of fruit development (first 2 months following fruit set), and by the increase in cell size attributable to cell enlargement during stage II of fruit development (Ferguson and Grafton-Cardwell 2014; Iglesias et al. 2007). In HLB-affected trees, the small size of mature fruit on symptomatic branches was reported to be caused by a low number of hypodermal cells in the peel, but not a reduction in cell size (Rosales and Burns 2011). Tang et al. (2020) provided evidence supporting the suppression of cell division and fruit growth by HLB; immature fruit (9 weeks after full bloom; most likely during stage I of fruit development) from severely HLB-symptomatic ‘Valencia’ sweet orange trees were significantly smaller than those from mildly affected trees. Levels of endogenous hormones were altered in immature fruit and leaves of sweet orange because of CLas infection (Martinelli et al. 2012; Nehela et al. 2018), which can also potentially impede cell division and/or expansion (Tang et al. 2019). Therefore, it can be postulated that CLas infection suppresses early fruit growth and development. Together with the relationship between FDF and fruit size demonstrated by the present study, the fate of a fruit (to drop or not) is likely determined before it reaches physiological maturity.
CLas-infection causes synthesis and accumulation of callose at the sieve pores of phloem in leaves (Achor et al. 2010; Kim et al. 2009). The expression of phloem proteins (e.g., PP2B15) is also upregulated in HLB-affected leaves, and their products contribute to phloem blockage (Achor et al. 2010; Etxeberria and Narciso 2015; Fan et al. 2013; Kim et al. 2009). The resulting phloem blockage disrupts the carbohydrate flow from source leaves to fruit, thus contributing to the starch accumulation seen in HLB symptomatic leaves (Etxeberria et al. 2009; Kim et al. 2009). Carbohydrate shortages and competition for the available carbohydrate supply are considered the dominant causes of immature fruitlet drop, which is commonly referred to as “June drop” (Goldschmidt 1999; Gómez-Cadenas et al. 2000; Ruiz et al. 2001). Therefore, it has been speculated that disruption of the carbohydrate flow seen in HLB-affected trees is the cause of the increased preharvest fruit drop associated with HLB. However, in the present study, the TSS content in the juice of tight fruit was only significantly higher than that of loose fruit at one timepoint for ‘Hamlin’ (November) and for ‘Valencia’ (March). Moreover, upon carbohydrate quantification, the concentrations of individual sugars (glucose, fructose, and inositol) were similar in loose and tight fruit from both ‘Hamlin’ and ‘Valencia’ sweet orange trees at mild and severe HLB symptom levels. This is consistent with the findings of previous work completed by Baldwin et al. (2018) and Tang et al. (2019, 2020), who also found no differences in the glucose and fructose contents in the juice of dropped and attached fruit. Although Rosales and Burns (2011) and Baldwin et al. (2018) reported that sucrose, a major translocation sugar, is generally lower in HLB-affected fruit than in healthy fruit, no distinctive pattern has been noted for HLB-affected dropped and retained fruit (loose vs. tight, respectively) across literature (Baldwin et al. 2018; Tang et al. 2019, 2020). In the present study sucrose was consistently and significantly higher in tight fruit than in loose fruit irrespective of symptom levels in both cultivars. It is worth noting that the tight fruit were also consistently larger than the loose fruit regardless of symptom levels of both cultivars. Because sucrose transport and catabolism to hexose sugars play large roles in controlling sink activity, and because fruit size is generally representative of sink strength (Sadka et al. 2019; Smith et al. 2018), it can be speculated that the fruit retained throughout the preharvest season are the stronger sinks (with higher growth), as represented by their larger sizes and higher sucrose contents compared with those of loose (and small) fruit.
The process of fruit abscission involves cell wall modifications within the AZ-C (Kim 2014; Merelo et al. 2017; Patterson 2001). Cellulase, polygalacturonase, pectate lyase, and pectin methylesterase activities result in the dissolution of the middle lamella connecting two adjacent cells, causing cell separation (Kim 2014; Merelo et al. 2017; Patterson 2001). The loose fruit had lower FDF value and higher expressions of such cell wall remodeling genes (CEL6, PG20, PL5) in AZ-C. This suggests that the abscission process had already been triggered in the AZ-C of loose fruit several weeks before the potential harvest of both cultivars. This is similar to the finding of Tang et al. (2019), who reported that loose fruit had higher expressions of cell wall hydrolytic genes in the AZ-C than tight fruit from HLB-affected ‘Valencia’. The plant hormone ethylene is known to trigger the cell wall hydrolytic enzymes, which causes abscission (González-Carranza et al. 2002, 2007; Kim 2014; Patterson 2001). Exogenously applied ethylene and its precursor 1-aminocyclopropane-1-carboxylate (ACC) can induce mature fruit abscission in healthy ‘Valencia’ sweet orange (Malladi and Burns 2008; Merelo et al. 2017; Yuan et al. 2005). The cell separation process of HLB-associated preharvest fruit drop (Tang et al. 2019) is similar to exogenous ethylene-induced abscission of healthy citrus (Burns and Lewandowski 2000; Cheng et al. 2015; Goren and Huberman 1976; Merelo et al. 2017). However, Tang et al. (2019) reported that the genes involving ethylene biosynthesis and signaling were not differentially expressed in tight and loose fruit 1 week before harvest, leading to the hypothesis that ethylene signaling that induces abscission could have occurred earlier during the preharvest period. In the present study, the ethylene biosynthesis gene, ACO, and ethylene responsive factor, ERF1 (transcription factor that regulates genes in response to ethylene), were highly expressed in the AZ-C of loose fruit as compared with those of tight fruit of ‘Hamlin’ in October; however, for ‘Valencia’, the gene encoding ethylene insensitive protein, EIN2 (a positive regulator of ethylene signaling), was highly expressed in the AZ-C of loose fruit as compared with that in the AZ-C of tight fruit in February (Guo and Ecker 2004) (i.e., earlier during the process). However, like Tang et al. (2019), no differences in the expressions of these genes were observed close to harvest for both ‘Hamlin’ and ‘Valencia’. This suggests that ethylene signaling, which enhances the process of abscission, occurs earlier during the preharvest fruit drop period when physical fruit drop is not substantial.
Concomitantly, NCED2 was highly expressed in the AZ-C of loose fruit compared with that in the AZ-C of tight fruit of both ‘Hamlin’ and ‘Valencia’ at early timepoints in October and February, respectively, irrespective of the HLB symptom level. NCED2 encodes for the enzyme 9-cis-epoxycarotenoid dioxygenase, which is involved in ABA biosynthesis (Seo and Koshiba 2002; Xiong and Zhu 2003). Therefore, the higher expression of NCED2 in loose fruit compared with that in tight fruit could suggest an increase in ABA accumulation in the loose fruit. Environmental signals, such as drought stress, can activate endogenous ABA biosynthesis (Xiong and Zhu 2003), resulting in an increase in ethylene production in citrus (Riov and Hausman 1987). By increasing the production of ACC, an ethylene precursor, ABA can stimulate ethylene production in citrus leaves (Abeles 1967; Riov et al. 1990). Thus, environmental signals can trigger ABA accumulation and accelerate the abscission process by increasing ethylene production. Such a series of events can be seen in the exacerbation of “June drop” in ‘Clementine’ mandarin under water deficit conditions (García-Tejero et al. 2010; Ginestar and Castel 1996; Romero et al. 2006). Relevantly, HLB-affected trees have lower root density than healthy trees (Graham et al. 2013; Johnson et al. 2014), resulting in reduced water uptake capacity (Hamido et al. 2017; Kadyampakeni et al. 2014), which may result in increased water stress. Therefore, it can be hypothesized that water deficits or limited water availability can result in ABA accumulation, which could enhance the abscission process, thus leading to preharvest fruit drop. This hypothesis is supported by the findings of Morgan (2016), who reported that HLB-affected trees under frequent irrigation showed less fruit drop than those under irregular irrigation. Furthermore, Tang et al. (2020) reported that HLB-affected ‘Valencia’ trees that exhibited severe symptoms had significantly lower water potentials than mild trees in March, but that no such differences were observed at the time of harvest (May). This is consistent with the present study in which leaf water potentials did not differ significantly between trees that exhibited different HLB symptom levels close to harvest (May) and leaf water potentials were correlated with the fruit size or FDF. However, this does not eliminate the possibility that differences in the water status occurred before the time of measurement. Therefore, it can be speculated that water deficits earlier during the season may trigger the abscission signal, leading to higher rates of preharvest fruit drop in HLB-affected trees later during the season.
Additionally, other stresses can contribute to preharvest fruit drop. APX expression was higher in the AZ-C of severely affected trees than in the AZ-C of mild trees of ‘Hamlin’ sweet orange in October, but they did not differ between tight and loose fruit. APX encodes an antioxidant enzyme, ascorbate peroxidase, that catalyzes the reduction of hydrogen peroxide, which is a reactive oxygen species that forms in response to oxidative stress (Racchi 2013). CLas-infected leaves have been shown to accumulate more hydrogen peroxide than noninfected leaves, indicating that HLB increases oxidative stress in affected plants (Ma et al. 2022; Pitino et al. 2017). Recently, it has been reported that low fruit drop rates of HLB-tolerant ‘LB8-9’ mandarin are possibly attributable to the advanced antioxidant mechanism that can mitigate pathogen-induced oxidative stress as compared with those of HLB-susceptible ‘Hamlin’ sweet orange (Tang and Vashisth 2020). Hence, it can be suggested that the higher APX expression in the fruit AZ-C of severely affected trees indicates a higher degree of oxidative stress in these fruit compared with those from mildly affected trees, and it may contribute to the higher preharvest fruit drop rates. Although this pattern was evident for ‘Hamlin’, it was not observed for ‘Valencia’ sweet orange. It is worth noting that the timeframe of the analysis was seasonally different between ‘Hamlin’ and ‘Valencia’. It is likely that the relationship between oxidative stress and fruit drop could be cultivar-specific or time-dependent and season-dependent.
Conclusion
Our results suggest that the signal for HLB-associated preharvest fruit drop occurs several weeks before substantial fruit drop is observed. This signal likely results from ABA accumulation and subsequent ethylene production. Although what triggers the initial upregulation of ABA biosynthesis remains unclear, it is possible that water deficits and oxidative stress play roles. These signals for fruit drop are also more evident in small fruit. It is likely that abiotic stress (water deficit) and biotic stress (CLas infection) make some fruit a weak sink, thus leading to the developmental defects that limit the fruit size and make fruit more likely to abscise during the preharvest period. Therefore, any strategies to prevent or reduce fruit drop should probably be performed earlier during the fruit developmental period. However, it remains to be determined whether the abscission process can be stopped after the abscission signal has been triggered (i.e., closer to the harvesting period). Preharvest fruit drop of sweet orange also increased with the increasing HLB severity and canopy loss; these results aligned with those of previous studies. The correlation between canopy density and preharvest fruit drop rates suggests that improving or maintaining tree health and, subsequently, improving or maintaining canopy density, should be priorities when preventing the increase in preharvest fruit drop associated with HLB.
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