Effect of Huanglongbing on the Volatile Organic Compound Profile of Fruit Juice and Peel Oil in ‘Ray Ruby’ Grapefruit

Along with orange and mandarin, grapefruit production in Florida has declined sharply due to Huanglongbing (HLB), or citrus greening disease, caused by Candidatus Liberibacter asiaticus (CLas). HLB affects the volatile profiles of juice and peel oil in oranges, but there is limited information on grapefruit. In this research, ‘Ray Ruby’ grapefruit were harvested in 2020 and 2021 from healthy (HLB−) and HLB-affected (HLB+) trees. Peel oil was extracted by hydrodistillation, and the volatiles were analyzed by direct injection of the oil samples into gas chromatography–mass spectrometry (GC-MS). Volatiles in the juice were analyzed by headspace (HS)-solid-phase microextraction (SPME) coupled with GC-MS. HLB significantly altered the volatile profiles of peel oil and juice in ‘Ray Ruby’ grapefruit. Juice samples of HLB+ fruits had lower decanal, nonanal, and octanal, important citrus juice flavor compounds. HLB+ samples also showed reduced content of nonterpene compounds, other aliphatic and terpene aldehydes, and terpene ketones. Ethanol, acetaldehyde, ethyl acetate, and ethyl butanoate were increased in HLB+ juice samples, indicating an HLB-induced stress response. The most abundant compounds D-limonene and β-caryophyllene, as well as other sesquiterpenes, were increased in HLB+ juice and peel oil samples. On the other hand, the oxidative/dehydrogenated terpenes were increased by HLB in peel oil but decreased in the juice sample. Nootkatone, the key grapefruit volatile was consistently reduced by HLB in both peel oil and juice samples. The impact of HLB on nootkatone deteriorated the quality of both juice and peel oil in grapefruits.


Introduction
Florida citrus production has plummeted since 2005 when Huanglongbing (HLB), or citrus greening disease, was first reported in the U.S. Total citrus production has been reduced by 80% from 291.8 million boxes in 2003/2004 to 57.8 million boxes last season (2020-2021) [1]. The impact on the grapefruit (Citrus paradisi) industry is even more severe, with a decrease of 90% from 40.9 to 4.1 million boxes [1,2]. Citrus canker disease (Xanthomonas citri subsp. citri) and associated eradication programs from the late 1990s to the early 2000s followed by catastrophic hurricanes in 2004 and 2005 were also responsible for the decrease in acreage and production [3,4].
HLB is caused by a gram-negative bacterium, 'Candidatus Liberibacter asiaticus' (CLas), which is transmitted by the Asian citrus psyllid (ACP, Diaphorina citri) [5]. The disease has spread through the major citrus-producing regions worldwide, causing high economic We used 37.85 L plastic pots (#10 Accelerator AP-10; Nursery Supplies, Chambersburg, PA, USA) filled with a medium consisting of (v/v) 50% clean washed silica sand, 15% Florida peat moss, 7.5% coconut fiber, 20% cypress sawdust, and 7.5% perlite (Harrell's, Lake Placid, FL, USA). The pots were placed on ceramic tiles to prevent tree roots from growing into the underlying native sandy soil. Each tree in the trial received routine irrigation and fertigation program according to Ferrarezi et al. [22].
Healthy grapefruit trees (HLB−) were cultivated under a protective screen that acted as a barrier against the ACP to prevent any transmission of the disease. HLB+ trees were grown in the open air and naturally exposed to a high ACP population [22]. Each treatment contained 24 trees and was equally divided into three replications.

'Candidatus Liberibacter asiaticus' Detection
Ten fully expanded leaves and leaf petioles with intact stems were collected from eight trees per replication. The detection analysis was conducted by quantitative real-time polymerase chain reaction (qRT-PCR) using the U.S. Department of Agriculture, Animal, and Plant Health Inspection Service approved primers [5,22]. The cycle threshold (Ct) to consider trees infected by HLB was below 32 [5].

Fruit Harvest and Measurements of Fruit Color, Size, Soluble Solids Contents, and Acidity
Fruits were harvested on 8 January 2020 and 7 January 2021. One fruit was harvested per tree, and eight fruits per replicate were collected. Fruits were washed using vegetable detergent, rinsed with deionized water, and air dried at room temperature for 30 min. The color was measured at three points around the fruit equator using a Chromameter (Minolta CR-400, Tokyo, Japan) calibrated to a white plate using the CIELAB system, and a*, b*, a*/b*, and hue values were reported [23]. A higher a*/b* value and/or lower hue value indicates a more orange or red color [6,24,25]. Individual fruit was measured for weight and size. Data reported are an average of eight individual fruits x three replicates.
The flavedo tissue (1-2 mm from the fruit surface) was manually taken using a sharp stainless-steel knife for peel oil extraction. The flavedo-removed fruits were then halved, and juice was gently extracted using a kitchen juicer with an automatic self-reversing reamer (Oster Model 3183, Household Appliance Sales and Service, Niles, IL, USA). Juice samples were centrifuged at 10,000× g for 15 min. The supernatant was used for soluble solids content (SSC) and titratable acidity (TA) measurements. SSC was determined by refractive index using a digital refractometer (Atago RX-5000cx, Tokyo, Japan). TA was Healthy grapefruit trees (HLB−) were cultivated under a protective screen that acted as a barrier against the ACP to prevent any transmission of the disease. HLB+ trees were grown in the open air and naturally exposed to a high ACP population [22]. Each treatment contained 24 trees and was equally divided into three replications.

'Candidatus Liberibacter asiaticus' Detection
Ten fully expanded leaves and leaf petioles with intact stems were collected from eight trees per replication. The detection analysis was conducted by quantitative real-time polymerase chain reaction (qRT-PCR) using the U.S. Department of Agriculture, Animal, and Plant Health Inspection Service approved primers [5,22]. The cycle threshold (Ct) to consider trees infected by HLB was below 32 [5].

Fruit Harvest and Measurements of Fruit Color, Size, Soluble Solids Contents, and Acidity
Fruits were harvested on 8 January 2020 and 7 January 2021. One fruit was harvested per tree, and eight fruits per replicate were collected. Fruits were washed using vegetable detergent, rinsed with deionized water, and air dried at room temperature for 30 min. The color was measured at three points around the fruit equator using a Chromameter (Minolta CR-400, Tokyo, Japan) calibrated to a white plate using the CIELAB system, and a*, b*, a*/b*, and hue values were reported [23]. A higher a*/b* value and/or lower hue value indicates a more orange or red color [6,24,25]. Individual fruit was measured for weight and size. Data reported are an average of eight individual fruits x three replicates.
The flavedo tissue (1-2 mm from the fruit surface) was manually taken using a sharp stainless-steel knife for peel oil extraction. The flavedo-removed fruits were then halved, and juice was gently extracted using a kitchen juicer with an automatic self-reversing reamer (Oster Model 3183, Household Appliance Sales and Service, Niles, IL, USA). Juice samples were centrifuged at 10,000× g for 15 min. The supernatant was used for soluble solids content (SSC) and titratable acidity (TA) measurements. SSC was determined by refractive index using a digital refractometer (Atago RX-5000cx, Tokyo, Japan). TA was measured by titration of 10 mL of supernatant with 0.1N NaOH to pH 8.1 using an autotitrator (808 Titrando, Metrohm, Riverview, FL, USA).

Peel Oil Extraction and Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
Peel oil was extracted by hydrodistillation [26]. The peel tissue, 200 g, from eight fruit in each replicate was homogenized in a blender with 500 g of ultrapure water and transferred to a 2000 mL round bottom flask fitted with a heating mantle. The oil sample was collected using a Pyrex Allihn Condenser (Corning, Inc., Corning, NY, USA) and a Clevenger-type receiver trap (Product #LG-11125-102, Wilmad-LabGlass, Vineland, NJ, USA) in a 2 h process until no more oil was obtained. The hydrodistillation system was cooled with an ethylene glycol-water (1:1 v/v) solution at −3 • C. The distilled oil mixture/emulsion was allowed to settle for 15 min. Then, the oil layer was collected and centrifuged at 8500× g at room temperature for 5 min to remove heavy impurities, and to separate it from water. The oil was then dried over anhydrous sodium sulfate and stored at −20 • C until analysis.
The volatile compositions in the oil samples were analyzed using a GC-MS system as previously reported [21]. The GC-MS (6890N GC, and 5975 MS, Agilent Technologies, Santa Clara, CA, USA) equipped with a capillary column (DB-5; 60 m length, 0.25 mm diameter, and 1 µm film thickness, J&W Scientific, Folsom, CA, USA) was used [21]. One microliter of peel oil was injected in a split/splitless injector (250 • C) in the split mode (split ratio 40:1). The oven was held at 40 • C for 0.5 min then increased to 225 • C at 4 • C min −1 and held for 13.25 min. The mass spectrometry data were recorded in the scan mode at an ionization energy of 70 eV. The mass range was from 40 to 400 at 2 scans s −1 . The inlet, ionizing source, and transfer line were kept at 250, 230, and 280 • C, respectively.

Juice Extraction and GC-MS Analysis
VOCs in juice samples were analyzed by headspace solid-phase microextraction (HS-SPME) coupled with GC-MS as described previously [11]. Freshly squeezed juice, 6 mL of each juice sample, was pipetted into a 20 mL vial and crimp-capped with a Teflon/silicone septum. A triplicate of each juice sample was prepared. Sample vials were stored at −20 • C until analysis.
Frozen sample vials were thawed under tap water and loaded into the autosampler (Model MPS2; Gerstel Inc., Linthicum, MD, USA) equipped with a cooled (4 • C) tray holder (Laird Tech, Göteborg, Sweden) controlled by a Peltier thermostat (CTC Analytics AG, Zwingen, Switzerland) for headspace sampling and GC-MS analysis. The analyses were conducted as described by Bai et al. [11].

Identification and Quantification of VOCs
GC-MS data were collected using the ChemStation G1701 AA data system (Hewlett-Packard, Palo Alto, CA, USA). A mixture of C-5 to C-20 n-alkanes were run at the beginning of each day to calculate retention indices (RIs) [11]. The volatile components were identified by matching their spectra with library entries [National Institute of Standards and Technology (NIST)/Environmental Protection Agency (EPA)/National Institutes of Health (NIH) Mass Spectral Library (NIST 14; WebBook, SRD69)] and authentic volatile compound standards, as well as by comparing their RIs with corresponding literature data [27]. For the quantification of the juice samples, peak size (total ion current) was used to compare the relative abundance between samples. To quantify D-limonene, which usually makes up more than 90% of total peel oil, peel oil samples were diluted to 3% with methanol. Standard curves were used to determine the concentration of each compound.

Statistical Analyses
Statistical analysis for fruit color, size, sugar, and acid was performed with statistical software (R version 4.1.0; R Core Team, 2021, http://www.r-project.org, accessed on 6 July 2021) using the ExpDes package. The data were tested for adequacy of normal distribution and homogeneity at 5% probability and evaluated using analysis of variance followed by Tukey's test (p ≤ 0.05). The volatile compounds data were analyzed with JMP software (version 11.2.2; SAS Institute, Cary, NC, USA). Differences were tested using Tukey's honest significant difference (HSD) (p ≤ 0.05). Cluster analysis (CA) and principal component analysis (PCA) were used to characterize the chemical profile of peel oil across HLB+/HLB−.

Huanglongbing Effect on Fruit Size, Color, SSC, and TA
Healthy grapefruit trees (HLB−) cultivated under the protective screen had a healthy canopy, fruits, and leaves, and there was no ACP under the screen [22]. The qPCR results showed that all leaf samples were CLas negative with Ct values > 40. On the other hand, all HLB+ trees grown in the open air were exposed to a high ACP population, and therefore a high CLas pressure, and had a compromised canopy, very few leaves, and greener, smaller, misshapen fruits, all typical symptoms of the HLB disease. The qPCR results showed that Ct values of CLas were 27.3 ± 0.64 in the leaf samples, confirming those trees were all infected by the bacterium [22]. HLB+ fruit samples exhibited a lower chromameter a*, a*/b* ratio and higher hue angle than healthy ones, indicating a greener and less orange fruit peel color in both the 2020 and 2021 seasons. Chromameter b* values did not statistically differ between the treatments but were slightly higher for HLB+ samples, confirming a less yellow and more blue peel color in HLB+ fruit samples (Table 1 and Figure 2). In 2021, the HLB+ fruits were less green than the ones of the 2020 season, as revealed by the higher peel a*/b* ratio and lower hue angle (Table 1). Regarding fruit size, HLB+ fruits were smaller in weight, length, and diameter in 2020 and 2021 compared to HLB− ones (Table 1 and Figure 2). The lower fruit size associated with the higher preharvest drop caused by HLB results in low fruit yield [7]. HLB+ grapefruit juice had a statistically reduced SSC and SSC/TA ratio (Table 1). However, there was no significant difference in TA (Table 1). At the point of evaluation, the samples, regardless of HLB+/− and harvest season, met the minimal maturity standards for grapefruit in Florida, USA [28], with the SSC of the juice above 7.5%, the SSC/TA ratio higher than 7.0, and more than 25% of the fruit surface with yellow or red color (Table 1). Overall, HLB+ fruits were smaller and greener with a lower SSC and SSC/TA ratio (Table 1 and Figure 2). These aspects are characteristics of HLB+ fruit and have been reported by several authors, particularly for sweet oranges, such as 'Valencia' and 'Hamlin', which were the most studied cultivars for the effect of HLB on fruit quality [4,6,8,10,29]. Usually, as the maturity progresses, the peel color of a healthy orange becomes less green and more orange, and TA in the juice decreases [12]. In citrus peel, chlorophylls are more abundant in dark green fruits. Carotenoids, however, follow the opposite trend [25]. Previous research showed that grapefruits grown inside the tree canopy or bagged accumulated more lycopene and carotenoids and less chlorophyll, which were evidenced by an acceleration of chloroplasts to chromoplast transition in the peel and the relative expression of genes related to chromoplast development was enhanced by light avoidance [30]. HLB+ had a much smaller tree canopy and fewer leaves [4,22], which could be one of the reasons HLB+ fruits were greener. HLB delays fruit development; thus, TA remains at high levels for a longer time. This high TA content is often reported for the juice of HLB+ oranges [4]. However, in this research, HLB did not significantly affect juice TA content in grapefruit juice (Table 1).  Overall, HLB+ fruits were smaller and greener with a lower SSC and SSC/TA ratio (Table 1 and Figure 2). These aspects are characteristics of HLB+ fruit and have been reported by several authors, particularly for sweet oranges, such as 'Valencia' and 'Hamlin', which were the most studied cultivars for the effect of HLB on fruit quality [4,6,8,10,29]. Usually, as the maturity progresses, the peel color of a healthy orange becomes less green and more orange, and TA in the juice decreases [12]. In citrus peel, chlorophylls are more abundant in dark green fruits. Carotenoids, however, follow the opposite trend [25]. Previous research showed that grapefruits grown inside the tree canopy or bagged accumulated more lycopene and carotenoids and less chlorophyll, which were evidenced by an acceleration of chloroplasts to chromoplast transition in the peel and the relative expression of genes related to chromoplast development was enhanced by light avoidance [30]. HLB+ had a much smaller tree canopy and fewer leaves [4,22], which could be one of the reasons HLB+ fruits were greener. HLB delays fruit development; thus, TA remains at high levels for a longer time. This high TA content is often reported for the juice of HLB+ oranges [4]. However, in this research, HLB did not significantly affect juice TA content in grapefruit juice (Table 1).
Monoterpene hydrocarbons were the predominant chemical class in all samples regardless of the year and treatment, with the content being 96.68% and 96.81% in HLB+ and 97.11% and 98.20% in HLB− peel oil samples in 2020 and 2021, respectively ( Table 2). D-limonene is the main volatile in citrus fruit and is an important contributor to the citrus flavor [31]. CA and PCA analysis showed that D-limonene was associated with HLB− samples (higher concentrations in HLB− samples, Figures 3 and 4). However, there were no significant differences ( Table 2). The results agreed with reports on orange peel oils [18,21].
Monoterpene hydrocarbons were the predominant chemical class in all samples regardless of the year and treatment, with the content being 96.68% and 96.81% in HLB+ and 97.11% and 98.20% in HLB− peel oil samples in 2020 and 2021, respectively ( Table 2). D-limonene is the main volatile in citrus fruit and is an important contributor to the citrus flavor [31]. CA and PCA analysis showed that D-limonene was associated with HLB− samples (higher concentrations in HLB− samples, Figures 3 and 4). However, there were no significant differences ( Table 2). The results agreed with reports on orange peel oils [18,21].
The other abundant compounds in 'Ray Ruby' grapefruit peel oil samples were βmyrcene, α-pinene, (E)-β-caryophyllene, and decanal (Table 2). β-Myrcene and α-pinene, major monoterpenes next to D-limonene, were found in significantly higher contents in HLB− samples in 2020 and 2021 (Table 2). This differed from most reports that showed HLB did not significantly affect monoterpene content in orange peel oils [18,21]. These volatiles have been found in high concentrations in citrus peel oils and have a large contribution to the peel oil aroma [32]. β-Myrcene is reported to have a green and mossy odor and is a much stronger odorant than D-limonene [33]. β-Myrcene is a versatile terpene used in different applications, mainly in the perfume and fragrance industries [34]. α-Pinene presents a pine tree resin aroma that can make a positive background contribution to the aroma of the sweet orange [33].  .10% z Calculated RI: retention index calculated from the retention time of each volatile and n-alkanes on the DB-5 column. y Reference RI: RI based on NIST GC RI, 5% equivalence nonpolar column (such as DB-5, BP-5, and HP-5). x HLB+ caused positive or negative changes. w composition less than 0.0005%. v "-" not available. u "++" compound induced by HLB+. * Significant difference at p ≤ 0.05 according to Tukey's HSD test.     Table 2.

Scores plot (A)
) harvests. VOCs corresponding to the numbers are listed in Table 2.
Sesquiterpene hydrocarbons were the second largest class in the peel oil, and the major VOCs in the class were (E)-β-caryophyllene, δ-cadinene, α-copaene, β-cubebene, and γ-muurolene, which were significantly increased by HLB affection in both years (Table 2). Similar observations were reported by Xu et al. [18] and Zhang et al. [35] in orange peel oil; stress from the bacterium preventing nutrient flow to the fruit may cause shifts in the biochemical pathways, creating a higher accumulation of those volatiles in the peel oil [18]. However, Sun et al. [21] reported that HLB decreased sesquiterpenes in orange peel oil.
In the 2020 harvest, HLB significantly increased the total aldehydes content in peel oil. However, there was no difference in the 2021 season ( Table 2). The individual analyses of the aldehydes revealed that citronellal, perilla aldehyde, β-sinensal, octanal, geranial, and neral had higher content in HLB+ samples in 2020 or both seasons, while undecanal had lower content (Table 2). Xu et al. [18] also found high citronellal and perilla aldehyde in oils of HLB symptomatic oranges.
Nootkatone content was consistently higher in HLB− peel oil samples, 7.9-and 2.7-fold higher in 2020 and 2021, respectively ( Table 2). Nootkatone is the most important and expensive aromatic compound in grapefruit [15]. In combination with other carbonylcontaining constituents, nootkatone is responsible for the flavor of good-quality grapefruit oil [36]. The concentration of nootkatone is used as the criterion for oil quality [15]. The accumulation of nootkatone in the peel is associated with the processes of maturation and senescence of the fruits [37]. It has been suggested that HLB+ fruits have a slow maturation due to a compromised vascular system, similar to immature fruits [9]. This can explain why the sesquiterpene ketone in HLB+ peel oil was lower than in healthy fruit peel oil. Nootkatone has been isolated from grapefruit peel and juice and can also be obtained from the oxidation of valencene [34,35]. Naturally produced nootkatone has a strong grapefruit odor, while the synthetic form shows a weak woody and spicy flavor, not typical of grapefruit flavor [34]. Grapefruit peel is the main source of nootkatone for the industry, and due to its limited production, it has a very high price [36]. Thus, a lower concentration of this sesquiterpene in the peel oil of HLB+ fruit has a major economic impact.
Previous studies reported that lower concentrations of linalool were found in orange peel oils derived from severely HLB+ fruit [18]. However, in the present study, HLB infection increased the accumulation of this compound in grapefruit peel oil in both seasons ( Table 2). This terpene alcohol is an oxygenated product of D-limonene, but unlike limonene, linalool accounts for characteristic citrus and floral odors, an important aroma for citrus [38]. Because of its flavorful and fragrant properties, linalool is used in the food, perfume, and cosmetics industries [39]. Besides that, linalool also exhibits strong antibacterial activity against bacterial strains [26].
A CA divided the compounds into four groups: group I with 11 compounds decreased due to HLB, including nootkatone and two abundant monoterpenes, β-myrcene and αpinene ( Figure 3); group II where 13 compounds were decreased by HLB in 2020 but increased by HLB in 2021; group III with 16 compounds increased by HLB in 2020 but decreased in 2021; and group IV with 35 compounds increased by HLB, including most sesquiterpene hydrocarbons, terpene aldehydes, terpene oxidations, and dehydrogenations ( Figure 3). More than 24 compounds were increased by HLB (35 increased-11 decreased, Figure 3). Some of the differences through the years in groups II and III may be due to climatic conditions, as the 2021 season was consistently rainy in the months preceding harvest (Figure 1).
PCA was performed to project the volatile components onto a two-component plot ( Figure 4). The PCA discriminated HLB+ from HLB− samples mainly on Component 2, explaining 29.5% of the variation ( Figure 4A,B). Volatiles from HLB+ samples were generally on the positive side of PC2, and HLB− samples were on the negative side of PC2 ( Figure 4A,B). The loading plot ( Figure 4B) in PCA confirmed that more compounds were associated with HLB+ fruits (Figure 4). The PCA discriminated the 2021 season samples from the 2020 season samples mainly on PC1, explaining 36.4% of the variation ( Figure 4A,B). The 2020 season had substantially low rainfall (Figure 1), which may have caused more stress, thus producing more oxidative and dehydrogenated terpenes (Table 2 and Figure 4).

Huanglongbing Effects on the Volatile Profile of 'Ray Ruby' Grapefruit Juice
A total of 87 volatiles were detected in HLB+ and HLB− 'Ray Ruby' grapefruit juice samples over two harvest seasons, and the components were classified into 12 chemical classes (Table 3 and Figure S2). In comparison to peel oil, the VOCs in the juice had a second major compound next to D-limonene, β-caryophyllene, a sesquiterpene hydrocarbon, and contained 12 more VOCs (a 16% increase), which were mostly low molecular weight aliphatic compounds (Tables 2 and 3).
The most abundant chemical class was monoterpene hydrocarbons with 13 components representing 47% to 55% of the total volatiles ( Table 3). The major monoterpene hydrocarbons were D-limonene followed by β-myrcene (Table 3). Unlike in the peel oil, they both increased in the juice of HLB+ fruit, as well as many other monoterpene compounds ( Figure 5), although the differences were not significant. β-myrcene has a strong green and metallic odor, which may negatively affect the aroma and flavor characteristics of citrus and orange juices when present in high amounts [11,40].
While sesquiterpene hydrocarbons constituted a small portion of the peel oil, 0.89-1.39% of total volatiles (Table 2), they were abundant in the juice, with relative abundances from 34 to 36% in 2020 and 31% in 2021 (Table 3). β-Caryophyllene has a woody-spicy, terpenic, sweet, and citrus flavor and is one of the key volatile components in grapefruit juice [41]. D-limonene and β-caryophyllene, the two major compounds in 'Ray Ruby' grapefruit juice volatiles, together accounted for nearly 70% of the total volatiles (Table 3). Both compounds decreased in HLB+ fruit in both harvest seasons ( Figure 5). Limonene and β-caryophyllene are reported to decrease in juice as the fruit matures [42]. This could be the explanation for the higher concentration of these volatiles in HLB+ juice, considering the slow maturation of HLB+ fruits, which are usually similar to immature fruit [9].
These terpene hydrocarbons have chemo-preventative functions, such as induction of glutathione-S-transferase activity and uridine diphosphoglucuronosyl transferase activity in the small intestine and liver [42,43]. D-limonene also has chemotherapeutic activity against pancreatic, mammary, and prostatic tumors [44] and helps to prevent the initiation and suppress the progression of mammary and liver cancer [45].
C8-C10 aldehydes were significantly lower in HLB+ fruit juice than in HLB− juice (Table 3, Figures 5 and 6). These long-chain aldehydes and nootkatone are critical for high-quality grapefruit juice [36,42]. The results confirmed that HLB negatively affected the juice flavor quality (Table 3, Figures 5 and 6).
The CA divided the compounds into four groups: group I with 48 out of 87 compounds decreased by HLB; group II with 10 compounds decreased by HLB in 2020 but increased by HLB in 2021; group III composed of 8 compounds increased by HLB in 2020 but decreased in 2021; and group IV with 21 compounds increased by HLB, including acetaldehydes, ethanol, and ethyl acetate ( Figure 5). Contrary to peel oil, in which more VOCs were increased by HLB, more VOCs were decreased in HLB+ juice (48 decreases-21 increases, Figure 5).       Table 3. C8-C10 aldehydes were significantly lower in HLB+ fruit juice than in HLB− juic (Table 3, Figures 5 and 6). These long-chain aldehydes and nootkatone are critical for high quality grapefruit juice [36,42]. The results confirmed that HLB negatively affected th juice flavor quality (Table 3, Figures 5 and 6).
The CA divided the compounds into four groups: group I with 48 out of 87 com pounds decreased by HLB; group II with 10 compounds decreased by HLB in 2020 bu increased by HLB in 2021; group III composed of 8 compounds increased by HLB in 202 but decreased in 2021; and group IV with 21 compounds increased by HLB, includin   Table 2.

Scores plot (A)
) harvests. VOCs corresponding to the numbers are listed in Table 3. HLB resulted in a significant reduction in aliphatic aldehydes and terpene ketones in the juice and an increase in aliphatic alcohols, aliphatic esters, and monoterpene hydrocarbons (Table 3, Figure 5). Long-chain aldehydes are known to contribute a lot to citrus juice aroma, with citrus-like, grassy, and soapy odors [33,[46][47][48]. In grapefruit, ketones also strongly contribute to the flavor, mainly due to nootkatone, the main fragrant component of grapefruit [42,48]. On the other hand, ethanol is the aliphatic alcohol derived from the fermentation process, which is responsible for a fermented flavor in fresh fruit and juice [12,46]. The decrease of C8-C10 aldehydes and increased ethanol and esters could result in an "off-flavor" in the juice [46]. Valencene is considered the precursor of nootkatone, and the levels of both compounds have been reported to increase as the fruit matures [42,49]. The fact HLB decreased nootkatone in the peel oil and juice confirmed that HLB slowed the maturation of fruits, similar to immature orange fruits [9].
In Valencia oranges, valencene accumulates throughout fruit maturation, but in grapefruit, it is further oxidized to nootkatone [50]. On the other hand, we did not identify the presence of valencene in the peel oil ( Table 2).
Consistent with our findings, previous studies also found that β-myrcene, D-limonene, and total terpenes had higher concentrations in HLB symptomatic Hamlin juice than healthy ones [8,9]. Previous studies also showed decreased levels of aldehyde compounds in Hamlin orange juice of HLB symptomatic fruits compared with the juice of healthy fruits [8,48].
PCA was performed to project the volatile juice components on a two-component plot ( Figure 6). The PCA discriminated HLB+ from HLB− samples on Component 2, explaining 24.9% of the variation ( Figure 6). Most juice volatiles were associated with HLB− samples, located on the positive side of PC2, while the VOCs compounds of HLB+ samples were on the negative side of PC2 ( Figure 6).
HLB caused increases in sesquiterpenes and decreases in valencene and nootkatone in both peel oil and juice samples (Figure 7). Valencene is a major sesquiterpene in oranges and some mandarins, but it was not detectable in other mandarins [12,21,49,51]. Valencene existed in grapefruit juice samples, although it was not the major sesquiterpene (Table 3 and Figure 7). However, valencene was not detected in the peel oil samples, although there were other sesquiterpenes in the samples (Table 2 and Figure 7). Sharon-Asa et al. [52] reported that Cstps1 encodes valencene synthase, and Yu et al. [51] confirmed that the gene expression level of Cstps1 and the abundance of valencene were closely correlated. The opposite responses to HLB between total sesquiterpenes and valencene in juice samples confirm the above observation again, suggesting a possible mechanism that valencene biosynthesis may be independent of other sesquiterpenes (Figure 7).
The enzymatic steps from valencene to nootkatone in planta remain unclear [50,53]. However, in recent years, the integration of heterologous metabolic pathways indicates that regioselective oxidation of valencene at C-2 to β-nootkatol through cytochrome P450 was coupled with P450 reductase (CPR) and dehydrogenase-catalyzed oxidation of β-nootkatol to nootkatone [53,54]. Our data indicate that in peel tissue, nootkatone could be converted from nonvalencene sesquiterpenes, or that valencene is not accumulated in peel oil but is directly converted to nootkatone (Table 2 and Figure 7).
Monoterpenes are the major components in peel oil (about 97-98%) and juice (about 50%) samples (Tables 2 and 3). HLB caused an increase of monoterpenes in peel oil samples but a decrease in juice samples (Figure 7, Tables 2 and 3). Monoterpenes are the major components in peel oil (about 97-98%) and juice (about 50%) samples (Tables 2 and 3). HLB caused an increase of monoterpenes in peel oil samples but a decrease in juice samples (Figure 7, Tables 2 and 3).
We focused on how HLB affects the volatile profiles in peel oil and juice. However, more complicated interactions mediated by volatiles exist among plants (including fruit), pathogens (including CLas), and insects (including the vector of CLas, ACP) [56][57][58][59]. Nootkatone, valencene, and many VOCs in citrus are insect repellents and/or antimicrobial agents [53,60,61]. The volatile profiling method has been used to compare the HLB tolerance of citrus cultivars [35,58,62]. Hijaz et al. [57] observed that the responses of the volatile profile in the citrus tree to ACP were more significant than to CLas infection. A better knowledge of the interactive effects of plant hosts and the insects and microbials on the volatile profile in the fruit may also help us better understand and estimate the tolerant plant selection, insect and disease control, and fruit quality control [56,59,63].

Conclusions
HLB decreased grapefruit quality, with smaller and greener fruit and a lower SSC and SSC/TA ratio. However, the responses of VOCs to HLB varied depending on the source (juice or peel oil) and harvest season. Nootkatone, the most important volatile component for grapefruit flavor, significantly decreased in juice and peel oil regardless of the harvest year. HLB disease resulted in a decrease of abundant monoterpenes, including Dlimonene, β-myrcene, and α-pinene in the peel oil; opposite trends were found in the juice samples. On the other hand, the oxidative/dehydrogenated terpenes were increased by HLB in peel oil but decreased in the juice sample. The long chain aldehydes, decanal, nonanal, and octanal also increased in HLB peel oil but decreased in the juice, resulting in likely lower flavor quality in the juice. Our results indicated that the volatile profiles of peel oil and juice had different responses to HLB than those in sweet orange fruit. This Figure 7. Summary of metabolic pathways leading to monoterpenes, sesquiterpenes, and nootkatones [49,52,53,55]. Bar graphs show changes (%) caused by HLB affection in peel oil and juice in 2020 and 2021. MEP, methylerythritol phosphate pathway; MVA, mevalonic acid pathway; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; Cstps1, valencene synthase.
We focused on how HLB affects the volatile profiles in peel oil and juice. However, more complicated interactions mediated by volatiles exist among plants (including fruit), pathogens (including CLas), and insects (including the vector of CLas, ACP) [56][57][58][59]. Nootkatone, valencene, and many VOCs in citrus are insect repellents and/or antimicrobial agents [53,60,61]. The volatile profiling method has been used to compare the HLB tolerance of citrus cultivars [35,58,62]. Hijaz et al. [57] observed that the responses of the volatile profile in the citrus tree to ACP were more significant than to CLas infection. A better knowledge of the interactive effects of plant hosts and the insects and microbials on the volatile profile in the fruit may also help us better understand and estimate the tolerant plant selection, insect and disease control, and fruit quality control [56,59,63].

Conclusions
HLB decreased grapefruit quality, with smaller and greener fruit and a lower SSC and SSC/TA ratio. However, the responses of VOCs to HLB varied depending on the source (juice or peel oil) and harvest season. Nootkatone, the most important volatile component for grapefruit flavor, significantly decreased in juice and peel oil regardless of the harvest year. HLB disease resulted in a decrease of abundant monoterpenes, including D-limonene, β-myrcene, and α-pinene in the peel oil; opposite trends were found in the juice samples. On the other hand, the oxidative/dehydrogenated terpenes were increased by HLB in peel oil but decreased in the juice sample. The long chain aldehydes, decanal, nonanal, and octanal also increased in HLB peel oil but decreased in the juice, resulting in likely lower flavor quality in the juice. Our results indicated that the volatile profiles of peel oil and juice had different responses to HLB than those in sweet orange fruit. This may affect the decision of fruit processors to favor processing for peel oil or juice depending on the fruit source received at the processing plant.