# A Comparison of the Volatile Components of Cold Pressed Hamlin and Valencia (Citrus sinensis (L.) Osbeck) Orange Oils Affected by Huanglongbing

Authors: Brittany M. Xu; George L. Baker; Paul J. Sarnoski; Renée M. Goodrich-Schneider
Journal: Journal of Food Quality
 (2017)

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## Abstract

Volatiles from huanglongbing (HLB) symptomatic and asymptomatic cold pressed orange oils from Florida Hamlin and Valencia fruit were assessed. Qualitative gas-liquid chromatography studies showed the presence of several compounds (β-longifolene, perillene, and 4-decenal) which are not commonly identified inCitrus sinensis (L.) Osbeck oils. Oils derived from huanglongbing symptomatic fruit had lower concentrations of linalool, decanal, citronellol, neral, geranial, carvone, dodecanal, and 2-decenal and higher concentrations of citronellal compared to asymptomatic fruit. A comparison to historic literature of orange oil investigations before HLB was of issue in Florida orange crops showed lower levels of linalool, decanal, neral, and geranial in Hamlin peel oil samples, as well as higher levels of dodecanal. Valencia peel oil samples showed lower concentrations of linalool and increased concentration of citronellol and dodecanal. As a result of huanglongbing (HLB) phenomena, the concentrations of several important volatiles found in Hamlin and Valencia peel oil profiles have changed compared to historic values. Differences in volatile concentrations of symptomatic and asymptomatic HLB affected peel oil compounds in orange fruit are identified.


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## Body

## 1. Introduction

Huanglongbing (HLB), also known as citrus greening disease, is a bacterial citrus disease. The causative bacterial species in the state of Florida isCandidatus Liberibacter asiaticus [1], which is vectored by the Asian citrus psyllidDiaphorina citri Kuwayama [2]. To date, an infected tree cannot be cured of HLB [1]. There are several noticeable anatomical and physiological changes which occur before orange fruit productivity drops, including root system decay, changes in the fruit, and asymmetrical leaf chlorosis, also known as yellow blotchy mottle [2]. Fruit which are symptomatic for HLB are somewhat green in color, small, and misshapen and contain aborted seeds [2]. HLB is an orange tree disease considered devastating to the citrus industry in Florida and other areas of the world, given the widespread infection of groves in a relatively short period of time, resulting in orange farmers to abandon or destroy their crop, year over year. Between the years of 2006 and 2011, over 6660 jobs and more than $3.63 billion in revenue were lost in Florida due to HLB [3].Cold pressed orange peel oil is a valuable commodity to the global beverage industry derived from a byproduct of orange juice production which may otherwise become animal feed or waste [4]. Wide variations in oil yield from Florida oranges have been found but are generally in the range of 0.11–0.58% of the total orange weight [5]. While commercial quantities of oil are present in oranges before the fruit is ready to harvest, oil content reaches a maximum when the fruit is fully mature [6]. The most common instrument for extracting cold pressed oil from oranges is the JBT (formerly FMC) inline extraction system [7]. With this equipment, the oil is extracted simultaneously with the juice from the orange peel. Orange oil is washed away from the orange peel with an excess of water to create a crude emulsion, which can be further processed into pure cold pressed oil 24–48 hours after extraction [8]. Cold pressed orange oil is used in beverages, including juices and juice concentrates. Orange oil is frequently added back to the concentrated product it is derived from or is added to other citrus juices [8]. Cold pressed orange oil gives juice sweet, fruity, and green top notes and is usually added to orange juice concentrate at a volume-to-volume ratio of 0.01% [8]. Cold pressed orange oil can also be used in desserts, soft drinks, chewing gum, and confections [7].While no previous studies have investigated the effect of HLB on cold pressed orange oil, several have studied the effects of HLB on the chemical constituents of orange juice [9, 10]. Dagulo et al. investigated the effect of HLB on orange juices made from HLB unaffected (control), asymptomatic, and symptomatic Valencia fruit [9]. Their results showed that juice produced from HLB symptomatic fruit has fewer overall esters, aldehydes, and total sesquiterpenes than juice extracted from control fruit. Juice produced from HLB symptomatic fruit in their studies contained increased levels of alcohols and terpenes. Overall, the chemical changes observed for juice produced from HLB symptomatic fruit were comparable to the chemical characteristics of juice produced from immature fruit and have a less favorable aroma profile compared to HLB unaffected fruit [9]. Moreover, they showed that orange juice produced from HLB asymptomatic fruit did not show consistent differences from control fruit [9].Baldwin et al. studied the effects of HLB on juices produced from symptomatic and control (unaffected) Valencia, Hamlin, and Midsweet oranges [10]. This study was conducted over the 2007 and 2008 harvest seasons, with multiple harvests per season. Their results showed that, during the 2007 season, hexanol levels were higher in juice derived from symptomatic Hamlin fruit than in juice produced from control orange fruit. Ethanol concentrations were higher in juice produced from HLB symptomatic Midsweet fruit, while hexanal, cis-3-hexenol, and linalool concentrations were higher in juice produced from control Midsweet fruit. Valencia juice did not show any variation in volatiles by disease status but did show differences between harvest times. During the 2008 season, no differences were seen between orange juices from Midsweet fruit. Hamlin showed higher levels of acetaldehyde, octanal, ethanol, cis-3-hexenol, and sabinene in control juice and higher levels of β-myrcene and ethyl hexanoate in juice produced from symptomatic fruit. Juice derived from symptomatic Valencia oranges showed higher levels of ethanol and sabinene and juice from control fruit showed higher levels of hexanol and ethyl acetate [10]. Overall, these data show that there can be season to season and cultivar differences in how HLB affects volatile concentrations in orange juices.The objective of this study was to investigate the volatile components of cold pressed oil from HLB symptomatic and asymptomatic oranges. No previous studies have been published which investigate these effects.


## 2. Materials and Methods

### 2.1. Harvesting and Orange Trees

Florida grown Hamlin and Valencia oranges were each harvested twice per year. Fruit were harvested from established groves at the University of Florida Citrus Research and Education Center in Lake Alfred, Florida. Fruit were harvested both early and late in the respective variety’s season for both Valencia and Hamlin oranges. The purpose of these different harvests was to test the effect HLB has on multiple cultivars of oranges at multiple points in the season. Disease stage and maturity of fruit were assessed by a trained horticulturist using established protocols [14]. Oranges were harvested by hand for the 2015-2016 season. Hamlin oranges were harvested on November 15, 2015, and January 14, 2016. Valencia oranges were harvested on February 11, 2016, and March 31, 2016.Approximately 30 trees per variety were harvested. The trees harvested were in adjacent rows (n=2–5). Approximately 300 kilograms each of asymptomatic (AS) and symptomatic (SY) oranges were collected during each harvest. SY fruit were noted to be mature but with characteristic outward greening symptoms including small fruit with reversed ripening where the stylar end remains green. While some trees contained both AS and SY fruit, many trees contained nearly 100% SY fruit. These trees commonly displayed overall sparse foliage on the tree and yellow blotchy mottle on the leaves. AS fruit appeared outwardly healthy, were overall larger, and did not display the reversed ripening common with HLB SY fruit. Significant quantities of HLB unaffected fruit can no longer be found in the state of Florida, as HLB infection rate at the grove level is near 100% [15]. Thus, only HLB SY and AS fruit were utilized for this study.


### 2.2. Cold Pressed Oil Extraction

Fruit were stored in a cold room at 4°C after harvest until processing. Processing of the raw oranges into cold pressed orange oil was performed in the Pilot Plant at the Citrus Research and Education Center in Lake Alfred, Florida, according to typical industry practices [7]. Before processing, fruit were sanitized with Fruit Cleaner 395 (JBT FoodTech, Lakeland, FL) and surface-dried. Juice and an oil emulsion were extracted simultaneously with a commercial John Bean Technologies (JBT) extractor (JBT FoodTech, Lakeland, FL). The main characteristic of this extractor is that the juice and oil are extracted simultaneously. The extractor was cleaned before and after use. The oil emulsion was collected in 190 liter tanks and put into cold storage at 4°C for approximately 40 hours. The crude emulsion was brought to room temperature over the course of 3 hours before being refined into cold pressed orange oil.A DeLaval table top centrifuge (DeLaval, Tumba, Sweden) specifically for oil was used to produce cold pressed orange oil. The centrifuge is equipped with a Dayton motor (Dayton Motor Company, Dayton, Texas) which operates at 1000 ×g. Raw material from the topmost oily portion of the emulsion was very slowly fed into the centrifuge by hand. Due to the large quantity of emulsion (~110 liters) produced, only the top part of the emulsion which contains the most oil was fed through the centrifuge. Oil was aliquoted into vials and stored at −20°C until use.


### 2.3. Gas Chromatography Conditions

Qualitative and quantitative gas chromatography was performed. In the qualitative experiment, cold pressed orange oils (CPOO) were analyzed by gas chromatography mass spectrometry and gas chromatography with a flame ionization detector (multidimensional GC/GC-MS (MDGC) by initial flame ionization detection (FID) and then “heart-cut” to an additional oven and detection by mass spectrometry (MS)) (Shimadzu GC-2010 Plus Gas Chromatograph, Shimadzu QP-2010 SE Mass Spectrophotometer).CPOO was diluted 1 : 5 with hexanes (Fisher HPLC grade), and approximately 1 mL of mixture was transferred to a brown glass sampling vial. An autoinjector (Shimadzu AOC-20i) was used to inject 2μL of sample mixture into the first GC and analyzed by FID. In the multidimensional gas chromatograph method, two ovens, an FID (Shimadzu Columbia, MD), a triple-quadrupole mass spectrometer, and a Dean’s switch (Shimadzu Columbia, MD) are utilized. In a typical MDGC setup, after initial detection by the FID, compounds can be “cut” by operational software using Dean’s switch to a second GC oven (with separate column and temperature programming) offering additional separation and detection by MS. Often, the purpose of MDGC is to separate insufficiently separated components. In this experiment, FID detection from the first GC was used to quantify all compounds in a first run and identification by MS in a second run cut via Dean’s switching to the second GC from beginning to end. GC/GC-MS conditions are listed below.The temperature program on the first GC was 50–250°C at a rate of 3°C/minute. The column used was an RTX-5 (Restek) column 30 meters in length, 0.25 mm internal diameter, and 0.25μm film thickness. Inlet temperature was 280°C. The column flow rate was 0.71 mL/minute. The inlet pressure was 190.9 kPa. The FID sampling rate was 40 milliseconds; FID inlet temperature was 300°C. The compounds were then cut to the second GC, which also had an RTX-5 column with the similar operating conditions as the first GC. The pressure in the second GC was 160.0 kPA, and the temperature program was the same as for the first GC. The column flow rate was 0.7 mL/min and the linear velocity was 23.7 cm/min. For the MS, scan mode was used. The ionization energy was 70 eV and the acquisition mass range was m/z 40–400. MS ionization was autotuned against perfluorotributylamine (PFTBA) for quality control based on Shimadzu engineering recommendations for maximum efficiency.Qualitative compound identification by MS was performed by matching compound MS output to database entries using similarity searching of MS fragmentation patterns against multiple compound libraries. The libraries used were the National Institute of Science and Technology (Gaithersburg, MD) Mass Spectrometry Spectra Database versions 11, 11s, 14, 14s, and Flavor and Fragrance Natural and Synthetic Compounds GC/MS library from Shimadzu (Columbia, MD). Compounds of interest were considered if it had a similarity match of at least 80%. However, the top match of similarity searches from “heart-cut” FID responses sent to the other RTX-5 column equivalent and GC oven by multidimensional switching were matched given delayed retention time calculations to account for flow rate through 30 meters of RTX-5 equivalent column.Each CPOO was analyzed in triplicate. Kovats Indices (KI) were calculated as a confirmation of identified compounds. Alkane standard measurement ranging from C6 to C35 was utilized for this purpose and detected by FID (SPEXOrganics, Metuchen, NJ). Six concentrations (4 to 66,700 ppm) were used to create a standard curve from the first GC by FID. Identified compounds were considered identified by LRI if the observed value was within 15 points of a value compared to reputable databases or literature [11–13].For each compound identified, the average total peak areas between SY and AS samples were compared. If the percent difference was ≥20%, this compound was flagged and considered for quantitative analysis. The following equation for percent difference was utilized:[(AS-SY)/(AS)]∗100, where AS is asymptomatic average total peak area and SY is symptomatic average total peak area.Using the criteria above, fifteen compounds were ultimately chosen for quantification. These compounds were purchased as analytical standards in the highest concentration available. Dodecanal, 2-decenal, citronellal, citronellyl acetate,α-terpinyl acetate, α-pinene, citral, perillaldehyde, decanal, linalool, and citronellol were purchased from Sigma Aldrich (St. Louis, MO). β-Myrcene and carvone were purchased from Acros Organics (Waltham, MA). (E,E)-2,4-Decadienal was purchased from Tokyo Chemical Industry (Tokyo, Japan). β-Myrcene was only available at a concentration of ~90%, so a correction factor was taken into account when calculating the concentration of this compound. Standard curves were used to determine concentration in parts per million (ppm).


### 2.4. Statistical Analysis

Student’st-tests were performed to determine if concentration of AS samples differed from SY samples based on disease stage or harvest time (early or late). Significance was set at α ≤ 0.01.


## 2.1. Harvesting and Orange Trees

Florida grown Hamlin and Valencia oranges were each harvested twice per year. Fruit were harvested from established groves at the University of Florida Citrus Research and Education Center in Lake Alfred, Florida. Fruit were harvested both early and late in the respective variety’s season for both Valencia and Hamlin oranges. The purpose of these different harvests was to test the effect HLB has on multiple cultivars of oranges at multiple points in the season. Disease stage and maturity of fruit were assessed by a trained horticulturist using established protocols [14]. Oranges were harvested by hand for the 2015-2016 season. Hamlin oranges were harvested on November 15, 2015, and January 14, 2016. Valencia oranges were harvested on February 11, 2016, and March 31, 2016.Approximately 30 trees per variety were harvested. The trees harvested were in adjacent rows (n=2–5). Approximately 300 kilograms each of asymptomatic (AS) and symptomatic (SY) oranges were collected during each harvest. SY fruit were noted to be mature but with characteristic outward greening symptoms including small fruit with reversed ripening where the stylar end remains green. While some trees contained both AS and SY fruit, many trees contained nearly 100% SY fruit. These trees commonly displayed overall sparse foliage on the tree and yellow blotchy mottle on the leaves. AS fruit appeared outwardly healthy, were overall larger, and did not display the reversed ripening common with HLB SY fruit. Significant quantities of HLB unaffected fruit can no longer be found in the state of Florida, as HLB infection rate at the grove level is near 100% [15]. Thus, only HLB SY and AS fruit were utilized for this study.


## 2.2. Cold Pressed Oil Extraction

Fruit were stored in a cold room at 4°C after harvest until processing. Processing of the raw oranges into cold pressed orange oil was performed in the Pilot Plant at the Citrus Research and Education Center in Lake Alfred, Florida, according to typical industry practices [7]. Before processing, fruit were sanitized with Fruit Cleaner 395 (JBT FoodTech, Lakeland, FL) and surface-dried. Juice and an oil emulsion were extracted simultaneously with a commercial John Bean Technologies (JBT) extractor (JBT FoodTech, Lakeland, FL). The main characteristic of this extractor is that the juice and oil are extracted simultaneously. The extractor was cleaned before and after use. The oil emulsion was collected in 190 liter tanks and put into cold storage at 4°C for approximately 40 hours. The crude emulsion was brought to room temperature over the course of 3 hours before being refined into cold pressed orange oil.A DeLaval table top centrifuge (DeLaval, Tumba, Sweden) specifically for oil was used to produce cold pressed orange oil. The centrifuge is equipped with a Dayton motor (Dayton Motor Company, Dayton, Texas) which operates at 1000 ×g. Raw material from the topmost oily portion of the emulsion was very slowly fed into the centrifuge by hand. Due to the large quantity of emulsion (~110 liters) produced, only the top part of the emulsion which contains the most oil was fed through the centrifuge. Oil was aliquoted into vials and stored at −20°C until use.


## 2.3. Gas Chromatography Conditions

Qualitative and quantitative gas chromatography was performed. In the qualitative experiment, cold pressed orange oils (CPOO) were analyzed by gas chromatography mass spectrometry and gas chromatography with a flame ionization detector (multidimensional GC/GC-MS (MDGC) by initial flame ionization detection (FID) and then “heart-cut” to an additional oven and detection by mass spectrometry (MS)) (Shimadzu GC-2010 Plus Gas Chromatograph, Shimadzu QP-2010 SE Mass Spectrophotometer).CPOO was diluted 1 : 5 with hexanes (Fisher HPLC grade), and approximately 1 mL of mixture was transferred to a brown glass sampling vial. An autoinjector (Shimadzu AOC-20i) was used to inject 2μL of sample mixture into the first GC and analyzed by FID. In the multidimensional gas chromatograph method, two ovens, an FID (Shimadzu Columbia, MD), a triple-quadrupole mass spectrometer, and a Dean’s switch (Shimadzu Columbia, MD) are utilized. In a typical MDGC setup, after initial detection by the FID, compounds can be “cut” by operational software using Dean’s switch to a second GC oven (with separate column and temperature programming) offering additional separation and detection by MS. Often, the purpose of MDGC is to separate insufficiently separated components. In this experiment, FID detection from the first GC was used to quantify all compounds in a first run and identification by MS in a second run cut via Dean’s switching to the second GC from beginning to end. GC/GC-MS conditions are listed below.The temperature program on the first GC was 50–250°C at a rate of 3°C/minute. The column used was an RTX-5 (Restek) column 30 meters in length, 0.25 mm internal diameter, and 0.25μm film thickness. Inlet temperature was 280°C. The column flow rate was 0.71 mL/minute. The inlet pressure was 190.9 kPa. The FID sampling rate was 40 milliseconds; FID inlet temperature was 300°C. The compounds were then cut to the second GC, which also had an RTX-5 column with the similar operating conditions as the first GC. The pressure in the second GC was 160.0 kPA, and the temperature program was the same as for the first GC. The column flow rate was 0.7 mL/min and the linear velocity was 23.7 cm/min. For the MS, scan mode was used. The ionization energy was 70 eV and the acquisition mass range was m/z 40–400. MS ionization was autotuned against perfluorotributylamine (PFTBA) for quality control based on Shimadzu engineering recommendations for maximum efficiency.Qualitative compound identification by MS was performed by matching compound MS output to database entries using similarity searching of MS fragmentation patterns against multiple compound libraries. The libraries used were the National Institute of Science and Technology (Gaithersburg, MD) Mass Spectrometry Spectra Database versions 11, 11s, 14, 14s, and Flavor and Fragrance Natural and Synthetic Compounds GC/MS library from Shimadzu (Columbia, MD). Compounds of interest were considered if it had a similarity match of at least 80%. However, the top match of similarity searches from “heart-cut” FID responses sent to the other RTX-5 column equivalent and GC oven by multidimensional switching were matched given delayed retention time calculations to account for flow rate through 30 meters of RTX-5 equivalent column.Each CPOO was analyzed in triplicate. Kovats Indices (KI) were calculated as a confirmation of identified compounds. Alkane standard measurement ranging from C6 to C35 was utilized for this purpose and detected by FID (SPEXOrganics, Metuchen, NJ). Six concentrations (4 to 66,700 ppm) were used to create a standard curve from the first GC by FID. Identified compounds were considered identified by LRI if the observed value was within 15 points of a value compared to reputable databases or literature [11–13].For each compound identified, the average total peak areas between SY and AS samples were compared. If the percent difference was ≥20%, this compound was flagged and considered for quantitative analysis. The following equation for percent difference was utilized:[(AS-SY)/(AS)]∗100, where AS is asymptomatic average total peak area and SY is symptomatic average total peak area.Using the criteria above, fifteen compounds were ultimately chosen for quantification. These compounds were purchased as analytical standards in the highest concentration available. Dodecanal, 2-decenal, citronellal, citronellyl acetate,α-terpinyl acetate, α-pinene, citral, perillaldehyde, decanal, linalool, and citronellol were purchased from Sigma Aldrich (St. Louis, MO). β-Myrcene and carvone were purchased from Acros Organics (Waltham, MA). (E,E)-2,4-Decadienal was purchased from Tokyo Chemical Industry (Tokyo, Japan). β-Myrcene was only available at a concentration of ~90%, so a correction factor was taken into account when calculating the concentration of this compound. Standard curves were used to determine concentration in parts per million (ppm).


## 2.4. Statistical Analysis

Student’st-tests were performed to determine if concentration of AS samples differed from SY samples based on disease stage or harvest time (early or late). Significance was set at α ≤ 0.01.


## 3. Results and Discussion

### 3.1. Qualitative Gas Chromatography

CPOOs from the 2015-2016 harvest year were evaluated by GC/GC-MS to determine volatile composition. CPOO from Early Hamlin, Late Hamlin, Early Valencia, and Late Valencia oranges were analyzed, and presumptive compound identification was performed by mass spectral match to several flavor and fragrance libraries, according to Shimadzu MDGC specifications by FID screening and identification by MS as previously described.Forty-four compounds were identified in Early Hamlin and 46 compounds were identified in Late Hamlin by initial FID detection and cut compounds in a second analysis of the gas-liquid source through a second oven and RTX-5 equivalent capillary GC column for MS identification (Table1).Table 1
Hamlin oils linear retention indices and percent peak areas.
Compound
LRI (observed)
LRI (literature)
EarlyASa  % peak area
Relative standard deviation
EarlySYb  % peak area
Relative standard deviation
Late AS % peak area
Relative standard deviation
Late SY % peak area
Relative standard deviation
Identifiedbyc
α-Thujene
922
928 [11]
Trace
7.14
Trace
2.73
Trace
7.85
Trace
6.54
MS, RI
α-Pinene
943
939 [11]
0.49
2.63
0.49
2.67
0.50
0.31
0.50
1.22
MS, ST, RI
Sabinene
975
972 [11]
0.14
5.21
0.17
3.24
0.14
4.52
0.17
5.20
MS, RI
β-Myrcene
995
992 [11]
1.42
3.26
1.43
3.17
1.38
0.67
1.42
0.71
MS, ST, RI
Octanal
1004
1006 [12]
0.69
2.18
0.62
2.66
0.67
2.65
0.62
2.75
MS, RI
δ-3-Carene
1020
1011 [12]
0.02
3.42
0.02
2.98
0.03
3.10
0.03
2.83
MS, RI
Limonene
1045
1033 [11]
96.7
2.60
96.7
3.00
96.6
0.86
96.6
0.66
MS, RI
γ-Terpinene
1083
1074 [11]
Trace
9.10
n
d
d
—
nd
—
nd
—
MS, RI
Terpinolene
1106
1091 [12]
0.01
2.09
0.01
1.84
0.01
0.79
0.01
1.04
MS, RI
Linalool
1110
1100 [11]
0.04
3.40
0.04
3.69
0.04
1.21
0.03
0.48
MS, ST, RI
Nonanal
1113
1104 [12]
0.01
1.49
0.01
1.38
0.01
0.34
0.01
1.03
MS, RI
Perillene
1123
1126 [12]
0.01
2.55
0.02
2.67
0.02
0.88
0.02
0.75
MS, RI
p-Mentha-2,8-dien-1-ol
1142
1132 [11]
Trace
8.96
Trace
4.48
nd
—
Trace
0.98
MS, RI
Limonene oxide
1150
1142 [11]
Trace
2.13
Trace
2.43
Trace
0.95
Trace
0.59
MS, RI
Citronellal
1161
1159 [11]
0.03
2.90
0.04
2.89
0.04
0.76
0.04
0.71
MS, ST, RI
1-Nonanol
1176
1171 [12]
Trace
4.12
Trace
5.12
Trace
4.45
Trace
4.21
MS, RI
α-Terpineol
1196
1195 [11]
Trace
4.46
Trace
3.56
Trace
4.25
nd
—
MS, RI
Decanal
1211
1209 [11]
0.07
2.78
0.05
2.58
0.09
0.80
0.08
0.80
MS, ST, RI
trans-Carveol
1217
1217 [11]
Trace
6.21
Trace
2.73
Trace
7.51
Trace
1.24
MS, RI
Citronellol
1233
1233 [11]
0.01
3.53
0.02
2.79
0.01
1.40
0.01
0.85
MS, ST, RI
Neral
1251
1247 [11]
0.03
2.55
0.03
2.56
0.03
0.86
0.02
0.97
MS, ST, RI
Carvone
1261
1255 [11]
0.01
4.08
0.01
2.47
Trace
2.54
Trace
1.25
MS, ST, RI
2-Decenal
1269
1262 [11]
nd
—
nd
—
Trace
3.87
Trace
3.75
MS, ST, RI
Geranial
1278
1277 [11]
0.04
2.17
0.03
2.38
0.04
0.55
0.03
0.94
MS, ST, RI
Perillaldehyde
1293
1279 [12]
0.01
2.34
0.01
2.56
0.01
2.37
0.01
2.54
MS, ST, RI
Undecanal
1311
1306 [12]
0.01
2.73
0.01
2.43
0.01
3.67
0.01
2.22
MS, RI
2,4-Decadienal
1327
1317 [11]
nd
—
nd
—
Trace
3.42
Trace
3.31
MS, ST, RI
Citronellyl acetate
1354
1357 [11]
0.01
3.25
0.01
2.66
0.01
4.90
0.01
5.80
MS, ST, RI
α-Terpinyl acetate
1366
1352 [11]
0.01
5.75
0.01
2.93
0.01
8.18
0.01
5.73
MS, ST, RI
α-Copaene
1385
1377 [11]
0.01
1.74
Trace
3.49
Trace
1.17
Trace
1.86
MS, RI
β-Cubebene
1398
1390 [12]
0.02
2.07
0.03
1.82
0.02
0.97
0.02
1.42
MS, RI
Dodecanal
1411
1407 [12]
0.04
3.06
0.04
1.59
0.04
8.27
0.05
6.47
MS, ST, RI
β-Longipinene
1413
1408 [13]
Trace
5.01
Trace
4.82
0.01
1.50
0.01
1.80
MS, RI
β-Caryophyllene
1446
1432 [12]
0.01
6.31
0.02
1.62
0.01
1.66
0.02
2.21
MS, RI
β-Humulene
1456
1454 [12]
0.01
7.10
0.01
4.31
0.01
7.62
0.01
7.09
MS, RI
α-Humulene
1461
1467 [11]
Trace
5.09
Trace
3.07
Trace
4.55
Trace
5.94
MS, RI
Isogermacrene-D
1484
1487 [11]
0.01
5.06
0.01
2.70
0.01
8.48
0.01
2.86
MS, RI
γ-Muurolene
1501
1499 [12]
Trace
3.38
Trace
1.39
Trace
0.59
Trace
3.22
MS, RI
Valencene
1510
1506 [12]
0.02
4.31
0.02
6.76
0.02
2.98
0.02
2.37
MS, RI
α-Farnesene
1523
1508 [12]
0.01
5.57
0.02
1.21
0.02
2.95
0.04
3.58
MS, RI
δ-Cadinene
1532
1524 [12]
Trace
4.49
Trace
1.46
Trace
2.56
Trace
6.00
MS, RI
α-Elemol
1546
1547 [11]
0.03
7.82
0.03
1.89
0.03
4.24
0.03
4.65
MS, RI
Caryophyllene oxide
1596
1581 [11]
Trace
3.85
0.01
0.86
Trace
2.30
Trace
1.95
MS, RI
β-Sinensal
1715
1706 [11]
0.01
9.39
0.01
3.54
0.01
8.62
0.01
4.22
MS, RI
Farnesol
1756
1742 [12]
nd
—
Trace
1.87
Trace
6.27
Trace
7.99
MS, RI
α-Sinensal
1766
1760 [12]
Trace
8.23
Trace
2.13
Trace
7.19
Trace
3.76
MS, RI
A
S
a = huanglongbing asymptomatic samples, SYb = huanglongbing symptomatic samples, forc “identified by,” MS = mass spectral library, ST = internal standard, RI = matching calculated retention index to literature value; ndd = not detected for this sample.All the compounds identified in Early Hamlin were identified in Late Hamlin. Late Hamlin additionally contained trace amounts of 2,4-decadienal and 2-decenal. Forty-seven compounds were identified in Early Valencia and 46 were identified in Late Valencia (Table2). All the compounds identified in Late Valencia were identified in Early Valencia; Early Valencia additionally contained δ-cadinene. Compounds are sorted by compound retention index, which is a standard method used to tentatively identify compounds.Table 2
Valencia oils linear retention indices and percent peak areas.
Compound
LRI (observed)
LRI (literature)
EarlyASa  % peak area
Relative standard deviation
EarlySYb  % peak area
Relative standard deviation
Late AS % peak area
Relative standard deviation
Late SY % peak area
Relative standard deviation
Identifiedbyc
α-Thujene
922
928 [11]
trace
3.62
trace
3.45
trace
2.87
trace
3.00
MS, RI
α-Pinene
943
939 [11]
0.51
2.71
0.50
0.98
0.48
2.70
0.49
1.03
MS, ST, RI
Sabinene
975
972 [11]
0.11
5.09
0.13
4.72
0.10
4.46
0.14
3.90
MS, RI
β-Myrcene
995
992 [11]
1.20
2.57
1.22
1.55
1.21
2.35
1.21
0.39
MS, ST, RI
Octanal
1004
1006 [12]
1.06
2.77
0.99
2.90
0.97
2.06
0.95
2.87
MS, RI
δ-3-Carene
1023
1011 [12]
0.12
3.01
0.09
2.94
0.08
3.86
0.07
3.64
MS, RI
Limonene
1045
1033 [11]
95.7
2.30
96.0
1.54
95.9
2.01
96.0
0.15
MS, RI
β-Ocimene
1075
1056 [11]
n
d
d
—
0.01
2.96
0.03
2.91
0.03
0.35
MS, RI
γ-Terpinene
1083
1074 [11]
trace
2.11
trace
1.55
0.01
2.70
0.01
0.65
MS, RI
Terpinolene
1106
1091 [12]
0.01
2.21
0.01
1.58
0.01
2.32
0.01
0.08
MS, RI
Linalool
1110
1100 [11]
0.23
2.00
0.20
0.50
0.23
1.71
0.20
0.36
MS, ST, RI
Nonanal
1113
1104 [11]
0.03
1.72
0.03
2.62
0.04
1.41
0.04
1.31
MS, RI
Perillene
1142
1126 [11]
trace
3.88
trace
3.96
trace
2.08
trace
1.37
MS, RI
Limonene oxide
1150
1142 [11]
trace
1.61
0.01
1.42
trace
1.68
0.01
0.30
MS, RI
Citronellal
1161
1159 [11]
0.04
1.90
0.05
1.49
0.04
2.10
0.07
0.13
MS, ST, RI
1-Nonanol
1176
1171 [12]
trace
2.28
trace
3.06
0.01
4.14
trace
2.97
MS, RI
α-Terpineol
1196
1195 [11]
trace
1.06
trace
1.53
trace
6.76
trace
1.89
MS, RI
4-Decenal
1200
1200 [11]
trace
2.98
0.01
2.13
trace
4.65
trace
3.83
MS, RI
Decanal
1211
1209 [11]
0.29
1.82
0.21
1.44
0.29
1.63
0.23
0.07
MS, ST, RI
trans-Carveol
1217
1217 [11]
trace
2.39
trace
2.07
trace
3.43
trace
1.76
MS, RI
Citronellol
1233
1233 [11]
0.01
3.49
0.01
2.40
0.02
4.45
0.01
1.25
MS, ST, RI
Neral
1251
1247 [11]
0.07
1.73
0.06
1.69
0.06
1.77
0.05
0.21
MS, ST, RI
Carvone
1261
1255 [11]
0.01
2.03
0.01
4.15
0.01
2.50
0.01
0.52
MS, ST, RI
2-Decenal
1269
1262 [11]
trace
4.64
trace
5.83
trace
3.18
trace
3.00
MS, ST, RI
Geranial
1278
1277 [11]
0.11
1.66
0.09
1.53
0.09
1.59
0.08
0.15
MS, ST, RI
Perillaldehyde
1293
1279 [12]
0.02
1.12
0.03
2.07
0.02
2.54
0.03
0.33
MS, ST, RI
Undecanal
1311
1306 [12]
0.02
4.02
0.02
0.85
0.02
1.84
0.02
1.05
MS, RI
2,4-Decadienal
1327
1317 [11]
trace
6.89
trace
6.18
trace
4.12
trace
5.07
MS, ST, RI
Citronellyl acetate
1354
1357 [11]
trace
3.32
trace
6.67
trace
3.06
trace
3.55
MS, ST, RI
α-Terpinyl acetate
1366
1352 [11]
trace
6.00
trace
4.75
trace
3.23
trace
5.03
MS, ST, RI
α-Copaene
1385
1377 [11]
trace
2.11
trace
2.17
trace
1.84
trace
0.78
MS, RI
β-Cubebene
1398
1390 [12]
0.02
1.19
0.02
1.41
0.02
1.18
0.02
0.27
MS, RI
Dodecanal
1411
1407 [12]
0.07
5.72
0.06
5.88
0.07
4.00
0.07
3.61
MS, ST, RI
β-Longipinene
1413
1408 [13]
trace
4.58
trace
3.68
0.01
2.63
0.01
0.86
MS, RI
β-Caryophyllene
1446
1432 [12]
0.01
2.70
0.01
2.87
0.02
4.78
0.02
0.33
MS, RI
B-Humulene
1456
1454 [12]
0.03
6.18
0.02
4.29
0.03
4.90
0.02
2.24
MS, RI
α-Humulene
1461
1467 [11]
trace
4.99
0.01
6.77
trace
6.18
0.01
2.62
MS, RI
Germacrene-D
1484
1487 [11]
trace
1.57
trace
5.34
trace
4.36
trace
4.30
MS, RI
γ-Muurolene
1501
1499 [12]
trace
1.35
trace
3.92
trace
2.74
trace
0.59
MS, RI
Valencene
1510
1506 [12]
0.02
1.54
0.03
5.45
0.03
2.49
0.03
0.43
MS, RI
α-Farnesene
1523
1508 [12]
0.02
0.70
0.02
3.76
0.06
1.28
0.04
1.17
MS, RI
δ-Cadinene
1532
1524 [12]
trace
4.30
trace
2.93
nd
—
nd
—
MS, RI
α-Elemol
1546
1547 [11]
0.03
5.88
0.03
3.00
0.03
4.73
0.03
5.84
MS, RI
Caryophyllene oxide
1623
1581 [11]
trace
6.26
trace
5.74
0.01
3.73
0.01
6.25
MS
β-Sinensal
1715
1706 [11]
0.02
6.89
0.03
5.24
0.02
3.99
0.03
7.26
MS, RI
Farnesol
1756
1742 [12]
trace
1.19
trace
2.75
trace
4.06
trace
1.80
MS, RI
α-Sinensal
1766
1760 [12]
0.01
1.57
0.01
2.00
0.01
5.14
0.01
4.21
MS, RI
A
S
a = huanglongbing asymptomatic samples, SYb = huanglongbing symptomatic samples, forc “identified by,” MS = mass spectral library, ST = internal standard, RI = matching calculated retention index to literature value; ndd = not detected for this sample.All the compounds identified in this study have been previously identified in CPOO, although three compounds are not commonly identified in CPOO extracted fromCitrus sinensis. These three compounds are perillene (3-(4-Methyl-3-pentenyl)furan), β-longifolene, and 4-decenal. The compound 4-decenal has previously been identified in orange essence oil, orange juice, Pontianak orange peel, and clementine peel [16–19]. One aspect which these four studies had in common is that 4-decenal was first identified by gas chromatography-olfactometry. Often 4-decenal is present in citrus at fairly low concentrations but is very odor active, having an odor which was called lemon and pine [19] or citrus and flower [18]. Because it is very odor active, 4-decenal is often more readily identified by gas chromatography-olfactometry than by GC-MS.β-Longifolene has previously been identified inCitrus sinensis orange essence oil [16]. Additionally, it has been reported inCitrus sphaerocarpa peel oil, fresh squeezed orange juice of an undisclosed variety, and oil derived fromCitrus sinensis flowers [20–22]. Perillene has previously been identified in Kenyan ValenciaCitrus sinensis CPOO, Australian finger lime(Citrus australasica) peel oil, and Neroli(Citrus aurantium) oil [23–25]. Perillene can be produced from biochemical conversion of β-myrcene, a common constituent in orange oil [26]. It is possible that this bioconversion mechanism leads to a measurable quantity of perillene in our samples.Qualitatively, the major compounds generally found in CPOO ofCitrus sinensis (L.) Osbeck were observed in our samples, with the exception of nerol and β-elemene. The compounds 4-decenal, perillene, and β-longifolene were tentatively identified by a combination of Kovats Indices and MS library match. These compounds are not commonly identified inCitrus sinensis (L.) Osbeck oils but have each been identified in citrus multiple studies. Further research is necessary to determine if presence of these compounds is biomarkers indicative of HLB positiveCitrus sinensis (L.) Osbeck oils.


### 3.2. Quantitative Gas Chromatography

Based on the results from the qualitative gas chromatography experiments, fifteen compounds were chosen for quantitation using GC-FID based on an indication that AS and SY samples may contain large differences in the concentration of these compounds (Tables3–6). While 13 of the compounds were included due to suspected differences based on the qualitative studies (≥20% difference in concentration between AS and SY samples), β-myrcene and α-pinene were also included because these terpenes are found at high concentrations in CPOO and contribute to orange aroma [27]. Chromatograms from each sample can be found in Figures 1–8.Table 3
Hamlin early asymptomatic versus symptomatic compound concentration.
Compound
A
S
a concentration (ppm)
S
Y
b concentration (ppm)
p value
Difference
α-Pinene
5267
4903
0.037
6.9%
β-Myrcene
12794
13918
0.089
7.9%
Linalool
660
486
0.0014c
26.4%
Citronellal
506
601
0.00072c
15.8%
Decanal
858
561
0.00052c
34.6%
Citronellol
129
141
0.043
8.5%
Neral
492
341
<0.0001c
30.6%
Carvone
69.9
60.7
0.0046c
13.2%
2-Decenal
n
d
d
nd
nd
nd
Geranial
732
536
0.0011c
26.8%
Perillaldehyde
140
138
0.440
1.4%
2,4-Decadienal
nd
nd
nd
nd
Citronellyl acetate
43.5
58.8
<0.0001c
26.0%
α-Terpinyl acetate
52.8
65.7
0.00095c
19.6%
Dodecanal
767
753
0.298
1.8%
A
S
a = huanglongbing asymptomatic samples; SYb = huanglongbing symptomatic samples; significantc differences at α ≤ 0.01; ndd = not detected.Table 4
Hamlin late asymptomatic versus symptomatic compound concentration.
Compound
A
S
a concentration (ppm)
S
Y
b concentration (ppm)
p value
Difference
α-Pinene
4757
4655
0.041
1.9%
β-Myrcene
12216
13601
0.720
3.6%
Linalool
447
242
<0.0001c
45.9%
Citronellal
648
725
<0.0001c
10.6%
Decanal
1302
716
<0.0001c
45.0%
Citronellol
128
123
0.0044
c
3.9%
Neral
438
231
<0.0001c
47.2%
Carvone
45.6
33.5
<0.0001c
26.5%
2-Decenal
n
d
d
nd
nd
nd
Geranial
650
386
<0.0001c
40.6%
Perillaldehyde
122
115
0.020
5.7%
2,4-Decadienal
nd
nd
nd
nd
Citronellyl acetate
50.2
57.5
0.0034c
12.7%
α-Terpinyl acetate
55.1
62.7
0.0090c
12.1%
Dodecanal
875
795
0.00066c
9.1%
A
S
a = huanglongbing asymptomatic samples; SYb = huanglongbing symptomatic samples; significantc differences at α ≤ 0.01; ndd = not detected.Table 5
Valencia early asymptomatic versus symptomatic compound concentration.
Compound
A
S
a concentration (ppm)
S
Y
b concentration (ppm)
p value
Difference
α-Pinene
4881
4704
0.170
3.6%
β-Myrcene
12149
11525
0.196
5.1%
Linalool
5312
4274
0.00020
c
19.5%
Citronellal
575
740
<0.0001c
22.3%
Decanal
3361
2630
<0.0001c
21.8%
Citronellol
69.1
57.6
0.019
16.6%
Neral
1203
953
<0.0001c
20.8%
Carvone
61.3
47.5
0.00025
c
22.5%
2-Decenal
35.0
21.9
0.00084
c
27.2%
Geranial
1556
1301
0.00011
c
16.4%
Perillaldehyde
266
302
0.00058
c
11.9%
2,4-Decadienal
37.8
27.5
0.064
27.2%
Citronellyl acetate
18.4
18.2
0.863
1.1%
α-Terpinyl acetate
30.1
27.7
0.462
8.0%
Dodecanal
1180
1082
0.0013
c
8.2%
A
S
a = huanglongbing asymptomatic samples; SYb = huanglongbing symptomatic samples; significantc differences at α ≤ 0.01.Table 6
Valencia late asymptomatic versus symptomatic compound concentration.
Compound
A
S
a concentration (ppm)
S
Y
b concentration (ppm)
p value
Difference
α-Pinene
4936
4664
0.030
5.5%
β-Myrcene
12251
11791
0.448
3.8%
Linalool
4260
4548
0.0083
c
6.3%
Citronellal
682
995
<0.0001c
31.5%
Decanal
3541
2712
<0.0001c
23.4%
Citronellol
134.9
113.7
<0.0001c
15.7%
Neral
1007
876
0.00025
c
13.0%
Carvone
81.1
60.3
<0.0001c
25.6%
2-Decenal
47.5
25.6
<0.0001c
46.1%
Geranial
1350
1165
<0.0001c
13.7%
Perillaldehyde
257
307
0.00011
c
16.3%
2,4-Decadienal
53.2
33.6
0.00091
c
36.8%
Citronellyl acetate
21.8
20.3
0.144
6.9%
α-Terpinyl acetate
35.4
34.1
0.517
3.7%
Dodecanal
1276
1194
0.00081
c
6.4%
A
S
a = huanglongbing asymptomatic samples; SYb = huanglongbing symptomatic samples; significantc differences at α ≤ 0.01.Figure 1
Chromatogram from Hamlin Early Asymptomatic.Note. 2-Decenal and 2,4-decadienal are not labeled on the chromatogram as they were not detected in this sample. Compound∗ has lower concentration compared to HES. Compound∗∗ has higher concentration compared to HES.Figure 2
Chromatogram from Hamlin Early Symptomatic.Note. 2-Decenal and 2,4-decadienal are not labeled on the chromatogram as they were not detected in this sample. Compound∗ has lower concentration compared to HEA. Compound∗∗ has higher concentration compared to HEA.Figure 3
Chromatogram from Hamlin Late Asymptomatic.Note. While 2-decenal and 2,4-decadienal were quantifiable in this sample, they were not labeled on the chromatogram as the peaks are too small to be seen. Compound∗ has lower concentration compared to HLS. Compound∗∗ has higher concentration compared to HLS.Figure 4
Chromatogram from Hamlin Late Symptomatic.Note. While 2-decenal and 2,4-decadienal were quantifiable in this sample, they were not labeled on the chromatogram as the peaks are too small to be seen. Compound∗ has lower concentration compared to HLA. Compound∗∗ has higher concentration compared to HLA.Figure 5
Chromatogram from Valencia Early Asymptomatic.Note. While 2,4-decadienal was quantifiable in this sample, it was not labeled on the chromatogram as the peak is too small to be seen. Compound∗ has lower concentration compared to VES. Compound∗∗ has higher concentration compared to VES.Figure 6
Chromatogram from Valencia Early Symptomatic.Note. While 2-decenal and 2,4-decadienal were quantifiable in this sample, they were not labeled on the chromatogram as the peaks are too small to be seen. Compound∗ has lower concentration compared to VEA. Compound∗∗ has higher concentration compared to VEA.Figure 7
Chromatogram from Valencia Late Asymptomatic.Note. While 2,4-decadienal was quantifiable in this sample, it was not labeled on the chromatogram as the peak is too small to be seen. Compound∗ has lower concentration compared to VLS. Compound∗∗ has higher concentration compared to VLS.Figure 8
Chromatogram from Valencia Late Symptomatic.Note. While 2-decenal and 2,4-decadienal were quantifiable in this sample, they were not labeled on the chromatogram as the peaks are too small to be seen. Compound∗ has lower concentration compared to VLA. Compound∗∗ has higher concentration compared to VLA.


#### 3.2.1. Effect of Disease Stage

Each of the 15 compounds was analyzed as analytical standards by GC-FID in duplicate at six concentrations to create a standard curve. Each CPOO sample was analyzed by GC-FID in triplicate, and the concentration of individual compounds was determined based on the equation derived from the standard curve. Tables3–6 show differences between AS and SY compounds for Early Hamlin, Late Hamlin, Early Valencia, and Late Valencia, respectively.For Early Hamlin (Table3), significant differences (α ≤ 0.01) in concentration between AS and SY oil were observed for linalool, citronellal, decanal, neral, carvone, geranial, citronellyl acetate, and α-terpinyl acetate. The largest percent differences were for linalool (26.4%), decanal (34.6%), neral (30.6%), geranial (26.8%), and citronellyl acetate (26.0%). Of the compounds for which there was a significant difference, AS concentration was higher for linalool, decanal, neral, carvone, and geranial. Conversely, SY concentration was significantly higher for citronellal, citronellyl acetate, and α-terpinyl acetate.For Late Hamlin (Table4), significant differences (α ≤ 0.01) in concentration between AS and SY oil were observed for linalool, citronellal, decanal, citronellol, neral, carvone, geranial, citronellyl acetate, α-terpinyl acetate, and dodecanal. The largest percent differences were for linalool (45.9%), decanal (45.0%), neral (47.2%), carvone (26.5%), and geranial (40.6%). Of the compounds for which there was a significant difference, AS concentration was higher for linalool, decanal, citronellol, neral, carvone, geranial, and dodecanal. Conversely, SY concentration was significantly higher for citronellal, citronellyl acetate, and α-terpinyl acetate.For Early Valencia (Table5), significant differences (α ≤ 0.01) in concentration between AS and SY oil were observed for linalool, citronellal, decanal, citronellol, neral, carvone, 2-decenal, geranial, perillaldehyde, and dodecanal. The largest percent difference was for 2-decenal (27.2%); this was the only significant percent difference which was ≥25.0%. Of the compounds for which there was a significant difference, AS concentration was higher for linalool, decanal, citronellol, neral, carvone, 2-decenal, geranial, and dodecanal. Conversely, SY concentration was significantly higher for citronellal and perillaldehyde.For Late Valencia (Table6) significant differences (α ≤ 0.01) in concentration between AS and SY oil were observed for linalool, citronellal, decanal, citronellol, neral, carvone, 2-decenal, geranial, perillaldehyde, 2,4-decadienal, and dodecanal. The largest percent differences were for citronellal (31.5%), carvone (25.6%), and 2-decenal (46.1%). Of the compounds for which there was a significant difference, AS concentration was higher for linalool, decanal, citronellol, neral, carvone, 2-decenal, geranial, 2,4-decadienal, and dodecanal. Conversely, SY concentration was significantly higher for citronellal and perillaldehyde.Some trends were gleaned from these results based on compound concentration and disease stage. Overall, no changes inα-pinene and β-myrcene concentration were observed based on disease stage. Compounds for which AS oil had significantly higher concentrations were linalool, decanal, citronellol, neral, geranial, carvone, dodecanal, and 2-decenal; the latter was only detectable in Valencia oil. Compounds for which SY oil had significantly higher concentration were citronellal for both Hamlin and Valencia samples, α-terpinyl acetate and citronellyl acetate for Hamlin samples, and perillaldehyde for Valencia samples.In HLB affected oranges both the physiology of the whole orange as well as the orange juice resemble that of less mature fruit [10]. Volatiles, including the dominant terpenoid class found in orange oil, are generally secondary metabolites. Unlike primary metabolites, secondary metabolites do not support the primary life functions of a plant, and a dearth of secondary metabolites will not directly result in plant death. The terpenoids in orange oil, primarily d-limonene, are secondary metabolites which play roles as chemical messengers [28]. It is possible that HLB affects oranges such that as the severity of HLB increases, volatile formation via secondary metabolism is inhibited in a substantial way. Previous studies on HLB affected orange juice [9, 10] show that overall many volatile compound concentrations decrease when oranges are HLB positive.Dagulo et al. investigated the effects of HLB on the volatiles in orange juice [9]. In this study, control (HLB unaffected), AS, and SY Valencia juice was analyzed by GC-MS to determine how HLB affects relative volatile concentrations. Of the 15 compounds studied qualitatively in our research, Dagulo et al. reported on eight of these compounds: α-pinene, β-myrcene, decanal, linalool, citronellol, carvone, citronellyl acetate, and perillaldehyde. When comparing SY to AS juice, α-pinene, β-myrcene, citronellyl acetate, and citronellol showed no significant differences between AS and SY juices. Our research supports the results seen concerning α-pinene and β-myrcene, but we found that citronellol was significantly higher in AS CPOO than in SY CPOO and citronellyl acetate was significantly higher in SY CPOO than AS CPOO. Dagulo et al. found that decanal and carvone had significantly higher concentrations in AS orange juice compared to SY orange juice. These results were corroborated by our study. Dagulo et al. found linalool and perillaldehyde to have significantly higher concentrations in SY orange juice compared to AS orange juice. These results were corroborated by our study for perillaldehyde but not for linalool. Overall, our observed relative volatile concentrations of α-pinene, β-myrcene, decanal, carvone, and perillaldehyde corroborated the research performed by Dagulo et al. However, we found different results for linalool, citronellyl acetate, and citronellol. While orange juice and CPOO share many of the same volatile compounds, they are ultimately different substances. While some of the volatiles found in orange juices are derived from the peel, many of the same compounds are independently found in the essence oil, a product which is derived from orange juice. All seven compounds of interest compared to the study by Dagulo et al. can be found in essence oil [29].The concentration increases for citronellal in both Hamlin and Valencia CPOO as well asα-terpinyl acetate and citronellyl acetate for Hamlin CPOO and perillaldehyde for Valencia CPOO are of interest. However, no studies supporting increases in citronellal or citronellyl acetate concentrations in plants during stress situations could be found. As this is the first study on the volatiles of HLB effected cold pressed orange oil, it is possible that this is a new development specific to HLB stress in citrus. Citronellal can be produced from citral (neral + geranial) [30]. It is possible that this pathway occurs more often in HLB SY fruit than in HLB AS fruit. A higher concentration of perillaldehyde in SY oils compared to AS Valencia oils was corroborated by the results seen in Valencia orange juice [9]. α-Terpinyl acetate has been shown to increase in concentration with water stress [31]. Additionally, while no specific data was given for α-terpinyl acetate or citronellyl acetate, some esters were shown to increase in concentration in HLB positive orange juice [10].Overall, it appears that, with increasing HLB severity, several important volatiles including linalool, decanal, citronellol, neral, geranial, carvone, dodecanal, and 2-decenal decrease in concentration. Citronellal in both Hamlin and Valencia samples,α-terpinyl acetate and citronellyl acetate for Hamlin samples, and perillaldehyde in Valencia samples show higher concentrations in HLB SY samples. It is possible that these compounds increased because the stress HLB causes the orange tree. These compounds may have all increased due to shifts in biochemical pathways which occurred in trees with more severe HLB symptoms, creating greater concentrations of these volatiles.


#### 3.2.2. Effect of Seasonality

In addition to disease stage, the effect of seasonality (early or late harvest) on the concentration of the 15 selected compounds was also analyzed. Tables7–10 show differences between early and late season compound concentrations for Hamlin AS, Hamlin SY, Valencia AS, and Valencia SY CPOO, respectively.Table 7
Hamlin asymptomatic early versus late compound concentration.
Compound
Early concentration (ppm)
Late concentration (ppm)
p value
Difference
α-Pinene
5267
4757
0.00048
a
9.7%
β-Myrcene
12807
14133
0.372
9.3%
Linalool
660
447
0.00024
a
32.3%
Citronellal
506
648
<0.0001a
21.9%
Decanal
858
1302
<0.0001a
34.1%
Citronellol
129
128
0.868
0.78%
Neral
492
438
0.0025a
11.0%
Carvone
69.9
45.6
<0.0001a
34.8%
2-Decenal
n
d
b
nd
nd
nd
Geranial
732
650
0.029
11.2%
Perillaldehyde
140
122
0.00071a
12.9%
2,4-Decadienal
nd
nd
nd
nd
Citronellyl acetate
43.5
50.2
0.0031a
13.3%
α-Terpinyl acetate
52.8
55.1
0.323
4.2%
Dodecanal
767
875
0.00054a
12.3%
S
i
g
n
i
f
i
c
a
n
t
a differences at α ≤ 0.01; ndb = not detected.Table 8
Hamlin symptomatic early versus late compound concentration.
Compound
Early concentration (ppm)
Late concentration (ppm)
p value
Difference
α-Pinene
4903
4655
0.039
5.1%
β-Myrcene
13911
13607
0.602
2.2%
Linalool
486
242
<0.0001a
50.2%
Citronellal
601
725
0.00011
a
17.1%
Decanal
561
716
0.0019
a
21.6%
Citronellol
141
123
0.00090a
12.8%
Neral
341
231
<0.0001a
37.5%
Carvone
60.7
33.5
<0.0001a
44.8%
2-Decenal
n
d
b
nd
nd
nd
Geranial
536
386
<0.0001a
28.0%
Perillaldehyde
138
115
0.00034a
16.7%
2,4-Decadienal
nd
nd
nd
nd
Citronellyl acetate
58.8
57.5
0.132
2.2%
α-Terpinyl acetate
65.7
62.7
0.035
4.6%
Dodecanal
753
795
0.013
5.3%
S
i
g
n
i
f
i
c
a
n
t
a differences at α ≤ 0.01; ndb = not detected.Table 9
Valencia asymptomatic early versus late compound concentration.
Compound
Early concentration (ppm)
Late concentration (ppm)
p value
Difference
α-Pinene
4881
4936
0.660
1.1%
β-Myrcene
12149
12251
0.729
0.83%
Linalool
5312
4260
0.00032
a
19.8%
Citronellal
575
682
0.00021
a
15.7%
Decanal
3361
3541
0.0095
a
5.1%
Citronellol
69.1
134.9
<0.0001a
48.8%
Neral
1203
1007
0.00026
a
16.3%
Carvone
61.3
81.1
<0.0001a
24.4%
2-Decenal
35.0
47.5
0.00029
a
26.3%
Geranial
1556
1350
0.00027
a
13.2%
Perillaldehyde
266
257
0.058
3.4%
2,4-Decadienal
37.8
53.2
0.026
28.9%
Citronellyl acetate
18.4
21.8
0.058
15.6%
α-Terpinyl acetate
30.1
35.4
0.137
15.0%
Dodecanal
1180
1276
0.0014a
7.5%
S
i
g
n
i
f
i
c
a
n
t
a differences at α ≤ 0.01.Table 10
Valencia symptomatic early versus late compound concentration.
Compound
Early concentration (ppm)
Late concentration (ppm)
p value
Difference
α-Pinene
4704
4664
0.349
0.85%
β-Myrcene
11525
11791
0.691
2.3%
Linalool
4274
4548
0.0025a
6.0%
Citronellal
740
995
<0.0001a
25.6%
Decanal
2630
2712
0.026
3.0%
Citronellol
57.6
113.7
<0.0001a
49.3%
Neral
953
876
0.0013
a
8.1%
Carvone
47.5
60.3
0.00018
a
21.2%
2-Decenal
21.9
25.6
0.040
14.4%
Geranial
1301
1165
0.00018
a
10.5%
Perillaldehyde
302
307
0.177
1.6%
2,4-Decadienal
27.5
33.6
0.0081
a
18.2%
Citronellyl acetate
18.2
20.3
0.061
10.3%
α-Terpinyl acetate
27.7
34.1
0.031
18.8%
Dodecanal
1082
1194
0.00025
a
9.6%
S
i
g
n
i
f
i
c
a
n
t
a differences at α ≤ 0.01.For Hamlin AS (Table7), significant differences (α ≤ 0.01) in concentration between early and late CPOO were observed for α-pinene, linalool, citronellal, decanal, neral, carvone, perillaldehyde, citronellyl acetate, and dodecanal. The largest percent differences were for linalool (32.3%), decanal (34.1%), and carvone (34.8%). Of the compounds for which there was a significant difference, early concentration was higher for α-pinene, linalool, neral, carvone, geranial, and perillaldehyde. Conversely, late concentration was significantly higher for citronellal, citronellyl acetate, and decanal.For Hamlin SY (Table8), significant differences (α ≤ 0.01) in concentration between early and late CPOO were observed for linalool, citronellal, decanal, citronellol, neral, carvone, geranial, and perillaldehyde. The largest percent differences were for linalool (50.2%), neral (37.5%), carvone (44.8%), and geranial (28.0%). Of the compounds for which there was a significant difference, early concentration was higher for linalool, citronellol, neral, carvone, geranial, and perillaldehyde. Conversely, late concentration was significantly higher for citronellal and decanal.For Valencia AS (Table9), significant differences (α ≤ 0.01) in concentration between early and late CPOO were observed for linalool, citronellal, decanal, citronellol, neral, carvone, 2-decenal, geranial, and dodecanal. The largest significant percent differences were for citronellol (48.8%) and 2-decenal (26.3%). Of the compounds for which there was a significant difference, early concentration was higher for linalool, neral, and geranial. Conversely, late concentration was significantly higher for citronellal, decanal, citronellol, carvone, 2-decenal, and dodecanal.For Valencia SY (Table10), significant differences (α ≤ 0.01) in concentration between early and late CPOO were observed for linalool, citronellal, citronellol, neral, carvone, geranial, 2,4-decadienal, and dodecanal. The largest significant percent differences were for citronellal (25.6%) and citronellol (49.3%). Of the compounds for which there was a significant difference, early concentration was higher for neral and geranial. Conversely, late concentration was significantly higher for linalool, citronellal, citronellol, carvone, 2,4-decadienal, and dodecanal.Some trends were gleaned from these results based on compound concentration and seasonality (early or late harvest). Overall,α-pinene, β-myrcene, citronellyl acetate, and α-terpinyl acetate showed few or no changes based on harvest time. Compounds for which early CPOO had significantly higher concentrations were linalool, neral, geranial, carvone in Hamlin samples, and perillaldehyde in Valencia samples. Compounds for which Late CPOO had significantly higher concentrations were citronellal, decanal, dodecanal, citronellol in Hamlin samples, and carvone in Valencia samples.To assist in explaining these results, the literature was investigated to determine what effect harvest time (early or late) could have on volatile concentrations in CPOO. Attaway et al. studied the terpenes in various citrus oils month-by-month over the course of one season [32]. The last month surveyed in this study for Hamlin oranges was December. Maturity of oranges can vary year to year, and it can be assumed that December would correspond similarly to late season oranges in our study and that October or November would correspond to early season oranges, as early season fruit was generally harvested 6–8 weeks before late season fruit for our research. Attaway et al. showed that α-pinene and β-myrcene either decreased slightly or remain unchanged (depending on whether October or November is considered “early”) from early to late season. Linalool and geranial either increased or remained the same (depending on whether October or November is considered “early”) from early to late season. Our data corroborated the concentrations of α-pinene and β-myrcene remaining the same but did not corroborate an increase in the concentration of linalool or geranial as the season progressed.Kesterson et al. found that the aldehyde content in Valencia CPOO increased from February to March [6]. These were the months our early and late season Valencia oranges were harvested. As decanal is the primary aldehyde in orange oil [6], an increase in aldehyde content for late season fruit corroborates the data shown by Kesterson et al. Overall, there is a lack of published literature to determine if the changes in volatile concentration during different harvest months observed in our study are similar or different than what would be expected before the onset of HLB.


### 3.3. Comparison to the Literature

As no control or HLB unaffected fruit was available at the time of this study, the percent areas for compounds found in AS and SY Hamlin and Valencia CPOO were compared to three published studies on orange oils. Since few studies report the effects of seasonality, early and late percent areas measured in triplicate were averaged, resulting in a single value for each compound for both Hamlin and Valencia fruit.While several studies [33–37] have reported on the relative concentration of compounds in Florida CPOO, these studies tend to be older, somewhat outdated, and incomplete. Therefore, studies usingCitrus sinensis (L.) Osbeck fruit from other countries were used for this comparison for a more complete, modern perspective. Mitiku et al. used both Hamlin and Valencia oranges from Ethiopia [38]. Qiao et al. [27] used Chinese Jinchen oranges, while Mondello et al. [39] used Italian oranges. For comparison to the latter two studies, the same literature values are used in both the Hamlin and Valencia tables, since Hamlin and Valencia varieties were not used in these two studies. Although we could calculate percent peak area to 0.0001% based on the GC-FID output, it is customary in literature to report any concentration less than 0.01% as “trace”; therefore, this methodology is utilized in Tables 11 and 12.Table 11
Hamlin peak area comparison to the literature data.
Compound
AS % area
SY % area
Literature % area (Mitiku)
Literature % areaQiaoa
Literature % areaMondellob
α-Pinene
0.50
0.50
0.41
0.76
0.64
β-Myrcene
1.40
1.42
1.77
1.88
2.03
Linalool
0.04
0.03
0.12
0.92
0.30
Citronellal
0.03
0.04
0.01
0.06
0.04
Decanal
0.08
0.07
0.10
0.21
0.17
Citronellol
0.01
0.01
trace
n
r
c
nr
Neral
0.03
0.02
0.04
0.06
0.06
Carvone
Trace
Trace
nr
nr
nr
2-Decenal
Trace
Trace
nr
nr
nr
Geranial
0.04
0.04
0.08
0.11
0.13
Perillaldehyde
0.01
0.01
Trace
0.03
Trace
2,4-Decadienal
Trace
Trace
Trace
nr
nr
Citronellyl acetate
Trace
Trace
Trace
0.01
0.01
α-Terpinyl acetate
Trace
Trace
Trace
nr
nr
Dodecanal
0.04
0.05
0.01
0.03
Trace
C
h
i
n
e
s
e
a variety Jinchen Citrus sinensis (L.) Osbeck; Italianb  Citrus sinensis(L.) Osbeck; nrc = not reported.Table 12
Valencia peak area comparison to the literature data.
Compound
AS % area
SY % area
Literature % area (Mitiku)
Literature % areaQiaoa
Literature % areaMondellob
α-Pinene
0.50
0.50
0.35
0.76
0.64
β-Myrcene
1.21
1.21
1.79
1.88
2.03
Linalool
0.25
0.22
0.35
0.92
0.30
Citronellal
0.04
0.06
0.03
0.06
0.04
Decanal
0.29
0.22
0.16
0.21
0.17
Citronellol
0.02
0.01
Trace
n
r
c
nr
Neral
0.07
0.05
0.09
0.06
0.06
Carvone
Trace
Trace
nr
nr
nr
2-Decenal
Trace
Trace
nr
nr
nr
Geranial
0.10
0.09
0.16
0.11
0.13
Perillaldehyde
0.02
0.03
Trace
0.03
Trace
2,4-Decadienal
Trace
Trace
Trace
nr
nr
Citronellyl acetate
Trace
Trace
Trace
0.01
0.01
α-Terpinyl acetate
Trace
Trace
Trace
nr
nr
Dodecanal
0.07
0.07
0.02
0.03
Trace
C
h
i
n
e
s
e
a variety Jinchen Citrus sinensis (L.) Osbeck; Italianb  Citrus sinensis(L.) Osbeck; nrc = not reported.Based on the three literature studies presented, in Hamlin CPOO (Table11), the concentrations of linalool, decanal, neral, and geranial are low in comparison to literature values. The concentration of dodecanal is high. Especially notable is the linalool content, which is approximately 10 times less than the average concentration seen in the three literature studies. It should be noted that Florida Hamlin CPOO has never been considered as high quality AS Valencia CPOO and was often not collected during orange juice processing [6]. The geranial deficit is also notable, with the content being 2.5 times less than the literature average. Dodecanal increased 2.3 times compared to the two reported literature concentrations. The deficits in decanal (2.1 times less) and neral (2.1 times less) and the increase in dodecanal are not as severe as the other changes yet are still notable.Valencia CPOO also showed notable changes compared to literature CPOO concentrations. Linalool (2.2 times less) had a notably lower concentration compared to the literature values. Citronellol and dodecanal (2.8 times more) had notably higher concentrations. Citronellol is generally either found in only trace quantities or is not quantifiable in CPOO, so the exact increase in the concentration of citronellol compared to literature values could not be determined. Geranial had a slightly lower (1.4 times less) and decanal a slightly higher (1.4 times more) concentration than the literature averages.Both linalool and geraniol are formed from the precursor geranyl diphosphate [40]. Geraniol, in turn, can be transformed either to citral (neral + geranial) or citronellol. Citronellol transformation from geraniol may increase during plant stress [41]. Additionally, α-terpinyl acetate can be derived from linalool [26]. During stress, certain volatile compounds can increase in concentration. Stress related volatile concentration change has not been studied in direct relation to HLB, so other forms of stress are considered. Water stress has been shown to increase the concentration of citronellol in aromatic grasses [41] and dodecanal in tea [42]. It is possible that stress from HLB causes oranges to increase production of citronellol and dodecanal. Overall, when compared to historical results, our samples show a large concentration deficit in linalool, small deficits in neral and geranial, and increases in citronellol and α-terpinyl acetate.


## 3.1. Qualitative Gas Chromatography

CPOOs from the 2015-2016 harvest year were evaluated by GC/GC-MS to determine volatile composition. CPOO from Early Hamlin, Late Hamlin, Early Valencia, and Late Valencia oranges were analyzed, and presumptive compound identification was performed by mass spectral match to several flavor and fragrance libraries, according to Shimadzu MDGC specifications by FID screening and identification by MS as previously described.Forty-four compounds were identified in Early Hamlin and 46 compounds were identified in Late Hamlin by initial FID detection and cut compounds in a second analysis of the gas-liquid source through a second oven and RTX-5 equivalent capillary GC column for MS identification (Table1).Table 1
Hamlin oils linear retention indices and percent peak areas.
Compound
LRI (observed)
LRI (literature)
EarlyASa  % peak area
Relative standard deviation
EarlySYb  % peak area
Relative standard deviation
Late AS % peak area
Relative standard deviation
Late SY % peak area
Relative standard deviation
Identifiedbyc
α-Thujene
922
928 [11]
Trace
7.14
Trace
2.73
Trace
7.85
Trace
6.54
MS, RI
α-Pinene
943
939 [11]
0.49
2.63
0.49
2.67
0.50
0.31
0.50
1.22
MS, ST, RI
Sabinene
975
972 [11]
0.14
5.21
0.17
3.24
0.14
4.52
0.17
5.20
MS, RI
β-Myrcene
995
992 [11]
1.42
3.26
1.43
3.17
1.38
0.67
1.42
0.71
MS, ST, RI
Octanal
1004
1006 [12]
0.69
2.18
0.62
2.66
0.67
2.65
0.62
2.75
MS, RI
δ-3-Carene
1020
1011 [12]
0.02
3.42
0.02
2.98
0.03
3.10
0.03
2.83
MS, RI
Limonene
1045
1033 [11]
96.7
2.60
96.7
3.00
96.6
0.86
96.6
0.66
MS, RI
γ-Terpinene
1083
1074 [11]
Trace
9.10
n
d
d
—
nd
—
nd
—
MS, RI
Terpinolene
1106
1091 [12]
0.01
2.09
0.01
1.84
0.01
0.79
0.01
1.04
MS, RI
Linalool
1110
1100 [11]
0.04
3.40
0.04
3.69
0.04
1.21
0.03
0.48
MS, ST, RI
Nonanal
1113
1104 [12]
0.01
1.49
0.01
1.38
0.01
0.34
0.01
1.03
MS, RI
Perillene
1123
1126 [12]
0.01
2.55
0.02
2.67
0.02
0.88
0.02
0.75
MS, RI
p-Mentha-2,8-dien-1-ol
1142
1132 [11]
Trace
8.96
Trace
4.48
nd
—
Trace
0.98
MS, RI
Limonene oxide
1150
1142 [11]
Trace
2.13
Trace
2.43
Trace
0.95
Trace
0.59
MS, RI
Citronellal
1161
1159 [11]
0.03
2.90
0.04
2.89
0.04
0.76
0.04
0.71
MS, ST, RI
1-Nonanol
1176
1171 [12]
Trace
4.12
Trace
5.12
Trace
4.45
Trace
4.21
MS, RI
α-Terpineol
1196
1195 [11]
Trace
4.46
Trace
3.56
Trace
4.25
nd
—
MS, RI
Decanal
1211
1209 [11]
0.07
2.78
0.05
2.58
0.09
0.80
0.08
0.80
MS, ST, RI
trans-Carveol
1217
1217 [11]
Trace
6.21
Trace
2.73
Trace
7.51
Trace
1.24
MS, RI
Citronellol
1233
1233 [11]
0.01
3.53
0.02
2.79
0.01
1.40
0.01
0.85
MS, ST, RI
Neral
1251
1247 [11]
0.03
2.55
0.03
2.56
0.03
0.86
0.02
0.97
MS, ST, RI
Carvone
1261
1255 [11]
0.01
4.08
0.01
2.47
Trace
2.54
Trace
1.25
MS, ST, RI
2-Decenal
1269
1262 [11]
nd
—
nd
—
Trace
3.87
Trace
3.75
MS, ST, RI
Geranial
1278
1277 [11]
0.04
2.17
0.03
2.38
0.04
0.55
0.03
0.94
MS, ST, RI
Perillaldehyde
1293
1279 [12]
0.01
2.34
0.01
2.56
0.01
2.37
0.01
2.54
MS, ST, RI
Undecanal
1311
1306 [12]
0.01
2.73
0.01
2.43
0.01
3.67
0.01
2.22
MS, RI
2,4-Decadienal
1327
1317 [11]
nd
—
nd
—
Trace
3.42
Trace
3.31
MS, ST, RI
Citronellyl acetate
1354
1357 [11]
0.01
3.25
0.01
2.66
0.01
4.90
0.01
5.80
MS, ST, RI
α-Terpinyl acetate
1366
1352 [11]
0.01
5.75
0.01
2.93
0.01
8.18
0.01
5.73
MS, ST, RI
α-Copaene
1385
1377 [11]
0.01
1.74
Trace
3.49
Trace
1.17
Trace
1.86
MS, RI
β-Cubebene
1398
1390 [12]
0.02
2.07
0.03
1.82
0.02
0.97
0.02
1.42
MS, RI
Dodecanal
1411
1407 [12]
0.04
3.06
0.04
1.59
0.04
8.27
0.05
6.47
MS, ST, RI
β-Longipinene
1413
1408 [13]
Trace
5.01
Trace
4.82
0.01
1.50
0.01
1.80
MS, RI
β-Caryophyllene
1446
1432 [12]
0.01
6.31
0.02
1.62
0.01
1.66
0.02
2.21
MS, RI
β-Humulene
1456
1454 [12]
0.01
7.10
0.01
4.31
0.01
7.62
0.01
7.09
MS, RI
α-Humulene
1461
1467 [11]
Trace
5.09
Trace
3.07
Trace
4.55
Trace
5.94
MS, RI
Isogermacrene-D
1484
1487 [11]
0.01
5.06
0.01
2.70
0.01
8.48
0.01
2.86
MS, RI
γ-Muurolene
1501
1499 [12]
Trace
3.38
Trace
1.39
Trace
0.59
Trace
3.22
MS, RI
Valencene
1510
1506 [12]
0.02
4.31
0.02
6.76
0.02
2.98
0.02
2.37
MS, RI
α-Farnesene
1523
1508 [12]
0.01
5.57
0.02
1.21
0.02
2.95
0.04
3.58
MS, RI
δ-Cadinene
1532
1524 [12]
Trace
4.49
Trace
1.46
Trace
2.56
Trace
6.00
MS, RI
α-Elemol
1546
1547 [11]
0.03
7.82
0.03
1.89
0.03
4.24
0.03
4.65
MS, RI
Caryophyllene oxide
1596
1581 [11]
Trace
3.85
0.01
0.86
Trace
2.30
Trace
1.95
MS, RI
β-Sinensal
1715
1706 [11]
0.01
9.39
0.01
3.54
0.01
8.62
0.01
4.22
MS, RI
Farnesol
1756
1742 [12]
nd
—
Trace
1.87
Trace
6.27
Trace
7.99
MS, RI
α-Sinensal
1766
1760 [12]
Trace
8.23
Trace
2.13
Trace
7.19
Trace
3.76
MS, RI
A
S
a = huanglongbing asymptomatic samples, SYb = huanglongbing symptomatic samples, forc “identified by,” MS = mass spectral library, ST = internal standard, RI = matching calculated retention index to literature value; ndd = not detected for this sample.All the compounds identified in Early Hamlin were identified in Late Hamlin. Late Hamlin additionally contained trace amounts of 2,4-decadienal and 2-decenal. Forty-seven compounds were identified in Early Valencia and 46 were identified in Late Valencia (Table2). All the compounds identified in Late Valencia were identified in Early Valencia; Early Valencia additionally contained δ-cadinene. Compounds are sorted by compound retention index, which is a standard method used to tentatively identify compounds.Table 2
Valencia oils linear retention indices and percent peak areas.
Compound
LRI (observed)
LRI (literature)
EarlyASa  % peak area
Relative standard deviation
EarlySYb  % peak area
Relative standard deviation
Late AS % peak area
Relative standard deviation
Late SY % peak area
Relative standard deviation
Identifiedbyc
α-Thujene
922
928 [11]
trace
3.62
trace
3.45
trace
2.87
trace
3.00
MS, RI
α-Pinene
943
939 [11]
0.51
2.71
0.50
0.98
0.48
2.70
0.49
1.03
MS, ST, RI
Sabinene
975
972 [11]
0.11
5.09
0.13
4.72
0.10
4.46
0.14
3.90
MS, RI
β-Myrcene
995
992 [11]
1.20
2.57
1.22
1.55
1.21
2.35
1.21
0.39
MS, ST, RI
Octanal
1004
1006 [12]
1.06
2.77
0.99
2.90
0.97
2.06
0.95
2.87
MS, RI
δ-3-Carene
1023
1011 [12]
0.12
3.01
0.09
2.94
0.08
3.86
0.07
3.64
MS, RI
Limonene
1045
1033 [11]
95.7
2.30
96.0
1.54
95.9
2.01
96.0
0.15
MS, RI
β-Ocimene
1075
1056 [11]
n
d
d
—
0.01
2.96
0.03
2.91
0.03
0.35
MS, RI
γ-Terpinene
1083
1074 [11]
trace
2.11
trace
1.55
0.01
2.70
0.01
0.65
MS, RI
Terpinolene
1106
1091 [12]
0.01
2.21
0.01
1.58
0.01
2.32
0.01
0.08
MS, RI
Linalool
1110
1100 [11]
0.23
2.00
0.20
0.50
0.23
1.71
0.20
0.36
MS, ST, RI
Nonanal
1113
1104 [11]
0.03
1.72
0.03
2.62
0.04
1.41
0.04
1.31
MS, RI
Perillene
1142
1126 [11]
trace
3.88
trace
3.96
trace
2.08
trace
1.37
MS, RI
Limonene oxide
1150
1142 [11]
trace
1.61
0.01
1.42
trace
1.68
0.01
0.30
MS, RI
Citronellal
1161
1159 [11]
0.04
1.90
0.05
1.49
0.04
2.10
0.07
0.13
MS, ST, RI
1-Nonanol
1176
1171 [12]
trace
2.28
trace
3.06
0.01
4.14
trace
2.97
MS, RI
α-Terpineol
1196
1195 [11]
trace
1.06
trace
1.53
trace
6.76
trace
1.89
MS, RI
4-Decenal
1200
1200 [11]
trace
2.98
0.01
2.13
trace
4.65
trace
3.83
MS, RI
Decanal
1211
1209 [11]
0.29
1.82
0.21
1.44
0.29
1.63
0.23
0.07
MS, ST, RI
trans-Carveol
1217
1217 [11]
trace
2.39
trace
2.07
trace
3.43
trace
1.76
MS, RI
Citronellol
1233
1233 [11]
0.01
3.49
0.01
2.40
0.02
4.45
0.01
1.25
MS, ST, RI
Neral
1251
1247 [11]
0.07
1.73
0.06
1.69
0.06
1.77
0.05
0.21
MS, ST, RI
Carvone
1261
1255 [11]
0.01
2.03
0.01
4.15
0.01
2.50
0.01
0.52
MS, ST, RI
2-Decenal
1269
1262 [11]
trace
4.64
trace
5.83
trace
3.18
trace
3.00
MS, ST, RI
Geranial
1278
1277 [11]
0.11
1.66
0.09
1.53
0.09
1.59
0.08
0.15
MS, ST, RI
Perillaldehyde
1293
1279 [12]
0.02
1.12
0.03
2.07
0.02
2.54
0.03
0.33
MS, ST, RI
Undecanal
1311
1306 [12]
0.02
4.02
0.02
0.85
0.02
1.84
0.02
1.05
MS, RI
2,4-Decadienal
1327
1317 [11]
trace
6.89
trace
6.18
trace
4.12
trace
5.07
MS, ST, RI
Citronellyl acetate
1354
1357 [11]
trace
3.32
trace
6.67
trace
3.06
trace
3.55
MS, ST, RI
α-Terpinyl acetate
1366
1352 [11]
trace
6.00
trace
4.75
trace
3.23
trace
5.03
MS, ST, RI
α-Copaene
1385
1377 [11]
trace
2.11
trace
2.17
trace
1.84
trace
0.78
MS, RI
β-Cubebene
1398
1390 [12]
0.02
1.19
0.02
1.41
0.02
1.18
0.02
0.27
MS, RI
Dodecanal
1411
1407 [12]
0.07
5.72
0.06
5.88
0.07
4.00
0.07
3.61
MS, ST, RI
β-Longipinene
1413
1408 [13]
trace
4.58
trace
3.68
0.01
2.63
0.01
0.86
MS, RI
β-Caryophyllene
1446
1432 [12]
0.01
2.70
0.01
2.87
0.02
4.78
0.02
0.33
MS, RI
B-Humulene
1456
1454 [12]
0.03
6.18
0.02
4.29
0.03
4.90
0.02
2.24
MS, RI
α-Humulene
1461
1467 [11]
trace
4.99
0.01
6.77
trace
6.18
0.01
2.62
MS, RI
Germacrene-D
1484
1487 [11]
trace
1.57
trace
5.34
trace
4.36
trace
4.30
MS, RI
γ-Muurolene
1501
1499 [12]
trace
1.35
trace
3.92
trace
2.74
trace
0.59
MS, RI
Valencene
1510
1506 [12]
0.02
1.54
0.03
5.45
0.03
2.49
0.03
0.43
MS, RI
α-Farnesene
1523
1508 [12]
0.02
0.70
0.02
3.76
0.06
1.28
0.04
1.17
MS, RI
δ-Cadinene
1532
1524 [12]
trace
4.30
trace
2.93
nd
—
nd
—
MS, RI
α-Elemol
1546
1547 [11]
0.03
5.88
0.03
3.00
0.03
4.73
0.03
5.84
MS, RI
Caryophyllene oxide
1623
1581 [11]
trace
6.26
trace
5.74
0.01
3.73
0.01
6.25
MS
β-Sinensal
1715
1706 [11]
0.02
6.89
0.03
5.24
0.02
3.99
0.03
7.26
MS, RI
Farnesol
1756
1742 [12]
trace
1.19
trace
2.75
trace
4.06
trace
1.80
MS, RI
α-Sinensal
1766
1760 [12]
0.01
1.57
0.01
2.00
0.01
5.14
0.01
4.21
MS, RI
A
S
a = huanglongbing asymptomatic samples, SYb = huanglongbing symptomatic samples, forc “identified by,” MS = mass spectral library, ST = internal standard, RI = matching calculated retention index to literature value; ndd = not detected for this sample.All the compounds identified in this study have been previously identified in CPOO, although three compounds are not commonly identified in CPOO extracted fromCitrus sinensis. These three compounds are perillene (3-(4-Methyl-3-pentenyl)furan), β-longifolene, and 4-decenal. The compound 4-decenal has previously been identified in orange essence oil, orange juice, Pontianak orange peel, and clementine peel [16–19]. One aspect which these four studies had in common is that 4-decenal was first identified by gas chromatography-olfactometry. Often 4-decenal is present in citrus at fairly low concentrations but is very odor active, having an odor which was called lemon and pine [19] or citrus and flower [18]. Because it is very odor active, 4-decenal is often more readily identified by gas chromatography-olfactometry than by GC-MS.β-Longifolene has previously been identified inCitrus sinensis orange essence oil [16]. Additionally, it has been reported inCitrus sphaerocarpa peel oil, fresh squeezed orange juice of an undisclosed variety, and oil derived fromCitrus sinensis flowers [20–22]. Perillene has previously been identified in Kenyan ValenciaCitrus sinensis CPOO, Australian finger lime(Citrus australasica) peel oil, and Neroli(Citrus aurantium) oil [23–25]. Perillene can be produced from biochemical conversion of β-myrcene, a common constituent in orange oil [26]. It is possible that this bioconversion mechanism leads to a measurable quantity of perillene in our samples.Qualitatively, the major compounds generally found in CPOO ofCitrus sinensis (L.) Osbeck were observed in our samples, with the exception of nerol and β-elemene. The compounds 4-decenal, perillene, and β-longifolene were tentatively identified by a combination of Kovats Indices and MS library match. These compounds are not commonly identified inCitrus sinensis (L.) Osbeck oils but have each been identified in citrus multiple studies. Further research is necessary to determine if presence of these compounds is biomarkers indicative of HLB positiveCitrus sinensis (L.) Osbeck oils.


## 3.2. Quantitative Gas Chromatography

Based on the results from the qualitative gas chromatography experiments, fifteen compounds were chosen for quantitation using GC-FID based on an indication that AS and SY samples may contain large differences in the concentration of these compounds (Tables3–6). While 13 of the compounds were included due to suspected differences based on the qualitative studies (≥20% difference in concentration between AS and SY samples), β-myrcene and α-pinene were also included because these terpenes are found at high concentrations in CPOO and contribute to orange aroma [27]. Chromatograms from each sample can be found in Figures 1–8.Table 3
Hamlin early asymptomatic versus symptomatic compound concentration.
Compound
A
S
a concentration (ppm)
S
Y
b concentration (ppm)
p value
Difference
α-Pinene
5267
4903
0.037
6.9%
β-Myrcene
12794
13918
0.089
7.9%
Linalool
660
486
0.0014c
26.4%
Citronellal
506
601
0.00072c
15.8%
Decanal
858
561
0.00052c
34.6%
Citronellol
129
141
0.043
8.5%
Neral
492
341
<0.0001c
30.6%
Carvone
69.9
60.7
0.0046c
13.2%
2-Decenal
n
d
d
nd
nd
nd
Geranial
732
536
0.0011c
26.8%
Perillaldehyde
140
138
0.440
1.4%
2,4-Decadienal
nd
nd
nd
nd
Citronellyl acetate
43.5
58.8
<0.0001c
26.0%
α-Terpinyl acetate
52.8
65.7
0.00095c
19.6%
Dodecanal
767
753
0.298
1.8%
A
S
a = huanglongbing asymptomatic samples; SYb = huanglongbing symptomatic samples; significantc differences at α ≤ 0.01; ndd = not detected.Table 4
Hamlin late asymptomatic versus symptomatic compound concentration.
Compound
A
S
a concentration (ppm)
S
Y
b concentration (ppm)
p value
Difference
α-Pinene
4757
4655
0.041
1.9%
β-Myrcene
12216
13601
0.720
3.6%
Linalool
447
242
<0.0001c
45.9%
Citronellal
648
725
<0.0001c
10.6%
Decanal
1302
716
<0.0001c
45.0%
Citronellol
128
123
0.0044
c
3.9%
Neral
438
231
<0.0001c
47.2%
Carvone
45.6
33.5
<0.0001c
26.5%
2-Decenal
n
d
d
nd
nd
nd
Geranial
650
386
<0.0001c
40.6%
Perillaldehyde
122
115
0.020
5.7%
2,4-Decadienal
nd
nd
nd
nd
Citronellyl acetate
50.2
57.5
0.0034c
12.7%
α-Terpinyl acetate
55.1
62.7
0.0090c
12.1%
Dodecanal
875
795
0.00066c
9.1%
A
S
a = huanglongbing asymptomatic samples; SYb = huanglongbing symptomatic samples; significantc differences at α ≤ 0.01; ndd = not detected.Table 5
Valencia early asymptomatic versus symptomatic compound concentration.
Compound
A
S
a concentration (ppm)
S
Y
b concentration (ppm)
p value
Difference
α-Pinene
4881
4704
0.170
3.6%
β-Myrcene
12149
11525
0.196
5.1%
Linalool
5312
4274
0.00020
c
19.5%
Citronellal
575
740
<0.0001c
22.3%
Decanal
3361
2630
<0.0001c
21.8%
Citronellol
69.1
57.6
0.019
16.6%
Neral
1203
953
<0.0001c
20.8%
Carvone
61.3
47.5
0.00025
c
22.5%
2-Decenal
35.0
21.9
0.00084
c
27.2%
Geranial
1556
1301
0.00011
c
16.4%
Perillaldehyde
266
302
0.00058
c
11.9%
2,4-Decadienal
37.8
27.5
0.064
27.2%
Citronellyl acetate
18.4
18.2
0.863
1.1%
α-Terpinyl acetate
30.1
27.7
0.462
8.0%
Dodecanal
1180
1082
0.0013
c
8.2%
A
S
a = huanglongbing asymptomatic samples; SYb = huanglongbing symptomatic samples; significantc differences at α ≤ 0.01.Table 6
Valencia late asymptomatic versus symptomatic compound concentration.
Compound
A
S
a concentration (ppm)
S
Y
b concentration (ppm)
p value
Difference
α-Pinene
4936
4664
0.030
5.5%
β-Myrcene
12251
11791
0.448
3.8%
Linalool
4260
4548
0.0083
c
6.3%
Citronellal
682
995
<0.0001c
31.5%
Decanal
3541
2712
<0.0001c
23.4%
Citronellol
134.9
113.7
<0.0001c
15.7%
Neral
1007
876
0.00025
c
13.0%
Carvone
81.1
60.3
<0.0001c
25.6%
2-Decenal
47.5
25.6
<0.0001c
46.1%
Geranial
1350
1165
<0.0001c
13.7%
Perillaldehyde
257
307
0.00011
c
16.3%
2,4-Decadienal
53.2
33.6
0.00091
c
36.8%
Citronellyl acetate
21.8
20.3
0.144
6.9%
α-Terpinyl acetate
35.4
34.1
0.517
3.7%
Dodecanal
1276
1194
0.00081
c
6.4%
A
S
a = huanglongbing asymptomatic samples; SYb = huanglongbing symptomatic samples; significantc differences at α ≤ 0.01.Figure 1
Chromatogram from Hamlin Early Asymptomatic.Note. 2-Decenal and 2,4-decadienal are not labeled on the chromatogram as they were not detected in this sample. Compound∗ has lower concentration compared to HES. Compound∗∗ has higher concentration compared to HES.Figure 2
Chromatogram from Hamlin Early Symptomatic.Note. 2-Decenal and 2,4-decadienal are not labeled on the chromatogram as they were not detected in this sample. Compound∗ has lower concentration compared to HEA. Compound∗∗ has higher concentration compared to HEA.Figure 3
Chromatogram from Hamlin Late Asymptomatic.Note. While 2-decenal and 2,4-decadienal were quantifiable in this sample, they were not labeled on the chromatogram as the peaks are too small to be seen. Compound∗ has lower concentration compared to HLS. Compound∗∗ has higher concentration compared to HLS.Figure 4
Chromatogram from Hamlin Late Symptomatic.Note. While 2-decenal and 2,4-decadienal were quantifiable in this sample, they were not labeled on the chromatogram as the peaks are too small to be seen. Compound∗ has lower concentration compared to HLA. Compound∗∗ has higher concentration compared to HLA.Figure 5
Chromatogram from Valencia Early Asymptomatic.Note. While 2,4-decadienal was quantifiable in this sample, it was not labeled on the chromatogram as the peak is too small to be seen. Compound∗ has lower concentration compared to VES. Compound∗∗ has higher concentration compared to VES.Figure 6
Chromatogram from Valencia Early Symptomatic.Note. While 2-decenal and 2,4-decadienal were quantifiable in this sample, they were not labeled on the chromatogram as the peaks are too small to be seen. Compound∗ has lower concentration compared to VEA. Compound∗∗ has higher concentration compared to VEA.Figure 7
Chromatogram from Valencia Late Asymptomatic.Note. While 2,4-decadienal was quantifiable in this sample, it was not labeled on the chromatogram as the peak is too small to be seen. Compound∗ has lower concentration compared to VLS. Compound∗∗ has higher concentration compared to VLS.Figure 8
Chromatogram from Valencia Late Symptomatic.Note. While 2-decenal and 2,4-decadienal were quantifiable in this sample, they were not labeled on the chromatogram as the peaks are too small to be seen. Compound∗ has lower concentration compared to VLA. Compound∗∗ has higher concentration compared to VLA.


### 3.2.1. Effect of Disease Stage

Each of the 15 compounds was analyzed as analytical standards by GC-FID in duplicate at six concentrations to create a standard curve. Each CPOO sample was analyzed by GC-FID in triplicate, and the concentration of individual compounds was determined based on the equation derived from the standard curve. Tables3–6 show differences between AS and SY compounds for Early Hamlin, Late Hamlin, Early Valencia, and Late Valencia, respectively.For Early Hamlin (Table3), significant differences (α ≤ 0.01) in concentration between AS and SY oil were observed for linalool, citronellal, decanal, neral, carvone, geranial, citronellyl acetate, and α-terpinyl acetate. The largest percent differences were for linalool (26.4%), decanal (34.6%), neral (30.6%), geranial (26.8%), and citronellyl acetate (26.0%). Of the compounds for which there was a significant difference, AS concentration was higher for linalool, decanal, neral, carvone, and geranial. Conversely, SY concentration was significantly higher for citronellal, citronellyl acetate, and α-terpinyl acetate.For Late Hamlin (Table4), significant differences (α ≤ 0.01) in concentration between AS and SY oil were observed for linalool, citronellal, decanal, citronellol, neral, carvone, geranial, citronellyl acetate, α-terpinyl acetate, and dodecanal. The largest percent differences were for linalool (45.9%), decanal (45.0%), neral (47.2%), carvone (26.5%), and geranial (40.6%). Of the compounds for which there was a significant difference, AS concentration was higher for linalool, decanal, citronellol, neral, carvone, geranial, and dodecanal. Conversely, SY concentration was significantly higher for citronellal, citronellyl acetate, and α-terpinyl acetate.For Early Valencia (Table5), significant differences (α ≤ 0.01) in concentration between AS and SY oil were observed for linalool, citronellal, decanal, citronellol, neral, carvone, 2-decenal, geranial, perillaldehyde, and dodecanal. The largest percent difference was for 2-decenal (27.2%); this was the only significant percent difference which was ≥25.0%. Of the compounds for which there was a significant difference, AS concentration was higher for linalool, decanal, citronellol, neral, carvone, 2-decenal, geranial, and dodecanal. Conversely, SY concentration was significantly higher for citronellal and perillaldehyde.For Late Valencia (Table6) significant differences (α ≤ 0.01) in concentration between AS and SY oil were observed for linalool, citronellal, decanal, citronellol, neral, carvone, 2-decenal, geranial, perillaldehyde, 2,4-decadienal, and dodecanal. The largest percent differences were for citronellal (31.5%), carvone (25.6%), and 2-decenal (46.1%). Of the compounds for which there was a significant difference, AS concentration was higher for linalool, decanal, citronellol, neral, carvone, 2-decenal, geranial, 2,4-decadienal, and dodecanal. Conversely, SY concentration was significantly higher for citronellal and perillaldehyde.Some trends were gleaned from these results based on compound concentration and disease stage. Overall, no changes inα-pinene and β-myrcene concentration were observed based on disease stage. Compounds for which AS oil had significantly higher concentrations were linalool, decanal, citronellol, neral, geranial, carvone, dodecanal, and 2-decenal; the latter was only detectable in Valencia oil. Compounds for which SY oil had significantly higher concentration were citronellal for both Hamlin and Valencia samples, α-terpinyl acetate and citronellyl acetate for Hamlin samples, and perillaldehyde for Valencia samples.In HLB affected oranges both the physiology of the whole orange as well as the orange juice resemble that of less mature fruit [10]. Volatiles, including the dominant terpenoid class found in orange oil, are generally secondary metabolites. Unlike primary metabolites, secondary metabolites do not support the primary life functions of a plant, and a dearth of secondary metabolites will not directly result in plant death. The terpenoids in orange oil, primarily d-limonene, are secondary metabolites which play roles as chemical messengers [28]. It is possible that HLB affects oranges such that as the severity of HLB increases, volatile formation via secondary metabolism is inhibited in a substantial way. Previous studies on HLB affected orange juice [9, 10] show that overall many volatile compound concentrations decrease when oranges are HLB positive.Dagulo et al. investigated the effects of HLB on the volatiles in orange juice [9]. In this study, control (HLB unaffected), AS, and SY Valencia juice was analyzed by GC-MS to determine how HLB affects relative volatile concentrations. Of the 15 compounds studied qualitatively in our research, Dagulo et al. reported on eight of these compounds: α-pinene, β-myrcene, decanal, linalool, citronellol, carvone, citronellyl acetate, and perillaldehyde. When comparing SY to AS juice, α-pinene, β-myrcene, citronellyl acetate, and citronellol showed no significant differences between AS and SY juices. Our research supports the results seen concerning α-pinene and β-myrcene, but we found that citronellol was significantly higher in AS CPOO than in SY CPOO and citronellyl acetate was significantly higher in SY CPOO than AS CPOO. Dagulo et al. found that decanal and carvone had significantly higher concentrations in AS orange juice compared to SY orange juice. These results were corroborated by our study. Dagulo et al. found linalool and perillaldehyde to have significantly higher concentrations in SY orange juice compared to AS orange juice. These results were corroborated by our study for perillaldehyde but not for linalool. Overall, our observed relative volatile concentrations of α-pinene, β-myrcene, decanal, carvone, and perillaldehyde corroborated the research performed by Dagulo et al. However, we found different results for linalool, citronellyl acetate, and citronellol. While orange juice and CPOO share many of the same volatile compounds, they are ultimately different substances. While some of the volatiles found in orange juices are derived from the peel, many of the same compounds are independently found in the essence oil, a product which is derived from orange juice. All seven compounds of interest compared to the study by Dagulo et al. can be found in essence oil [29].The concentration increases for citronellal in both Hamlin and Valencia CPOO as well asα-terpinyl acetate and citronellyl acetate for Hamlin CPOO and perillaldehyde for Valencia CPOO are of interest. However, no studies supporting increases in citronellal or citronellyl acetate concentrations in plants during stress situations could be found. As this is the first study on the volatiles of HLB effected cold pressed orange oil, it is possible that this is a new development specific to HLB stress in citrus. Citronellal can be produced from citral (neral + geranial) [30]. It is possible that this pathway occurs more often in HLB SY fruit than in HLB AS fruit. A higher concentration of perillaldehyde in SY oils compared to AS Valencia oils was corroborated by the results seen in Valencia orange juice [9]. α-Terpinyl acetate has been shown to increase in concentration with water stress [31]. Additionally, while no specific data was given for α-terpinyl acetate or citronellyl acetate, some esters were shown to increase in concentration in HLB positive orange juice [10].Overall, it appears that, with increasing HLB severity, several important volatiles including linalool, decanal, citronellol, neral, geranial, carvone, dodecanal, and 2-decenal decrease in concentration. Citronellal in both Hamlin and Valencia samples,α-terpinyl acetate and citronellyl acetate for Hamlin samples, and perillaldehyde in Valencia samples show higher concentrations in HLB SY samples. It is possible that these compounds increased because the stress HLB causes the orange tree. These compounds may have all increased due to shifts in biochemical pathways which occurred in trees with more severe HLB symptoms, creating greater concentrations of these volatiles.


### 3.2.2. Effect of Seasonality

In addition to disease stage, the effect of seasonality (early or late harvest) on the concentration of the 15 selected compounds was also analyzed. Tables7–10 show differences between early and late season compound concentrations for Hamlin AS, Hamlin SY, Valencia AS, and Valencia SY CPOO, respectively.Table 7
Hamlin asymptomatic early versus late compound concentration.
Compound
Early concentration (ppm)
Late concentration (ppm)
p value
Difference
α-Pinene
5267
4757
0.00048
a
9.7%
β-Myrcene
12807
14133
0.372
9.3%
Linalool
660
447
0.00024
a
32.3%
Citronellal
506
648
<0.0001a
21.9%
Decanal
858
1302
<0.0001a
34.1%
Citronellol
129
128
0.868
0.78%
Neral
492
438
0.0025a
11.0%
Carvone
69.9
45.6
<0.0001a
34.8%
2-Decenal
n
d
b
nd
nd
nd
Geranial
732
650
0.029
11.2%
Perillaldehyde
140
122
0.00071a
12.9%
2,4-Decadienal
nd
nd
nd
nd
Citronellyl acetate
43.5
50.2
0.0031a
13.3%
α-Terpinyl acetate
52.8
55.1
0.323
4.2%
Dodecanal
767
875
0.00054a
12.3%
S
i
g
n
i
f
i
c
a
n
t
a differences at α ≤ 0.01; ndb = not detected.Table 8
Hamlin symptomatic early versus late compound concentration.
Compound
Early concentration (ppm)
Late concentration (ppm)
p value
Difference
α-Pinene
4903
4655
0.039
5.1%
β-Myrcene
13911
13607
0.602
2.2%
Linalool
486
242
<0.0001a
50.2%
Citronellal
601
725
0.00011
a
17.1%
Decanal
561
716
0.0019
a
21.6%
Citronellol
141
123
0.00090a
12.8%
Neral
341
231
<0.0001a
37.5%
Carvone
60.7
33.5
<0.0001a
44.8%
2-Decenal
n
d
b
nd
nd
nd
Geranial
536
386
<0.0001a
28.0%
Perillaldehyde
138
115
0.00034a
16.7%
2,4-Decadienal
nd
nd
nd
nd
Citronellyl acetate
58.8
57.5
0.132
2.2%
α-Terpinyl acetate
65.7
62.7
0.035
4.6%
Dodecanal
753
795
0.013
5.3%
S
i
g
n
i
f
i
c
a
n
t
a differences at α ≤ 0.01; ndb = not detected.Table 9
Valencia asymptomatic early versus late compound concentration.
Compound
Early concentration (ppm)
Late concentration (ppm)
p value
Difference
α-Pinene
4881
4936
0.660
1.1%
β-Myrcene
12149
12251
0.729
0.83%
Linalool
5312
4260
0.00032
a
19.8%
Citronellal
575
682
0.00021
a
15.7%
Decanal
3361
3541
0.0095
a
5.1%
Citronellol
69.1
134.9
<0.0001a
48.8%
Neral
1203
1007
0.00026
a
16.3%
Carvone
61.3
81.1
<0.0001a
24.4%
2-Decenal
35.0
47.5
0.00029
a
26.3%
Geranial
1556
1350
0.00027
a
13.2%
Perillaldehyde
266
257
0.058
3.4%
2,4-Decadienal
37.8
53.2
0.026
28.9%
Citronellyl acetate
18.4
21.8
0.058
15.6%
α-Terpinyl acetate
30.1
35.4
0.137
15.0%
Dodecanal
1180
1276
0.0014a
7.5%
S
i
g
n
i
f
i
c
a
n
t
a differences at α ≤ 0.01.Table 10
Valencia symptomatic early versus late compound concentration.
Compound
Early concentration (ppm)
Late concentration (ppm)
p value
Difference
α-Pinene
4704
4664
0.349
0.85%
β-Myrcene
11525
11791
0.691
2.3%
Linalool
4274
4548
0.0025a
6.0%
Citronellal
740
995
<0.0001a
25.6%
Decanal
2630
2712
0.026
3.0%
Citronellol
57.6
113.7
<0.0001a
49.3%
Neral
953
876
0.0013
a
8.1%
Carvone
47.5
60.3
0.00018
a
21.2%
2-Decenal
21.9
25.6
0.040
14.4%
Geranial
1301
1165
0.00018
a
10.5%
Perillaldehyde
302
307
0.177
1.6%
2,4-Decadienal
27.5
33.6
0.0081
a
18.2%
Citronellyl acetate
18.2
20.3
0.061
10.3%
α-Terpinyl acetate
27.7
34.1
0.031
18.8%
Dodecanal
1082
1194
0.00025
a
9.6%
S
i
g
n
i
f
i
c
a
n
t
a differences at α ≤ 0.01.For Hamlin AS (Table7), significant differences (α ≤ 0.01) in concentration between early and late CPOO were observed for α-pinene, linalool, citronellal, decanal, neral, carvone, perillaldehyde, citronellyl acetate, and dodecanal. The largest percent differences were for linalool (32.3%), decanal (34.1%), and carvone (34.8%). Of the compounds for which there was a significant difference, early concentration was higher for α-pinene, linalool, neral, carvone, geranial, and perillaldehyde. Conversely, late concentration was significantly higher for citronellal, citronellyl acetate, and decanal.For Hamlin SY (Table8), significant differences (α ≤ 0.01) in concentration between early and late CPOO were observed for linalool, citronellal, decanal, citronellol, neral, carvone, geranial, and perillaldehyde. The largest percent differences were for linalool (50.2%), neral (37.5%), carvone (44.8%), and geranial (28.0%). Of the compounds for which there was a significant difference, early concentration was higher for linalool, citronellol, neral, carvone, geranial, and perillaldehyde. Conversely, late concentration was significantly higher for citronellal and decanal.For Valencia AS (Table9), significant differences (α ≤ 0.01) in concentration between early and late CPOO were observed for linalool, citronellal, decanal, citronellol, neral, carvone, 2-decenal, geranial, and dodecanal. The largest significant percent differences were for citronellol (48.8%) and 2-decenal (26.3%). Of the compounds for which there was a significant difference, early concentration was higher for linalool, neral, and geranial. Conversely, late concentration was significantly higher for citronellal, decanal, citronellol, carvone, 2-decenal, and dodecanal.For Valencia SY (Table10), significant differences (α ≤ 0.01) in concentration between early and late CPOO were observed for linalool, citronellal, citronellol, neral, carvone, geranial, 2,4-decadienal, and dodecanal. The largest significant percent differences were for citronellal (25.6%) and citronellol (49.3%). Of the compounds for which there was a significant difference, early concentration was higher for neral and geranial. Conversely, late concentration was significantly higher for linalool, citronellal, citronellol, carvone, 2,4-decadienal, and dodecanal.Some trends were gleaned from these results based on compound concentration and seasonality (early or late harvest). Overall,α-pinene, β-myrcene, citronellyl acetate, and α-terpinyl acetate showed few or no changes based on harvest time. Compounds for which early CPOO had significantly higher concentrations were linalool, neral, geranial, carvone in Hamlin samples, and perillaldehyde in Valencia samples. Compounds for which Late CPOO had significantly higher concentrations were citronellal, decanal, dodecanal, citronellol in Hamlin samples, and carvone in Valencia samples.To assist in explaining these results, the literature was investigated to determine what effect harvest time (early or late) could have on volatile concentrations in CPOO. Attaway et al. studied the terpenes in various citrus oils month-by-month over the course of one season [32]. The last month surveyed in this study for Hamlin oranges was December. Maturity of oranges can vary year to year, and it can be assumed that December would correspond similarly to late season oranges in our study and that October or November would correspond to early season oranges, as early season fruit was generally harvested 6–8 weeks before late season fruit for our research. Attaway et al. showed that α-pinene and β-myrcene either decreased slightly or remain unchanged (depending on whether October or November is considered “early”) from early to late season. Linalool and geranial either increased or remained the same (depending on whether October or November is considered “early”) from early to late season. Our data corroborated the concentrations of α-pinene and β-myrcene remaining the same but did not corroborate an increase in the concentration of linalool or geranial as the season progressed.Kesterson et al. found that the aldehyde content in Valencia CPOO increased from February to March [6]. These were the months our early and late season Valencia oranges were harvested. As decanal is the primary aldehyde in orange oil [6], an increase in aldehyde content for late season fruit corroborates the data shown by Kesterson et al. Overall, there is a lack of published literature to determine if the changes in volatile concentration during different harvest months observed in our study are similar or different than what would be expected before the onset of HLB.


## 3.2.1. Effect of Disease Stage

Each of the 15 compounds was analyzed as analytical standards by GC-FID in duplicate at six concentrations to create a standard curve. Each CPOO sample was analyzed by GC-FID in triplicate, and the concentration of individual compounds was determined based on the equation derived from the standard curve. Tables3–6 show differences between AS and SY compounds for Early Hamlin, Late Hamlin, Early Valencia, and Late Valencia, respectively.For Early Hamlin (Table3), significant differences (α ≤ 0.01) in concentration between AS and SY oil were observed for linalool, citronellal, decanal, neral, carvone, geranial, citronellyl acetate, and α-terpinyl acetate. The largest percent differences were for linalool (26.4%), decanal (34.6%), neral (30.6%), geranial (26.8%), and citronellyl acetate (26.0%). Of the compounds for which there was a significant difference, AS concentration was higher for linalool, decanal, neral, carvone, and geranial. Conversely, SY concentration was significantly higher for citronellal, citronellyl acetate, and α-terpinyl acetate.For Late Hamlin (Table4), significant differences (α ≤ 0.01) in concentration between AS and SY oil were observed for linalool, citronellal, decanal, citronellol, neral, carvone, geranial, citronellyl acetate, α-terpinyl acetate, and dodecanal. The largest percent differences were for linalool (45.9%), decanal (45.0%), neral (47.2%), carvone (26.5%), and geranial (40.6%). Of the compounds for which there was a significant difference, AS concentration was higher for linalool, decanal, citronellol, neral, carvone, geranial, and dodecanal. Conversely, SY concentration was significantly higher for citronellal, citronellyl acetate, and α-terpinyl acetate.For Early Valencia (Table5), significant differences (α ≤ 0.01) in concentration between AS and SY oil were observed for linalool, citronellal, decanal, citronellol, neral, carvone, 2-decenal, geranial, perillaldehyde, and dodecanal. The largest percent difference was for 2-decenal (27.2%); this was the only significant percent difference which was ≥25.0%. Of the compounds for which there was a significant difference, AS concentration was higher for linalool, decanal, citronellol, neral, carvone, 2-decenal, geranial, and dodecanal. Conversely, SY concentration was significantly higher for citronellal and perillaldehyde.For Late Valencia (Table6) significant differences (α ≤ 0.01) in concentration between AS and SY oil were observed for linalool, citronellal, decanal, citronellol, neral, carvone, 2-decenal, geranial, perillaldehyde, 2,4-decadienal, and dodecanal. The largest percent differences were for citronellal (31.5%), carvone (25.6%), and 2-decenal (46.1%). Of the compounds for which there was a significant difference, AS concentration was higher for linalool, decanal, citronellol, neral, carvone, 2-decenal, geranial, 2,4-decadienal, and dodecanal. Conversely, SY concentration was significantly higher for citronellal and perillaldehyde.Some trends were gleaned from these results based on compound concentration and disease stage. Overall, no changes inα-pinene and β-myrcene concentration were observed based on disease stage. Compounds for which AS oil had significantly higher concentrations were linalool, decanal, citronellol, neral, geranial, carvone, dodecanal, and 2-decenal; the latter was only detectable in Valencia oil. Compounds for which SY oil had significantly higher concentration were citronellal for both Hamlin and Valencia samples, α-terpinyl acetate and citronellyl acetate for Hamlin samples, and perillaldehyde for Valencia samples.In HLB affected oranges both the physiology of the whole orange as well as the orange juice resemble that of less mature fruit [10]. Volatiles, including the dominant terpenoid class found in orange oil, are generally secondary metabolites. Unlike primary metabolites, secondary metabolites do not support the primary life functions of a plant, and a dearth of secondary metabolites will not directly result in plant death. The terpenoids in orange oil, primarily d-limonene, are secondary metabolites which play roles as chemical messengers [28]. It is possible that HLB affects oranges such that as the severity of HLB increases, volatile formation via secondary metabolism is inhibited in a substantial way. Previous studies on HLB affected orange juice [9, 10] show that overall many volatile compound concentrations decrease when oranges are HLB positive.Dagulo et al. investigated the effects of HLB on the volatiles in orange juice [9]. In this study, control (HLB unaffected), AS, and SY Valencia juice was analyzed by GC-MS to determine how HLB affects relative volatile concentrations. Of the 15 compounds studied qualitatively in our research, Dagulo et al. reported on eight of these compounds: α-pinene, β-myrcene, decanal, linalool, citronellol, carvone, citronellyl acetate, and perillaldehyde. When comparing SY to AS juice, α-pinene, β-myrcene, citronellyl acetate, and citronellol showed no significant differences between AS and SY juices. Our research supports the results seen concerning α-pinene and β-myrcene, but we found that citronellol was significantly higher in AS CPOO than in SY CPOO and citronellyl acetate was significantly higher in SY CPOO than AS CPOO. Dagulo et al. found that decanal and carvone had significantly higher concentrations in AS orange juice compared to SY orange juice. These results were corroborated by our study. Dagulo et al. found linalool and perillaldehyde to have significantly higher concentrations in SY orange juice compared to AS orange juice. These results were corroborated by our study for perillaldehyde but not for linalool. Overall, our observed relative volatile concentrations of α-pinene, β-myrcene, decanal, carvone, and perillaldehyde corroborated the research performed by Dagulo et al. However, we found different results for linalool, citronellyl acetate, and citronellol. While orange juice and CPOO share many of the same volatile compounds, they are ultimately different substances. While some of the volatiles found in orange juices are derived from the peel, many of the same compounds are independently found in the essence oil, a product which is derived from orange juice. All seven compounds of interest compared to the study by Dagulo et al. can be found in essence oil [29].The concentration increases for citronellal in both Hamlin and Valencia CPOO as well asα-terpinyl acetate and citronellyl acetate for Hamlin CPOO and perillaldehyde for Valencia CPOO are of interest. However, no studies supporting increases in citronellal or citronellyl acetate concentrations in plants during stress situations could be found. As this is the first study on the volatiles of HLB effected cold pressed orange oil, it is possible that this is a new development specific to HLB stress in citrus. Citronellal can be produced from citral (neral + geranial) [30]. It is possible that this pathway occurs more often in HLB SY fruit than in HLB AS fruit. A higher concentration of perillaldehyde in SY oils compared to AS Valencia oils was corroborated by the results seen in Valencia orange juice [9]. α-Terpinyl acetate has been shown to increase in concentration with water stress [31]. Additionally, while no specific data was given for α-terpinyl acetate or citronellyl acetate, some esters were shown to increase in concentration in HLB positive orange juice [10].Overall, it appears that, with increasing HLB severity, several important volatiles including linalool, decanal, citronellol, neral, geranial, carvone, dodecanal, and 2-decenal decrease in concentration. Citronellal in both Hamlin and Valencia samples,α-terpinyl acetate and citronellyl acetate for Hamlin samples, and perillaldehyde in Valencia samples show higher concentrations in HLB SY samples. It is possible that these compounds increased because the stress HLB causes the orange tree. These compounds may have all increased due to shifts in biochemical pathways which occurred in trees with more severe HLB symptoms, creating greater concentrations of these volatiles.


## 3.2.2. Effect of Seasonality

In addition to disease stage, the effect of seasonality (early or late harvest) on the concentration of the 15 selected compounds was also analyzed. Tables7–10 show differences between early and late season compound concentrations for Hamlin AS, Hamlin SY, Valencia AS, and Valencia SY CPOO, respectively.Table 7
Hamlin asymptomatic early versus late compound concentration.
Compound
Early concentration (ppm)
Late concentration (ppm)
p value
Difference
α-Pinene
5267
4757
0.00048
a
9.7%
β-Myrcene
12807
14133
0.372
9.3%
Linalool
660
447
0.00024
a
32.3%
Citronellal
506
648
<0.0001a
21.9%
Decanal
858
1302
<0.0001a
34.1%
Citronellol
129
128
0.868
0.78%
Neral
492
438
0.0025a
11.0%
Carvone
69.9
45.6
<0.0001a
34.8%
2-Decenal
n
d
b
nd
nd
nd
Geranial
732
650
0.029
11.2%
Perillaldehyde
140
122
0.00071a
12.9%
2,4-Decadienal
nd
nd
nd
nd
Citronellyl acetate
43.5
50.2
0.0031a
13.3%
α-Terpinyl acetate
52.8
55.1
0.323
4.2%
Dodecanal
767
875
0.00054a
12.3%
S
i
g
n
i
f
i
c
a
n
t
a differences at α ≤ 0.01; ndb = not detected.Table 8
Hamlin symptomatic early versus late compound concentration.
Compound
Early concentration (ppm)
Late concentration (ppm)
p value
Difference
α-Pinene
4903
4655
0.039
5.1%
β-Myrcene
13911
13607
0.602
2.2%
Linalool
486
242
<0.0001a
50.2%
Citronellal
601
725
0.00011
a
17.1%
Decanal
561
716
0.0019
a
21.6%
Citronellol
141
123
0.00090a
12.8%
Neral
341
231
<0.0001a
37.5%
Carvone
60.7
33.5
<0.0001a
44.8%
2-Decenal
n
d
b
nd
nd
nd
Geranial
536
386
<0.0001a
28.0%
Perillaldehyde
138
115
0.00034a
16.7%
2,4-Decadienal
nd
nd
nd
nd
Citronellyl acetate
58.8
57.5
0.132
2.2%
α-Terpinyl acetate
65.7
62.7
0.035
4.6%
Dodecanal
753
795
0.013
5.3%
S
i
g
n
i
f
i
c
a
n
t
a differences at α ≤ 0.01; ndb = not detected.Table 9
Valencia asymptomatic early versus late compound concentration.
Compound
Early concentration (ppm)
Late concentration (ppm)
p value
Difference
α-Pinene
4881
4936
0.660
1.1%
β-Myrcene
12149
12251
0.729
0.83%
Linalool
5312
4260
0.00032
a
19.8%
Citronellal
575
682
0.00021
a
15.7%
Decanal
3361
3541
0.0095
a
5.1%
Citronellol
69.1
134.9
<0.0001a
48.8%
Neral
1203
1007
0.00026
a
16.3%
Carvone
61.3
81.1
<0.0001a
24.4%
2-Decenal
35.0
47.5
0.00029
a
26.3%
Geranial
1556
1350
0.00027
a
13.2%
Perillaldehyde
266
257
0.058
3.4%
2,4-Decadienal
37.8
53.2
0.026
28.9%
Citronellyl acetate
18.4
21.8
0.058
15.6%
α-Terpinyl acetate
30.1
35.4
0.137
15.0%
Dodecanal
1180
1276
0.0014a
7.5%
S
i
g
n
i
f
i
c
a
n
t
a differences at α ≤ 0.01.Table 10
Valencia symptomatic early versus late compound concentration.
Compound
Early concentration (ppm)
Late concentration (ppm)
p value
Difference
α-Pinene
4704
4664
0.349
0.85%
β-Myrcene
11525
11791
0.691
2.3%
Linalool
4274
4548
0.0025a
6.0%
Citronellal
740
995
<0.0001a
25.6%
Decanal
2630
2712
0.026
3.0%
Citronellol
57.6
113.7
<0.0001a
49.3%
Neral
953
876
0.0013
a
8.1%
Carvone
47.5
60.3
0.00018
a
21.2%
2-Decenal
21.9
25.6
0.040
14.4%
Geranial
1301
1165
0.00018
a
10.5%
Perillaldehyde
302
307
0.177
1.6%
2,4-Decadienal
27.5
33.6
0.0081
a
18.2%
Citronellyl acetate
18.2
20.3
0.061
10.3%
α-Terpinyl acetate
27.7
34.1
0.031
18.8%
Dodecanal
1082
1194
0.00025
a
9.6%
S
i
g
n
i
f
i
c
a
n
t
a differences at α ≤ 0.01.For Hamlin AS (Table7), significant differences (α ≤ 0.01) in concentration between early and late CPOO were observed for α-pinene, linalool, citronellal, decanal, neral, carvone, perillaldehyde, citronellyl acetate, and dodecanal. The largest percent differences were for linalool (32.3%), decanal (34.1%), and carvone (34.8%). Of the compounds for which there was a significant difference, early concentration was higher for α-pinene, linalool, neral, carvone, geranial, and perillaldehyde. Conversely, late concentration was significantly higher for citronellal, citronellyl acetate, and decanal.For Hamlin SY (Table8), significant differences (α ≤ 0.01) in concentration between early and late CPOO were observed for linalool, citronellal, decanal, citronellol, neral, carvone, geranial, and perillaldehyde. The largest percent differences were for linalool (50.2%), neral (37.5%), carvone (44.8%), and geranial (28.0%). Of the compounds for which there was a significant difference, early concentration was higher for linalool, citronellol, neral, carvone, geranial, and perillaldehyde. Conversely, late concentration was significantly higher for citronellal and decanal.For Valencia AS (Table9), significant differences (α ≤ 0.01) in concentration between early and late CPOO were observed for linalool, citronellal, decanal, citronellol, neral, carvone, 2-decenal, geranial, and dodecanal. The largest significant percent differences were for citronellol (48.8%) and 2-decenal (26.3%). Of the compounds for which there was a significant difference, early concentration was higher for linalool, neral, and geranial. Conversely, late concentration was significantly higher for citronellal, decanal, citronellol, carvone, 2-decenal, and dodecanal.For Valencia SY (Table10), significant differences (α ≤ 0.01) in concentration between early and late CPOO were observed for linalool, citronellal, citronellol, neral, carvone, geranial, 2,4-decadienal, and dodecanal. The largest significant percent differences were for citronellal (25.6%) and citronellol (49.3%). Of the compounds for which there was a significant difference, early concentration was higher for neral and geranial. Conversely, late concentration was significantly higher for linalool, citronellal, citronellol, carvone, 2,4-decadienal, and dodecanal.Some trends were gleaned from these results based on compound concentration and seasonality (early or late harvest). Overall,α-pinene, β-myrcene, citronellyl acetate, and α-terpinyl acetate showed few or no changes based on harvest time. Compounds for which early CPOO had significantly higher concentrations were linalool, neral, geranial, carvone in Hamlin samples, and perillaldehyde in Valencia samples. Compounds for which Late CPOO had significantly higher concentrations were citronellal, decanal, dodecanal, citronellol in Hamlin samples, and carvone in Valencia samples.To assist in explaining these results, the literature was investigated to determine what effect harvest time (early or late) could have on volatile concentrations in CPOO. Attaway et al. studied the terpenes in various citrus oils month-by-month over the course of one season [32]. The last month surveyed in this study for Hamlin oranges was December. Maturity of oranges can vary year to year, and it can be assumed that December would correspond similarly to late season oranges in our study and that October or November would correspond to early season oranges, as early season fruit was generally harvested 6–8 weeks before late season fruit for our research. Attaway et al. showed that α-pinene and β-myrcene either decreased slightly or remain unchanged (depending on whether October or November is considered “early”) from early to late season. Linalool and geranial either increased or remained the same (depending on whether October or November is considered “early”) from early to late season. Our data corroborated the concentrations of α-pinene and β-myrcene remaining the same but did not corroborate an increase in the concentration of linalool or geranial as the season progressed.Kesterson et al. found that the aldehyde content in Valencia CPOO increased from February to March [6]. These were the months our early and late season Valencia oranges were harvested. As decanal is the primary aldehyde in orange oil [6], an increase in aldehyde content for late season fruit corroborates the data shown by Kesterson et al. Overall, there is a lack of published literature to determine if the changes in volatile concentration during different harvest months observed in our study are similar or different than what would be expected before the onset of HLB.


## 3.3. Comparison to the Literature

As no control or HLB unaffected fruit was available at the time of this study, the percent areas for compounds found in AS and SY Hamlin and Valencia CPOO were compared to three published studies on orange oils. Since few studies report the effects of seasonality, early and late percent areas measured in triplicate were averaged, resulting in a single value for each compound for both Hamlin and Valencia fruit.While several studies [33–37] have reported on the relative concentration of compounds in Florida CPOO, these studies tend to be older, somewhat outdated, and incomplete. Therefore, studies usingCitrus sinensis (L.) Osbeck fruit from other countries were used for this comparison for a more complete, modern perspective. Mitiku et al. used both Hamlin and Valencia oranges from Ethiopia [38]. Qiao et al. [27] used Chinese Jinchen oranges, while Mondello et al. [39] used Italian oranges. For comparison to the latter two studies, the same literature values are used in both the Hamlin and Valencia tables, since Hamlin and Valencia varieties were not used in these two studies. Although we could calculate percent peak area to 0.0001% based on the GC-FID output, it is customary in literature to report any concentration less than 0.01% as “trace”; therefore, this methodology is utilized in Tables 11 and 12.Table 11
Hamlin peak area comparison to the literature data.
Compound
AS % area
SY % area
Literature % area (Mitiku)
Literature % areaQiaoa
Literature % areaMondellob
α-Pinene
0.50
0.50
0.41
0.76
0.64
β-Myrcene
1.40
1.42
1.77
1.88
2.03
Linalool
0.04
0.03
0.12
0.92
0.30
Citronellal
0.03
0.04
0.01
0.06
0.04
Decanal
0.08
0.07
0.10
0.21
0.17
Citronellol
0.01
0.01
trace
n
r
c
nr
Neral
0.03
0.02
0.04
0.06
0.06
Carvone
Trace
Trace
nr
nr
nr
2-Decenal
Trace
Trace
nr
nr
nr
Geranial
0.04
0.04
0.08
0.11
0.13
Perillaldehyde
0.01
0.01
Trace
0.03
Trace
2,4-Decadienal
Trace
Trace
Trace
nr
nr
Citronellyl acetate
Trace
Trace
Trace
0.01
0.01
α-Terpinyl acetate
Trace
Trace
Trace
nr
nr
Dodecanal
0.04
0.05
0.01
0.03
Trace
C
h
i
n
e
s
e
a variety Jinchen Citrus sinensis (L.) Osbeck; Italianb  Citrus sinensis(L.) Osbeck; nrc = not reported.Table 12
Valencia peak area comparison to the literature data.
Compound
AS % area
SY % area
Literature % area (Mitiku)
Literature % areaQiaoa
Literature % areaMondellob
α-Pinene
0.50
0.50
0.35
0.76
0.64
β-Myrcene
1.21
1.21
1.79
1.88
2.03
Linalool
0.25
0.22
0.35
0.92
0.30
Citronellal
0.04
0.06
0.03
0.06
0.04
Decanal
0.29
0.22
0.16
0.21
0.17
Citronellol
0.02
0.01
Trace
n
r
c
nr
Neral
0.07
0.05
0.09
0.06
0.06
Carvone
Trace
Trace
nr
nr
nr
2-Decenal
Trace
Trace
nr
nr
nr
Geranial
0.10
0.09
0.16
0.11
0.13
Perillaldehyde
0.02
0.03
Trace
0.03
Trace
2,4-Decadienal
Trace
Trace
Trace
nr
nr
Citronellyl acetate
Trace
Trace
Trace
0.01
0.01
α-Terpinyl acetate
Trace
Trace
Trace
nr
nr
Dodecanal
0.07
0.07
0.02
0.03
Trace
C
h
i
n
e
s
e
a variety Jinchen Citrus sinensis (L.) Osbeck; Italianb  Citrus sinensis(L.) Osbeck; nrc = not reported.Based on the three literature studies presented, in Hamlin CPOO (Table11), the concentrations of linalool, decanal, neral, and geranial are low in comparison to literature values. The concentration of dodecanal is high. Especially notable is the linalool content, which is approximately 10 times less than the average concentration seen in the three literature studies. It should be noted that Florida Hamlin CPOO has never been considered as high quality AS Valencia CPOO and was often not collected during orange juice processing [6]. The geranial deficit is also notable, with the content being 2.5 times less than the literature average. Dodecanal increased 2.3 times compared to the two reported literature concentrations. The deficits in decanal (2.1 times less) and neral (2.1 times less) and the increase in dodecanal are not as severe as the other changes yet are still notable.Valencia CPOO also showed notable changes compared to literature CPOO concentrations. Linalool (2.2 times less) had a notably lower concentration compared to the literature values. Citronellol and dodecanal (2.8 times more) had notably higher concentrations. Citronellol is generally either found in only trace quantities or is not quantifiable in CPOO, so the exact increase in the concentration of citronellol compared to literature values could not be determined. Geranial had a slightly lower (1.4 times less) and decanal a slightly higher (1.4 times more) concentration than the literature averages.Both linalool and geraniol are formed from the precursor geranyl diphosphate [40]. Geraniol, in turn, can be transformed either to citral (neral + geranial) or citronellol. Citronellol transformation from geraniol may increase during plant stress [41]. Additionally, α-terpinyl acetate can be derived from linalool [26]. During stress, certain volatile compounds can increase in concentration. Stress related volatile concentration change has not been studied in direct relation to HLB, so other forms of stress are considered. Water stress has been shown to increase the concentration of citronellol in aromatic grasses [41] and dodecanal in tea [42]. It is possible that stress from HLB causes oranges to increase production of citronellol and dodecanal. Overall, when compared to historical results, our samples show a large concentration deficit in linalool, small deficits in neral and geranial, and increases in citronellol and α-terpinyl acetate.


## 4. Conclusions

This study illuminated some of the effects huanglongbing has on the quality of cold pressed orange oil. The only compounds identified which are not normally identified in cold pressed orange oil areβ-longifolene, perillene, and 4-decenal. These compounds have all been previously identified in other orange products. It is possible that β-longifolene, perillene, and 4-decenal are biomarkers for huanglongbing, but further research is necessary for confirmation. Nerol and β-elemene are commonly reported in cold pressed orange oil but were not found in our samples. β-Elemene may have coeluted with β-cubebene. Nerol may have coeluted with citronellol or may not have been present.Results showed that, based on disease stage,α-pinene and β-myrcene showed few changes in concentration. Linalool, decanal, citronellol, neral, geranial, carvone, dodecanal, and 2-decenal (only detectable in Valencia) had higher concentrations in asymptomatic samples compared to symptomatic samples. Perillaldehyde in Valencia oil, α-terpinyl acetate, and citronellyl acetate in Hamlin oil and citronellal in both showed higher concentrations in symptomatic than asymptomatic samples. It is possible that the biochemical pathways which form volatiles in orange oils are disrupted by huanglongbing, causing some compounds to change concentration based on the severity of huanglongbing infection. Concentrations of several important compounds including linalool, geranial, and neral were significantly lower than what is typically seen in the literature while dodecanal and citronellol had increased concentrations. Future studies on huanglongbing should focus on the concentrations of these five volatiles. There are additional differences based on harvest time and harvest year, so it is important to keep in mind that there are more effects than merely disease stage at play when differences in physiochemical properties are noted.



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*Source: 6793986-2017-11-15.xml*