Abstract
Citrus greening, or huanglongbing (HLB), caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CaLas), threatens the global citrus industry. Field observations have demonstrated that some citrus cultivars are more tolerant to the CaLas pathogen than others. ‘Parson Brown’ is an early maturing sweet orange variety that has consistently exhibited minimal leaf and fruit drop in the field compared with the ‘Hamlin’ sweet orange under similar conditions. This study aimed to understand performance of the ‘Parson Brown’ cultivar in several locations across the citrus production regions of Florida. Results indicated that the CaLas bacterial titer in both cultivars were similar with the quantitative polymerase chain reaction cycle threshold values ranging between 24.99 and 28.61 in ‘Hamlin’ and between 25.48 and 30.89 in ‘Parson Brown’. Leaves from the ‘Parson Brown’ trees however demonstrated higher chlorophyll content and total phenolic compounds in most of the locations. We also detected higher relative expression of CsPR1 and CsPR2 transcripts in ‘Parson Brown’ leaves in the first sampling period (March) and the fourth period (November). Additionally, Phloem protein 2 transcripts were downregulated in ‘Parson Brown’ leaves compared with ‘Hamlin’ at all locations. The ‘Hamlin’ juice had higher acidity, whereas ‘Parson Brown’ juice demonstrated a higher Brix to acidity ratio and juice color. The oil content in the juice ranged between 0.020% and 0.042%, and there was variation in the oil content between the locations, which could indicate clonal differences. ‘Parson Brown’ juice however contained higher limonin and nomilin content than ‘Hamlin’ juice in most of the locations. Taken together, the current results confirmed the enhanced tolerance of ‘Parson Brown’ trees to HLB when compared with ‘Hamlin’ in all sampled locations.
HLB, or the citrus greening disease, has become endemic in many countries. In the United States, it is endemic in Florida and is now spreading to California and Texas. HLB causes stunted growth, yellow shoots, blotchy mottled leaves, yellowing of leaf veins, twig dieback, leaf yellowing, misshapen fruits, and bitter-tasting fruits (Bové 2006). Sustainability of citrus production has been jeopardized by this disease, as evidenced by a more than 50% reduction in citrus groves in the United States (Singerman et al. 2021). Many strategies for HLB mitigation have been widely investigated, including nutrition management, application of antimicrobials and plant defense inducers, biocontrol, control of insect vectors, general grove management, and eradication of HLB-infected trees (Yang and Ancona 2022).
The ‘Parson Brown’ sweet orange is an early-season orange that has been observed to produce normal-sized fruit and little fruit drop even under endemic HLB conditions (Castle and Baldwin 2010). As late as the mid-20th century, there were ∼15 clones of ‘Parson Brown’ registered with the Florida Department of Agriculture Citrus Budwood Program (England 2015). It is possible that several of these clones have been planted throughout the citrus growing belt in Florida, but current records are scarce on the exact location of the specific clones grown. One of the original clones currently remains active with DPI (F-56-2). ‘Parson Brown’ is seedy with a relatively thick peel that is slightly rough or pebbly. It has been grown largely for its supposedly better juice flavor and color compared with ‘Hamlin’, but its lower yield was the primary reason precluding its widespread adoption in the Florida citrus industry (Castle and Baldwin 2010). In earlier studies, ‘Parson Brown’ was evaluated as a scion in rootstock evaluation trials and occasionally as a rootstock (Cochen and Reitz 1963; Cohen 1973). The harvest season of sweet orange fruits ranges between mid-November and May in Florida, depending on the specific cultivar (Bai et al. 2016). The typical fruit traits that are preferred by the juice processing plants are high soluble solids content (SSC, Brix) >8.5%, titratable acidity (TA or acid) >0.4%, SSC/TA ratio >10.25, and juice content >45 mL/100 g (4.5 gal per 1.6-bushel box) (Hardenburg et al. 1990). In Florida, at the beginning of the season, TSS/acidity must reach values of 8% with a ratio of 9 or 10.5, whereas at the end of the season this ratio may exceed 20. Moreover, acidity should not be below 0.4% to 0.5% to avoid insipid taste (Ladaniya 2008). In recent years, HLB has resulted in increased orange juice bitterness due to increased nomilin and limonin content (Paula et al. 2018).
Many studies have reported HLB tolerance in some citrus species, such as citron and its hybrids (e.g., lemons), and in some trifoliate oranges and their hybrids (Peng et al. 2020). Sugar belle, a recently released mandarin hybrid, and finger lime have also been observed to be HLB-tolerant (Dutt et al. 2021; Killiny et al. 2018; Weber et al. 2022), as were several wild sexually compatible cultivars, such as Citrus ichangensis ‘2586’ (Wu et al. 2020) and C. latipes (Folimonova et al. 2009; Hijaz et al. 2016).
Plants have developed several stress tolerance techniques over time. These include biochemical, molecular, and physiological changes to perceive pathogen attack and adapt to biotic stress (Kumar and Verma 2018). These mechanisms assist plants in mitigating stress and maintaining their growth and development under stress conditions. Reactive oxygen species (ROS) production in different subcellular compartments is an important mechanism for biotic stress tolerance (Balint‐Kurti 2019). As an intercellular defensive response toward plant pathogens, there is a wide spectrum of defense compounds, such as phenolic compounds, terpenoids, alkaloids, nonprotein amino acids, benzoxazinoids, and cyanogenic glucosides (Hijaz et al. 2020; Killiny and Hijaz 2016) and are typically accompanied by the production of pathogenesis-related proteins, which are part of a systemic acquired response (Albrecht and Bowman 2008).
Studies have been conducted to identify specific genes and proteins that are activated or expressed in leaf tissues in response to HLB (Martinelli et al. 2012; Weber et al. 2022). Trees have primarily evolved two defensive reaction mechanisms: induced systematic resistance (ISR) and systemic acquired resistance (SAR), both of which are activated by pathogens (Van Loon 1997). SAR is a type of resistance that is nonspecific in nature and can be induced by the generation of mobile signal(s) after primary pathogen infection. SAR signals generated in the local infected tissue systematically translocate throughout the plant via phloem tissues. Moreover, this can confer long-term protection against a broad spectrum of microorganisms (Kuć 1982). The activation of innate plant resistance mechanisms, such as SAR, is being used for developing HLB tolerance in citrus (Dutt et al. 2015; Qiu et al. 2020; Soares et al. 2022).
In the grove, the ‘Parson Brown’, an early season sweet orange cultivar seemed to better tolerate HLB with field trees ranging in age from 25 years to 35 years. Similarly aged ‘Hamlin’, the most widely planted early maturing sweet orange in Florida, were very rare, with most having perished from HLB, and most groves had trees that were comparably much younger. The objective of the current study was to understand whether ‘Parson Brown’, irrespective of the many clones scattered across the citrus belt is truly tolerant to HLB, and to identify genetic and physiological factors influencing this tolerance mechanism.
Materials and Methods
Plant materials
Eight groves were surveyed in this study located throughout the citrus growing belt in Florida: Haines City (Polk County), Lake Wales (two locations; Polk County), Sebring (Highlands County), Lorida (Highlands County), Fort Pierce (St. Lucie County), Ortona (Glades County), and Immokalee (Collier County; Fig. 1). In each location, sampled ‘Parson Brown’ and ‘Hamlin’ trees were either in the same block or in adjacent blocks. Trees were sampled at a quarterly interval (March, June, September, and November).
A map of Florida indicating the approximate location of the sites sampled in this study and marked with a blue asterisk (*). The corresponding county is highlighted in orange.
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
A map of Florida indicating the approximate location of the sites sampled in this study and marked with a blue asterisk (*). The corresponding county is highlighted in orange.
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
A map of Florida indicating the approximate location of the sites sampled in this study and marked with a blue asterisk (*). The corresponding county is highlighted in orange.
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Physiological and biochemical variables
A total of 12 biological replicates were collected from each grove and three technical replicates were sampled from every individual tree. Fifteen mature leaves were harvested from each replicate of ‘Parson Brown’ or ‘Hamlin’ trees, frozen and ground in liquid nitrogen. The ground leaves were stored at –20 °C before use in biochemical assays.
Photosynthetic pigments and starch quantification.
Chlorophyll a, chlorophyll b, carotenoids and total chlorophyll were estimated according to (Lichtenthaler and Wellburn 1983). Starch content was determined according to Rosales and Burns (2011) with some modifications. Fresh tissues (100 mg) were ground to a powder, suspended, and homogenized in 700 μL of distilled water. Leaf samples and a standard were boiled in water for 10 min and moved to cold water to cool. Samples were vortexed and centrifuged for 2 min at 6000 rpm. Supernatant aliquots were collected into new tubes. A 300 μL of supernatant was extracted with 900 μL of 100% ethanol. The mixture was vortexed and centrifuged for 10 min at 10,000 rpm. The supernatant was discarded, and 1 mL of distilled water was added to dissolve the pellet. Fifty microliters of KI:I2 (8 mM:50 mM) was added. Quantification of starch was accomplished by monitoring the color change in a spectrophotometer at 594 nm. Rice starch (Sigma Aldrich, St Louis, MO, USA) was used as a standard.
2,2-diphenyl-1-picryl-hydrazyl-hydrate radical scavenging capacity and total phenolic compounds.
Total phenolic compounds were estimated according to Singleton and Rossi (1965). In brief, 100 mg of fresh leaf was extracted in 1 mL of 80% ethanol and then centrifuged for 20 min at 10,000 rpm. The supernatant was incubated overnight at room temperature until evaporation and complete dryness. Distilled water (5 mL) was added to the plate to dissolve the dried gel. Two hundred microliter aliquots were diluted to 3 mL in distilled water. The folin reagent was added at 0.5 mL for 3 min, and then 2 mL of (20%) Na2CO3 solution was added to each tube. The color change was recorded after 60 min at 650 nm. The standard curve of phenol was prepared by measuring 1 mL of a series of ethanolic gallic solutions at different concentrations from 0 to 1.00 mg/mL. The phenol contents were expressed as mg gallic acid per 100 g fresh weight tissue.
CaLas assessment in ‘Parson Brown’ and ‘Hamlin’ leaves
To diagnose the CaLas titer in the citrus trees, genomic DNA was isolated periodically from the leaf petioles and midveins of fully expanded leaf tissues using the GeneJET Plant Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). The same leaves used for biochemical assays were used for CaLas diagnosis. DNA was normalized to 25 ng/μL before performing qualitative polymerase chain reaction (qPCR) using a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific). Detection of CaLas genomic DNA was determined by qPCR using TaqMan™ Gene Expression Master Mix and CQUL primers (Table 1) to amplify a fragment of the CaLas rplJ/rplL ribosomal protein gene (Wang et al. 2006).
TAQMAN based primer sequences used to amplify an 87-bp fragment of the Candidatus Liberibacter asiaticus rplJ/rplL ribosomal protein gene.
Fruit quality assessment
General fruit and juice traits.
Twelve trees were used to collect <35 fruits from respective replicates at harvest in December 2021. Fruits were cleaned and mechanically juiced using a JBT Citrus Juice Extractor. Juice color, SSC, and TA were measured the same day. Fruit juice color was measured in a spectrophotometer (Macbeth Color-Eye 3100; Kollmorgh Instruments Corp., Baltimore, MD, USA) according to Lee (2000). Juice SSC made from fresh fruit was measured using Brix acid unit (BAU) by Florida Department of Agriculture and Consumer Services using a few drops of juice with temperature and acid corrections. The percentage of TA (percent anhydrous citric acid) was determined by titrating juice samples to pH 8.3 with NaOH using an automatic titrator (HI931; Hanna Instruments, Smithfield, RI, USA). The empirical soluble solids content/acid ratio, calculated by dividing the soluble solids content by the titratable acidity, is one of the most used indicators of juice quality (Kimball 1991).
Oil content determination.
The oil content in the juice was analyzed by a bromate titration method (Scott and Veldhuis 1966). In brief, the juice sample (25 mL) and 2-propanol (25 mL) with a few boiling stones were distilled until the solvent ceased to reflux. Forty milliliters of the oil were collected and mixed with Hydrochloric acid (HCl), 33.33% (v/v), with 0.0005% (w/v) methyl orange. The oil content was determined by titrating the distilled fraction with 0.025 N bromide-bromate solution until reaching a clear color. The amounts of bromide-bromate solution used were recorded and multiplied by 0.004 to obtain the percentage of oil in each juice sample.
Limonoid analysis.
High-performance liquid chromatography–mass spectrometry was used to separate and determine limonoid compounds. A 16-mL well-mixed juice sample was added to 4 mL of acetonitrile in a 50-mL Falcon tube, sonicated for 10 min, and centrifuged at 10,000 gn for 5 min. Ten milliliters of supernatant was passed to waste through a Hypersep C18 Sep-Pak cartridge (500 mg/6 mL) that was preconditioned with 3 mL of acetonitrile followed by 3 mL of water. Three milliliters of 20% acetonitrile were passed to the waste. The cartridge was eluted with 3 mL of 60% Acetonitrile and captured in a 15 mL centrifuge tube. The sample was mixed and filtered through a 0.45 um nylon filter into an autosampler vial for analysis. Two microliters of filtered sample were used as the injection volume. The main fragment ions, 471 m/z for limonin and 515 m/z for nomilin, were used for quantification. Standards were purchased from Sigma Aldrich.
Gene expression assessment
RNA was isolated from 100 mg of finely ground leaf tissues using a Direct-zol™ RNA Miniprep according to the manufacturer’s protocol. qPCR was performed with a final volume of 10 μl using the SYBR green kit according to the manufacturer’s instructions. Each sample was tested twice in 12 replicates, and the data were analyzed using Applied Biosystems (Waltham, MA, USA) software version 3.0.1. The cycle threshold (Ct) value of the PCR curve was analyzed and compared with ‘Hamlin’ as a control tree at a particular sampling time under each location. The relative expression of the selected genes was calculated by the 2−ΔΔCT method (Livak and Schmittgen 2001). The actin housekeeping gene was used as an endogenous control. The primer sequences of the genes evaluated in this study are outlined in Table 2.
List of the primer sequences used in SYBR Green based real-time polymerase chain reaction assay.
Experimental design and statistical analysis
To investigate the performance of ‘Parson Brown’ and ‘Hamlin’ sweet oranges under the HLB endemic conditions in eight locations, analysis of variance was conducted in JMP Pro version 16 (SAS Institute, Cary, NC, USA). The combinations of location and cultivar were designed as a factorial experiment with two factors (eight locations) and two cultivars. P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations at ‘Parson Brown’ is indicated by differing uppercase letters; mean separation between locations at ‘Hamlin’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). Differences were significantly different when P values were less than 5%.
Results
Visual observations
The ‘Parson Brown’ trees in most locations had a full canopy, whereas ‘Hamlin’ trees had typical symptoms of advanced HLB infection with lower canopy volume (Fig. 2). Fruit drop was also pronounced on the ‘Hamlin’ trees, whereas ‘Parson Brown’ trees had minimal fruit drop. The average age of the ‘Parson Brown’ trees was 25 or more, with the oldest grove being planted in the early 1980s (>40 years); the most ‘Hamlin’ had been replanted and were younger, with the oldest being no more than 15 years. It was difficult to find ‘Hamlin’ trees of similar age as ‘Parson Brown’ trees.
Comparison between a ‘Parson Brown’ and a ‘Hamlin’ tree of similar age and located in the same grove.
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Comparison between a ‘Parson Brown’ and a ‘Hamlin’ tree of similar age and located in the same grove.
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Comparison between a ‘Parson Brown’ and a ‘Hamlin’ tree of similar age and located in the same grove.
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
CaLas diagnosis
In the first sampling time during March, Ct-values of CaLas ranged from 26.36 to 29.36 in ‘Hamlin’ leaves in all locations, whereas it ranged from 25.96 to 29.20 in ‘Parson Brown’ leaves (Table 3). When the same trees were sampled in November, we detected an increase in CaLas titer in the Lake Wales 2, Lorida, Fort Pierce, and Lake Wales 1 locations, where the Ct-values ranged between 26.36 and 30.88 during March and 24.99 and 28.61 during November in ‘Hamlin’ leaf petioles. The CaLas titer in ‘Parson Brown’ ranged from 25.96 to 29.20 during March and 26.36 and 30.88 during November in leaf petioles. There was a significant difference in the Ct-values between the selected locations.
Quantification of Candidatus Liberibacter asiaticus bacterial titers following quantitative polymerase chain reaction from leaf petioles and midveins of ‘Hamlin’ and ‘Parson Brown’ trees of the eight sampled locations.
Physiological and biochemical variables
Chlorophyll pigments and starch accumulation in leaves.
Significant variance analysis of physiological parameters among two early sweet orange cultivars and different locations was implemented using a two-way analysis of variance (Table 4). In general, chlorophyll a (CHL a), chlorophyll b (CHL b), carotenoids, and total chlorophyll content showed a significant difference between the two cultivars (‘Hamlin’ and ‘Parson Brown’), the locations and the interaction between the cultivars and locations (P < 0.005). The foliar CHL a content was higher in the ‘Parson Brown’ samples collected from Fort Pierce, Lake Wales 2, Sebring, and Lorida (5.64, 5.94, 6.10, and 5.85 mg·g−1 FW, respectively) compared with ‘Hamlin’ trees. Only ‘Hamlin’ samples collected from the Immokalee field trial showed higher content in CHL a content (4.67 mg·g−1 FW) compared with the foliar content of ‘Parson Brown’ trees (3.93 mg·g−1 FW) in the same location. There was no significant difference in the foliar CHL a content in ‘Hamlin’ and ‘Parson Brown’ samples collected from Lake Wales 1, Haines City and Ortona. The foliar CHL b content showed a significant increase in the ‘Parson Brown’ samples collected from Fort Pierce, Ortona, Sebring and Lorida (2.20, 1.95, 2.37, and 2.06 mg·g−1 FW, respectively) compared with ‘Hamlin’ trees under the same conditions. The chlorophyll pigment content was significantly increased in ‘Hamlin’ samples only in the samples collected from Immokalee compared with the other locations. The content of foliar carotenoids of ‘Parson Brown’ samples collected from Lake Wales 1, Haines City, Ortona, and Immokalee showed an increase in carotenoid content compared with ‘Hamlin’ under the same conditions. The same observations were recorded regarding the total pigment content. The foliar total CHL content showed a significant increase in the ‘Parson Brown’ samples collected from Fort Pierce, Ortona, Sebring, and Lorida (7.85, 6.97, 8.48, and 7.90 mg·g−1 FW, respectively) compared with ‘Hamlin’ trees (5.99, 6.08, 7.20, and 6.74 mg·g−1 FW, respectively) under the same conditions (Fig. 3). There was no significant difference in foliar starch content between ‘Hamlin’ and ‘Parson Brown’ in all locations (Fig. 4).
Significance analysis of some physiological traits of ‘Hamlin’ and ‘Parson Brown’ trees under eight locations using a two-way analysis of variance assay.
Foliar chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll content in ‘Hamlin’ and ‘Parson Brown’ trees under eight locations. P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Foliar chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll content in ‘Hamlin’ and ‘Parson Brown’ trees under eight locations. P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Foliar chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll content in ‘Hamlin’ and ‘Parson Brown’ trees under eight locations. P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Foliar starch content in ‘Hamlin’ and ‘Parson Brown’ trees under eight locations. P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Foliar starch content in ‘Hamlin’ and ‘Parson Brown’ trees under eight locations. P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Foliar starch content in ‘Hamlin’ and ‘Parson Brown’ trees under eight locations. P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Total phenolic compound content and free radical scavenging activity.
The total phenolic compound (TPC) content was determined in ‘Parson Brown’ and ‘Hamlin’ leaves to assess the induced oxidation response (Fig. 5). There was variation between the different locations (P = 0.0012) and the interaction between cultivars and locations (P = 0.01). The TPC content ranged between 64.30 and 91.49 mg gallic acid/g FW in ‘Hamlin’ and between 70.04 and 83.00 mg gallic acid/g FW in ‘Parson Brown’ leaves. ‘Parson Brown’ had an obvious increase in TPC content compared with ‘Hamlin’ in Ortona (83.00 and 69.21 mg gallic acid/g FW, respectively). ‘Hamlin’ samples recorded a significant increase in TPC content compared with ‘Parson Brown’ in Immokalee (78.59 and 70.04 mg gallic acid/g FW, respectively). The DPPH-radical scavenging activity significantly increased in the ‘Parson Brown’ samples collected from Haines City, Ortona, and Sebring (52.20%, 54.06%, and 47.7%) compared with ‘Hamlin’ samples (45.46%, 30.34%, and 33.80%, respectively).
Foliar total phenolic compounds (TPC) content and free radical scavenging activity in ‘Hamlin’ and ‘Parson Brown’ trees under eight locations. P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing lowercase letters; mean separation location for ‘Parson Brown’ is indicated by differing uppercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Foliar total phenolic compounds (TPC) content and free radical scavenging activity in ‘Hamlin’ and ‘Parson Brown’ trees under eight locations. P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing lowercase letters; mean separation location for ‘Parson Brown’ is indicated by differing uppercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Foliar total phenolic compounds (TPC) content and free radical scavenging activity in ‘Hamlin’ and ‘Parson Brown’ trees under eight locations. P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing lowercase letters; mean separation location for ‘Parson Brown’ is indicated by differing uppercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Juice quality assessment
Juice parameters.
To assess the fruit quality of ‘Hamlin’ and ‘Parson Brown’, we measured Pounds juice per box (lbs), titratable acidity, total Brix, Brix to acidity ratio, lbs. solid per box, and juice color in the juice samples at the CREC packinghouse (Table 5). Analysis of variance indicated a P value <0.0001 for cultivars, <0.0001 for locations, and <0.0001 for the interaction between cultivar × locations for all the tested parameters. Pounds of juice per box ranged between 47.27 and 53.54 in ‘Hamlin’ fruits in all locations, whereas it ranged from 50.94 to 53.87 in ‘Parson Brown’ fruits. Compared with ‘Hamlin’, in Haines City, Immokalee and Sebring, ‘Parson Brown’ showed a significant increase in lbs. juice per box values.
Juice parameters in ‘Hamlin’ and ‘Parson Brown’ trees.
It was observed that ‘Hamlin’ juice had a higher titratable acidity percentage than ‘Parson Brown’ juice at all sites except Fort Pierce. The titratable acidity percentage ranged from 44.67 to 81.00 in ‘Hamlin’ fruits at all locations, whereas it ranged from 42.33 to 57.17 in ‘Parson Brown’ fruits. There was no significant difference between ‘Hamlin’ and ‘Parson Brown’ fruits regarding total Brix. The ratio of Brix to acidity showed a significant increase in ‘Parson Brown’ fruits compared with ‘Hamlin’ fruits under all field locations. It ranged from 13.01 to 20.28 in ‘Hamlin’ fruits in all the locations, whereas it ranged from 16.87 to 22.84 in ‘Parson Brown’ fruits. Pounds solids per box ranged between 4.32 and 5.40 in ‘Hamlin’ and 3.90 and 5.35 in ‘Parson Brown’. According to juice analysis data, ‘Parson Brown’ juice was darker in color than ‘Hamlin’ juice. The highest value of juice color of ‘Parson Brown’ juice was recorded in Fort Pierce (35.07), followed by Sebring and Ortona (34.16 and 34.30); however, the highest value of juice color of ‘Hamlin’ juice was recorded in Lorida (33.93).
Oil content.
Oil content was estimated in juice samples, and analysis of variance showed a P value <0.0001 for cultivars, <0.0001 for locations, and <0.0011 for the interaction between cultivar × locations. The oil content % ranged from 0.007 to 0.0011 in ‘Hamlin’ juice at all locations, whereas it ranged from 0.020 to 0.042 in ‘Parson Brown’ juice (Fig. 6).
Oil content percentage in ‘Hamlin’ and ‘Parson Brown’ juice under eight locations. P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Oil content percentage in ‘Hamlin’ and ‘Parson Brown’ juice under eight locations. P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Oil content percentage in ‘Hamlin’ and ‘Parson Brown’ juice under eight locations. P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Limonoid analysis.
‘Parson Brown’ juice consistently had higher levels of limonin content than ‘Hamlin’ juice from the Haines City, Lake Wales 1, Lake Wales 2, Lorida, and Immokalee locations. The limonin content ranged from 2.26 to 3.83 ppm in ‘Hamlin’ juice at all locations except in Sebring, which showed a peak at 5.26 ppm, whereas it ranged from 3.31 to 4.90 in ‘Parson Brown’ juice. There is a variation in the nomilin content between ‘Hamlin’ and ‘Parson Brown’ juice (Table 6). The content of nomilin ranged from 0.19 to 0.85 ppm in ‘Hamlin’ juice; however, it ranged from 0.32 to 0.97 ppm in ‘Parson Brown’ juice. There was a significant difference in the content of limonin and nomilin in ‘Hamlin’ juice and the content of nomilin in ‘Parson Brown’ juice in the selected locations. No significant difference was recorded when the content of limonin was compared in ‘Parson Brown’ juice from the different locations.
Content of Limonin and Nomilin in ‘Hamlin’ and ‘Parson Brown’ Juice.
Gene expression analysis
SAR: Differential expression of pathogenesis-related genes.
In general, we recorded higher relative expression of the CsPR1 transcript in PB leaves in the first sampling in March and the fourth sampling in November compared with ‘Hamlin’ in most locations (Fig. 7, Table 7). The second and third samplings indicated a different trend when the relative expression of the CsPR1 transcript in the eight locations was compared. Two locations (Lake Wales 1 and Lake Wales 2) showed overexpression of CsPR1 at all sampling times. There was a significant reduction in the relative expression of CsPR1 in the third sampling time in September in Haines City, Fort Pierce, Lorida, Ortona, and Immokalee compared with the records in the other sampling times in the same locations. No significant difference was observed in the samples collected at Ortona in the second and third sampling times.
The differential expression of CsPR1 transcript in ‘Parson Brown’ relative to ‘Hamlin’ expression of eight locations. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). (*) means significant difference between ‘Hamlin’ and ‘Parson Brown’ under each site. The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
The differential expression of CsPR1 transcript in ‘Parson Brown’ relative to ‘Hamlin’ expression of eight locations. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). (*) means significant difference between ‘Hamlin’ and ‘Parson Brown’ under each site. The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
The differential expression of CsPR1 transcript in ‘Parson Brown’ relative to ‘Hamlin’ expression of eight locations. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). (*) means significant difference between ‘Hamlin’ and ‘Parson Brown’ under each site. The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Significance analysis of CsPR1 relative expression of ‘Parson Brown’ leaves compared with ‘Hamlin’ sweet orange in four seasons using a two-way analysis of variance assay.
The relative expression of the CsPR2 transcript of ‘Parson Brown’ showed an increase in the fourth sampling time in November in the samples collected from all the sites (Fig. 8, Table 8). There was a significant reduction in the expression of CsPR2 in the third sampling time compared with the fourth sampling time. There was no significant difference between the sampling time in the Fort Pierce trial compared with ‘Hamlin’ in the first three times, whereas the relative expression of CsPR2 increased in November.
The differential expression of CsPR2 transcript in ‘Parson Brown’ relative to ‘Hamlin’ expression of eight locations. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). (*) means significant difference between ‘Hamlin’ and ‘Parson Brown’ under each site. The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
The differential expression of CsPR2 transcript in ‘Parson Brown’ relative to ‘Hamlin’ expression of eight locations. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). (*) means significant difference between ‘Hamlin’ and ‘Parson Brown’ under each site. The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
The differential expression of CsPR2 transcript in ‘Parson Brown’ relative to ‘Hamlin’ expression of eight locations. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). (*) means significant difference between ‘Hamlin’ and ‘Parson Brown’ under each site. The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Significance analysis of CsPR2 relative expression of ‘Parson Brown’ leaves compared with ‘Hamlin’ sweet orange in four seasons using a two-way analysis of variance assay.
Differential expression of the phloem protein gene.
The relative expression of phloem protein (CsPP2–B15) transcripts was downregulated in ‘Parson Brown’ samples compared with ‘Hamlin’ (Fig. 9), especially in the Fort Pierce, Lorida, and Lake Wales 1 locations.
The differential expression of CsPP2-B15 transcript in ‘Parson Brown’ relative to ‘Hamlin’ expression under eight locations. The error bar indicates SE (n = 12). P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
The differential expression of CsPP2-B15 transcript in ‘Parson Brown’ relative to ‘Hamlin’ expression under eight locations. The error bar indicates SE (n = 12). P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
The differential expression of CsPP2-B15 transcript in ‘Parson Brown’ relative to ‘Hamlin’ expression under eight locations. The error bar indicates SE (n = 12). P values were used to compare mean separation between the two cultivars in a particular location. Mean separation between locations for ‘Hamlin’ is indicated by differing uppercase letters; mean separation location for ‘Parson Brown’ is indicated by differing lowercase letters by Tukey’s honestly significant difference test (P ≤ 0.05). The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
Discussion
Most citrus sweet orange cultivars are highly sensitive to HLB, and the trees decline a few years after infection. This study was initiated following anecdotal reports that ‘Parson Brown’ trees could survive over 25 years with minimal leaf and fruit drop compared with ‘Hamlin’ sweet orange under endemic HLB conditions. Our observations revealed that in most of the locations, ‘Parson Brown’ and ‘Hamlin’ trees had comparable CaLas Ct-values indicating a similar infection rate of HLB. While ‘Hamlin’ trees had severe fruit drop, we observed minimal fruit drop from the ‘Parson Brown’ trees. Progression of HLB severity increases the fruit drop rate in most sweet oranges in Florida (Tang et al. 2019), but this was not visually obseved in blocks containing the ‘Parson Brown’ trees.
The current research also documented an increase in the foliar chlorophyll content levels exhibited by ‘Parson Brown’ trees when compared with ‘Hamlin’ (Table 4). Chlorophyll (Chl) is a key component for photosynthesis and is required for the absorption of sunlight (Hörtensteiner and Kräutler 2011). CaLas-infected trees usually exhibit yellow chlorotic mottled leaves (Inoue et al. 2020). Chlorophyll breakdown can also occur as a response to biotic and abiotic stresses (Mahmoud et al. 2021a, 2022) or many macro- and micronutrient deficiencies (Uthman et al. 2022). The inhibition of chlorophyll biosynthesis or degradation can lead to ROS production and cell death (Hörtensteiner and Kräutler 2011). Enhanced chlorophyll content can result in improved photosynthesis and better tree health. There was no significant difference in the starch content between ‘Parson Brown’ and ‘Hamlin’ in most of the locations. The starch content could be related to HLB infection or differences in the age of the leaves or the trees.
HLB activates ROS production and triggers the expression of defense genes as signal molecules (Weber et al. 2022). Previous studies reported that CaLas infection causes overproduction of free oxygen radicals that can potentially cause toxic build-up leading to programmed death. Generally, DPPH activity is a key parameter to evaluate the activity of ROS scavenging in plant cells (Mahmoud et al. 2021b). Our results indicated that ‘Parson Brown’ has a higher scavenging capacity than ‘Hamlin’. Furthermore, a significant increase in the transcript levels of the CsAPX1, CsSOD1, and CsSOD2 genes contributed to reducing oxidative stress (Supplemental Fig. 1). To gain a better understanding of the enhanced HLB tolerance trait in ‘Parson Brown’, we evaluated changes in the differential expression of the CsPR1 and CsPR2 transcripts. PR1 and PR2 were induced by the CaLas infection in citrus plants (Soares et al. 2022). PR genes are key to the plant’s innate immune system. Enhanced PR1 and PR2 gene expression is indicative of a robust Systemic Acquired Resistance (SAR) process in the plant (Kim et al. 2015). Overall, the relative gene expression of CsPR1 and CsPR2 was mainly upregulated in ‘Parson Brown’ leaves during March and December compared with ‘Hamlin’. The CaLas levels also tend to be higher during this time (Monzo and Stansly 2017). Induction of SAR can boost plant defense against abiotic and biotic stress. Detection of these PR1 and PR2 genes can be indicative of SAR activity and have been used as molecular markers (Ali et al. 2018). Following pathogen attack, plants activate defense signaling pathways which involve the salicylic acid and jasmonic acid hormones. These chemicals stimulate the transcription of NPR1 (nonexpressor of pathogen-related gene 1), which subsequently activates the SAR gene products locally as well as systematically, leading to SAR (Ali et al. 2018). Therefore, this occurrence could potentially contribute to the improved tolerance to HLB seen in ‘Parson Brown’ trees, as evidenced by the increased expression of PR1 and PR2 genes. However, information concerning fruit drop and fruit yield will be presented in a forthcoming report. We also observed a downregulation in the relative expression of the phloem limited PP2-B15 transcript in the ‘Parson Brown’. Some phloem proteins (PP) play an important role in callose deposition in the phloem and are abundantly found in plugged sieve pores (Qingli et al. 2018) and PP2 genes are usually upregulated in diseased trees (Musetti et al. 2010; Ghosh et al. 2018). Our findings agree with Qingli et al. (2018), who found upregulation of CsPP2-B15 expression in the infected leaves of HLB susceptible Jincheng orange (Citrus sinensis Osbeck) and downregulation in the tolerant sour pomelo (Citrus grandis Osbeck). CsPP2-B15 was also found to be downregulated in tolerant Citrus ichangensis (Wu et al. 2020). Because enhanced PP2 expression in citrus is always indicative of HLB susceptibility, we can surmise from our results that lower PP2-B15 levels in ‘Parson Brown’ may also provide indirect evidence on its ability to better withstand infection, albeit by a yet undetermined process.
To obtain a clear picture of the flavor quality in the juice, several factors responsible for sweetness, and bitterness (SSC, TA, SSC/TA, oil content, limonin, and nomilin) were evaluated in ‘Parson Brown’ and ‘Hamlin’ juice as well as peel oil. These factors can influence orange juice flavor and can in turn affect the quality of the juice. The TSS content in the juice is considered as an important selection criterion that affects the flavor of sweet orange fruits (Ramos et al. 2021). The increase in juiciness and fruitiness may benefit the processing of citrus fruits, resulting in increased juice yields. Changes in TA and the SSC/TA ratio are derived predominantly from a TA decrease due to reduction of citric acid levels (the dominant acid in citrus fruit). The ‘Parson Brown’ juice was comparable to ‘Hamlin’ with better color, and this cultivar was originally selected for its better juice color (Castle and Baldwin 2010).
The maximum USDA-designated oil content of grade A orange juice is 0.035%, whereas 0.01% to 0.025% is preferred in most orange juices (Bai et al. 2016; Barrett et al. 2004). Excessive peel oil in orange juice tends to be bitter or burning in taste (Rich 1990). The oil content in all the ‘Hamlin’ juice samples was grade A, whereas ‘Parson Brown’ had a higher and variable oil content percentage, which depended on the location from which the fruit was harvested. There are many clones of the ‘Parson Brown’ being grown in Florida, and thus clonal differences may be partly responsible for this observed variation. Growth or regrowth of fruit and peel directly correlates to the enlargement of oil glands (Rich 1990; Turner 1999), and juice oils are overwhelmingly from these oil glands (Mottram 1991). The bitter limonoids limonin and nomilin generally increased in ‘Parson Brown’. Limonin is the primary compound that contributes to bitterness in orange juice and levels can vary on a yearly basis, with levels as high as 6.24 ppm being recorded in some commercially cultivated juice oranges in Florida. Both limonin and nomilin levels increase several folds in citrus fruit from HLB-affected trees (Paula et al. 2018). Considering the increased levels of limonin and nomilin in the Florida-derived sweet orange juice, all our juice samples could be deemed acceptable to the processors under current endemic HLB conditions.
Conclusion
The present study evaluated ‘Parson Brown’ and ‘Hamlin’ trees that were HLB-positive and had similar CaLas titers in their phloem tissues. It was observed that regardless of location, clone, or rootstock, ‘Parson Brown’ trees demonstrated enhanced systemic acquired resistance (SAR) activity, as indicated by increased production of CsPR1 and CsPR2 transcripts. The downregulation of the CsPP2-B15 transcript indirectly suggest an active protective mechanism in the phloem of ‘Parson Brown’ trees, which may contribute to their overall better health observed in all locations investigated. Although ‘Parson Brown’ fruits had higher oil content in the juice, it is important to note that new extraction methods are commercially available to remove this oil while maintaining juice quality. On the basis of the study results, it can be concluded that ‘Parson Brown’ has the potential to serve as a suitable alternative to ‘Hamlin’ for early-season orange juice processing.
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The differential expression of antioxidants transcript in ‘Parson Brown’ relative to ‘Hamlin’. The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
The differential expression of antioxidants transcript in ‘Parson Brown’ relative to ‘Hamlin’. The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
The differential expression of antioxidants transcript in ‘Parson Brown’ relative to ‘Hamlin’. The error bar indicates SE (n = 12).
Citation: HortScience 58, 10; 10.21273/HORTSCI17241-23
