Effect of Abscission Zone Formation on Orange (Citrus sinensis) Fruit

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Effect of abscission zone formation on orange (Citrus sinensis) fruit/juice quality for trees affected by Huanglongbing (HLB) Elizabeth Baldwin, Anne Plotto, Jinhe Bai, John A. Manthey, Wei Zhao, Smita Raithore, and Mike Irey J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05635 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 8, 2018

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Journal of Agricultural and Food Chemistry

Effect of abscission zone formation on orange (Citrus sinensis) fruit/juice quality for trees affected by Huanglongbing (HLB)

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Elizabeth Baldwin1*, Anne Plotto1, Jinhe Bai1, John Manthey1, Wei Zhao1, Smita Raithore2 and Mike Irey3

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USDA-ARS Horticultural Research Laboratory, Fort Pierce, FL 34945

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Symrise AG, Teterboro, NJ 07608

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Southern Gardens Citrus Nursery LLC, Clewiston, FL 33440

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*Corresponding author, [email protected]

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This article is a US Government work and is in the public domain in the USA. Mention of a trademark or proprietary product is for identification only and does not imply a guarantee or warranty of the product by the US Department of Agriculture. The US Department of Agriculture prohibits discrimination in all its programs and activities on the basis of race, color, national origin, gender, religion, age, disability, political beliefs, sexual orientation, and marital or family status.

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Abstract: Orange trees affected by huanglongbing (HLB) exhibit excessive fruit drop, and fruit

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loosely attached to the tree may have inferior flavor. Fruit were collected from healthy and

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HLB-infected (Candidatus liberibacter asiaticus) ‘Hamlin’ and ‘Valencia’ trees. Prior to harvest,

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the trees were shaken, fruit that dropped collected, tree-retained fruit harvested and all fruit

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juiced. For chemical analyses, sugars and acids were generally lowest in HLB dropped (HLB-D)

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fruit juice compared to non-shaken healthy (H), healthy retained (H-R) and healthy dropped fruit

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(H-D) in early season (December) but not for the late season (January)’Hamlin’ or ‘Valencia’

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except for sugar/acid ratio. The bitter limonoids, many flavonoids and terpenoid volatiles were

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generally higher in HLB juice, especially HLB-D juice, compared to the other samples. The

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lower sugars, higher bitter limonoids, flavonoids and terpenoid volatiles in HLB-D fruit, loosely

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attached to the tree, contributed to off-flavor as was confirmed by sensory analyses.

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Keywords: flavor, aroma volatiles, flavonoids, limonoids, qPCR, sensory, HLB

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INTRODUCTION The citrus industry has been devastated by citrus greening or huanglongbing (HLB)

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disease 1-3, which has reduced yields and fruit/juice quality 1, 4-9. Florida orange production has

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fallen by over 70% due to the hurricanes of 2004 and 2005, citrus canker disease and HLB

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disease 10. In recent years production, not related to hurricanes and canker, has declined by over

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50%. In Florida, HLB disease is thought to be caused by a gram negative bacteria Candidatus

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liberibacter asiaticus (CLas), vectored by a psyllid (Diaphorina citri) 1. Orange (Citrus sinensis)

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fruit quality is impacted by HLB disease, which causes the fruit to become smaller and greener

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and these symptomatic fruit are more likely to have reduced sugars, sometimes higher acids and

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higher bitter limonoids and astringent flavonoids, especially earlier in the harvest season 4.

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Aroma volatiles are also changed with less esters and sesquiterpenes and more terpene aroma

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volatiles in HLB symptomatic versus healthy fruit juice 6. Flavor of some orange varieties is

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more impacted by HLB than others in terms of lower sugars and higher limonoids and

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flavonoids, but all tested so far are affected. Sensory studies confirmed the flavor chemical

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profile in that juice from fruit symptomatic for HLB disease, were perceived to be less sweet,

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more bitter and more astringent than juice from healthy fruit7.

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The reduction in yield due to HLB is partially attributed to the premature fruit drop,

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perhaps due to carbohydrate or water deficiency, resulting in loss of leaves and root mass typical

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of HLB decline. On the other hand, it could also be related to secondary infection, such as has

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been recently related to the fungus Lasiodiplodia theobromae (formerly known as Diplodia

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natalensis) 11, 12, hereafter referred to as Diplodia, the causal organism of citrus stem end rot.

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Diplodia normally infects the flower blossom and remains quiescent until after harvest, causing

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postharvest stem end rot. Apparently, in the case of HLB in Florida, Diplodia infects the orange



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fruit abscission zone on HLB-weakened trees, as well as the fruit itself while still on the tree,

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rather than postharvest, and induces the fruit to produce ethylene gas which promotes abscission

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zone formation, thus resulting in a loosening of fruit on the tree and eventually fruit drop 11, 13.

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This may affect the quality of fruit loosely held on the tree since partial abscission zone

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formation may affect the transportation of hormones and nutrients to the fruit.

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The objectives of this study were to determine if fruit that are ready to abscise, and are

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thus loose on the tree with an abscission zone at least partially formed, would be of equal or

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lower quality compared to fruit held tightly on the tree for either healthy (Clas negative) or HLB-

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affected (Clas positive) trees. If so, then development of treatments to prevent the fruit

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abscission zone from forming may prevent fruit drop and improve fruit quality. If Diplodia is

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exacerbating the abscission rather than the tree shedding fruit due to lack of resources due to

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decline from HLB, then perhaps judicious use of appropriate fungicides would be warranted,

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preventing fruit drop and improving fruit quality.

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MATERIALS AND METHODS Fruit material. Six-year old ‘Hamlin’ orange trees (Citrus sinensis (L.) Osbeck), about

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2.5-3.0 m tall, on 'Swingle' citrumelo (C. paradisi Macf. × Poncirus trifoliata (L) Raf.)

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rootstock, were located in a commercial grove located in southern Florida. Eighteen trees were

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selected for the experiment, of which nine were CLas negative (healthy) and non-symptomatic

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for the disease and the other nine were HLB symptomatic and CLas positive (HLB), as tested by

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qPCR using the method of Li et al. 14. The selected trees were similar in size, in close proximity

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to each other, and all were grown under similar agro-climatic conditions and received common


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cultural practices and the grower’s standard pest and disease management. Each set of three trees

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for either healthy or HLB-affected represented a field replication, so there were three composite

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replications of three trees. Fruit were harvested in December, 2014 and again in January, 2015

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(during commercial harvest season). The ground under the trees was cleaned of fruit and leaves

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just before shaking the trees, and trees were shaken manually. Trees were shaken until enough

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fruit fell on the ground to obtain a decent sample (89-199). Healthy trees required a more

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vigorous shaking to obtain enough dropped fruit. The dropped fruit (D) from the trees upon

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shaking were collected, and the retained fruit (R) were hand-picked off the trees. One set of

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healthy fruit were harvested from three additional trees in the same area that were not shaken (H)

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for comparison to healthy retained (H-R), healthy dropped (H-D), HLB retained (HLB-R) and

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HLB dropped (HLB-D) fruit. This was repeated for 6-year old ‘Valencia’ trees (same rootstock)

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in the same groves in April, 2015. To recap, there were 89-199 fruit from three trees per

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replicate for all treatments (H, H-R, H-D, HLB-R, HLB-D) and varieties/harvests, which were

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later juiced, resulting in three composite juice replicates per treatment/variety/harvest.

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For simulated industrial processing, a JBT 391 Extractor with “Premium Setting” was

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used for ‘Valencia’ and “Standard Industry Setting” used for ‘Hamlin’ fruit extraction as is a

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customary industry practice due to peel oil differences between the two varieties. Juice was

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passed through a pressure filtration finisher with screen size 0.51 mm and then pasteurized at 90

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°C for 10 s (1.2 L per min) and cooled to 10 °C using a pilot pasteurizer (UHT/HTST Lab

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25EHV Hybrid - Microthermics, Inc.; Raleigh, NC). Juices were cold filled into 1L glass bottles

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and cooled further to 5 °C using an ice bath and frozen at -10 °C until analyzed for chemical or

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sensorial characteristics (1-2 months). There were 89-199 fruit per replicate (representing 3

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trees) with three composite replicates per treatment. Each replicate fruit sample was processed



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and pasteurized separately. For ‘Hamlin’ fruit, the weight of the December 14th harvest ranged

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from 26.8-29.0 kg for the different fruit types; for the January 2015 harvest, from 17.2-24.5 kg;

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and for the ‘Valencia’ April harvest, from 26.8-28.1 kg. This resulted in three composite juiced

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fruit samples (representing juiced fruit from three trees = 89-199 juiced fruit). These composite

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juiced fruit replicates were used for all chemical and biochemical analyses.

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Fruit color and size. Prior to processing, color of 20 randomly selected fruits per

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replicate, with three replicates per fruit type (total 60 fruits) was measured at three points around

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the equator of the fruit using a Chromameter (Minolta CR-300, Tokyo, Japan), calibrated to a

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white plate using the CIE L*, a*, b* system including chroma and hue 15, however only a*, b*

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and hue values were used. Fruit size of the same 20 fruits per replicate with three replicates per

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treatment (total 60 fruit) was measured for horizontal diameter (equator of the fruit), and by the

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sizing machinery at the processing plant (JBT sizer set as 7.94 cm), with

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3 replicates per treatment of 84-199 fruit per replicate. The fruit used for color and diameter

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measurements were later added back to the appropriate replicated fruit samples for processing.

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Sugar and acid analyses. For quality determination, soluble solids content (SS) and

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titratable acidity (TA) of the juice replicates (comprised of 89-199 juiced fruit) was determined

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prior to individual sugars and acids analyses. SS, determined by refractive index, was measured

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with a digital ATAGO PR-101 refractometer (Atago Co, Tokyo, Japan), and TA was calculated

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from titration of 10mL of juice with 0.1 mol· L-1 NaOH to a pH 8.1 endpoint using a 808

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Titrando (Metrohm, Riverview, FL, USA). Individual sugars (sucrose, glucose and fructose)

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were analyzed with a high performance liquid chromatography (HPLC) system following an

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optimized extraction of the juice samples 16 and their values summed for total sugars (TS). Juice

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samples were centrifuged (Eppendorf microfuge, Westbury, NY) at 11,952 x g for 20 min at 6 ACS Paragon Plus Environment

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10 °C. A total of 10 mL of the filtered solution was passed through a C-18 Sep-Pak

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(Waters/Millipore), and the eluate was filtered with a 0.45 µm Millipore (Siemens-Millipore,

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Shrewbury, MA) filter before analysis by HPLC with a Sugar-Pak™ column (10 µm, 6.5 mm x

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300 mm) (Waters, Milford, MA) operated at 90 °C in a CH-30 column heater and a TC-50

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controller (FIAtron, Milwaukee, WI) and an Agilent 1100 series refractive index detector

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(Agilent Technologies, Santa Clara, CA). Quantification was based on the external standard

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method (EZChrom Elite software, Version 3.3.2. SP2, Santa Clara, CA) using standards for

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sucrose, glucose and fructose. All results are expressed as g·100 mL-1 of juice. Sucrose

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equivalence (SE) was calculated by assigning sweetness values to glucose and fructose by

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multiplying their concentrations by 0.74 and 1.73, respectively, to normalize them to the

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sweetness value of sucrose 17. Organic acids (citric and malic) along with total ascorbic acid (TAA) were also analyzed

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by HPLC of the same extracts that were prepared for the individual sugars. Chromatographic

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separation was done with an AltechOA1000 Prevail organic acid column (9 µm, 300 mm x

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6.5 mm) (Grave Davison Discovery Sciences, Deerfield, IL) with a Spectra System UV 6000 LP

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photo diode array detector (Thermo Fisher Scientific, Waltham, MA). Quantification was based

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on the calibration curves for standards of citric, malic, ascorbic and dehydroascorbic acids,

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expressed as g·100 mL-1 of juice. Ascorbic and dehydro ascorbic acids were combined for TAA

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16

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. Flavonoid and limonoid analyses. Concentrations of limonin and nomilin in orange

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juice were determined by high performance liquid chromatography mass spectrometry (HPLC-

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MS) modifying a previous method 18. Triplicate samples of pasteurized orange juice (35g) were

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centrifuged at 27,000 x g for 30 min. Supernatants were collected and pellets were carefully re7 ACS Paragon Plus Environment


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suspended with 35mL deionized water for further centrifugation. The final pellets collected

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(supernatant discarded) were vacuum dried at 55 °C. Prior to analysis, the collected supernatant

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samples (970 µL), from the first centrifugation, were spiked with 30 µL mangiferin (internal

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standard, 0.18 mg/mL) and analyzed without further processing. For pellet sample preparation,

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the vacuum dried samples were ground to fine powder under liquid nitrogen. The ground pellet

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(100 mg) was then extracted with 3mL dimethyl sulfoxide (DMSO) by shaking for 18h with a

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platform shaker (New Brunswick Scientific, Enfield, CT) at 110 rpm at 25 °C. The extracts were

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centrifuged at 7500 x g for 15 min to remove any solid particulates. The supernatant (970 µL)

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was placed in a vial containing 30 µL mangiferin (internal standard, 0.18 mg·mL-1) prior to

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analysis by HPLC-MS. The HPLC-MS system used to analyze samples consisted of a Waters

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2695 Alliance HPLC (Waters, Medford, MA) connected in parallel with a Waters 996 (Photo

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Diode Array) PDA detector and a Waters/Micromass ZQ single quadrupole mass spectrometer

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equipped with an electrospray ionization source as previously described by Baldwin et al. 4.

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Aroma volatile analysis. Three mL of juice was transferred to a 10 mL crimp-capped

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vial, rapidly frozen in liquid nitrogen then stored at -80 °C. Frozen samples were thawed under

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running tap water and inserted into a Gerstel multipurpose autosampler for headspace injection

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onto an Agilent 6890 (Agilent Technologies) GC equipped with Stabilwax and HP-5 low bleed

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columns. The flow rate was split equally to the two columns at 17 mL·min-1 at 40 ºC with an

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increase in temperature at 6 °C⋅min-1 up to 180 ºC, where the temperature was held constant for

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an additional 5.8 min. The GC peaks for the aroma volatile compounds were quantified using

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standard curves as determined by enrichment of deodorized orange juice by known

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concentrations of authentic volatile compound standards as described for orange juice samples

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by Baldwin et al. 4. Compound identification was confirmed using Solid Phase Micro


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Extraction (SPME) fibers with mass spectroscopy (MS). Confirmation by MS was accomplished

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by solid phase micro-extraction (SPME, 50/30 um DVB/CAR/PDMS, Supelco Bellefonte, PA)

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as reported by Wang et al. 19. The instrument and settings for SPME injection: GC-MS (model

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6890 GC + 5973N MS; Agilent) with non-polar column (0.25 mm x 60 m, 0.50 µ film thickness,

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DB-5, Agilent). Volatile compounds were identified by the comparison of retention indices and

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mass spectra with library entries (NIST/EPA/NIH Mass spectral Library version 2.0; National

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Institute of Standards and Technology, Gaithersburg, MD).

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DNA extraction and qPCR detection of CLas from juice. DNA was extracted from

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500 µL of each of the three replicates of orange juice per treatment/variety using a modified

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CTAB method as described in a patent publication 20. DNA quality (260/280 and 260/230 ratio)

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and quantity were assessed by spectrophotometry (Nano Drop, Thermo Scientific, Waltham,

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MA). CLas detection was accomplished by qPCR. Specific primers targeting CLas 16S rRNA

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gene (Li primers) 14 or CLas hyv1 (LJ primers) 21 were synthesized by Integrated DNA

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Technologies, Inc. (Coralville, IA). PCR mixtures with a total volume of 15 µl contained 7.5 µl

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of TaqMan PCR master mix (Applied Biosystems) for Li primers, or SYBR Green PCR Master

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Mix (Applied Biosystems) for LJ primers, 250 nM each primer, 150 nM probe (for Li primers)

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and 100 ng of template DNA. Real-time PCR amplifications were performed in a 7500 real-time

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PCR system (Applied Biosystems, Foster City, CA). The qPCR parameters were as follows: 95

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°C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min, with fluorescence

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signal capture at each stage of 60 °C. For SYBR® Green Real-Time PCR (with LJ primers), the

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default Melt Curve (disassociation) stage is continued after the 40 cycles of PCR. Cycle

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threshold (Ct) values were analyzed using ABI 7500 Software version 2.0.6 (Applied



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Biosystems, Inc., Carlsbad, CA) with a manually set threshold at 0.02 and automated baseline

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settings.

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Sensory analysis. Difference tests and descriptive analysis were performed on thawed

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juice samples with untrained and trained panelists, respectively. For difference tests, 55

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untrained panelists, staff of the U.S. Horticultural Research Laboratory in Fort Pierce, Florida

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were presented two pairs of samples: the first pair was juice from Healthy trees, the second pair

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was juice from HLB trees. Each pair was either retained/dropped or retained/retained or

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dropped/dropped. The design allowed for 16 possible combinations of order of presentation of

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the two pairs. Panelists were asked to taste sequentially each pair, and determine whether the two

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samples in each pair were the same or different. The pair of healthy samples was always

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presented before the pair of HLB samples due to the lingering off-flavor of some of the HLB

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samples. Panelists were also asked to give comments explaining their choice in a free comment

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question. Juice was served as 18 mL in 30 mL cups at 14 °C. Each replication was served on a

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separate day. For the trained descriptive panel, 13 panelists were trained for 55+ hours to

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describe orange juice. The core of the trained panelists had tasted orange juice as affected by

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HLB for more than 3 years. Panelists were presented the five different treatments in a

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randomized order. Field replications were served as separate tasting sessions and panelists rated

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each sample one at a time. Reference standards for each of the descriptors were also presented 22.

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Juice samples were served as 45 mL in 118 mL cups at 14 °C. All tasting took place in isolated

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booths under red lighting. Data were recorded using Compusense® five v.5.6 (Compusense,

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Guelf, ON, Canada).

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Statistical analysis. For chemical and physical analyses, analysis of variance (ANOVA) for each attribute/compound was conducted using the ANOVA procedure in SAS (Version 9.3; 10 ACS Paragon Plus Environment

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SAS Institute, Gary, NC, USA). Mean separation was determined by Tukey’s test at the 5%

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level. For multivariate statistical analyses, principal component analysis (PCA) was performed

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using Senpaq v. 5.01 (Qi Statistics, Reading, UK) to test the separation among treatments based

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on the physical, chemical and biological measurements taken for this study. PCA was performed

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using the correlation matrix option to account for different scales in the variables.

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For sensory data analysis, the simple-difference test, results were tabulated and a Chi-

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Square analysis was calculated for each pair using Compusense® v. 5.6. Data from the

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descriptive trained panel were analyzed by ANOVA in a mixed model where “Panelists” is the

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random variable, and the main effect is tested against the interaction “Panelist x Sample”. A

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PCA was also performed. Both ANOVA and PCA were performed using Senpaq v. 5.01 and the

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PCA was also performed using the correlation matrix option.

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RESULTS AND DISCUSSION

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Healthy and HLB-affected ‘Hamlin’ and ‘Valencia’ orange trees, grown in south Florida,

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were shaken and fruit that fell were collected while fruit that remained on the tree were harvested

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with fruit also harvested from healthy, not-shaken trees. In a previous study, qPCR analysis of

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the fruit abscission zones of the fruit juiced in this study revealed that the dropped fruit from

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HLB trees compared to healthy trees had higher titers of Clas and especially L. theobromae, or

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Diplodia, a fungus that normally causes postharvest stem end rot 12. This fungus is now infecting

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the HLB-weakened fruit abscission zone on the tree, as well as invading the fruit, and reducing

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fruit/stem pull force, contributing to fruit drop as well as increased postharvest stem end rot, as

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described in previous studies 11, 12. Fruit that dropped from HLB trees (HLB-D) were the only

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ones that produced ethylene, a ripening and fruit abscission hormone, as described in a previous 11 ACS Paragon Plus Environment


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paper where an extra set of fruit from the same trees were tested for ethylene production 13.

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These fruit did not retain their calix, abscising under the calix compared to the HLB-R fruit that

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were harvested from the tree, for which the breakpoint was above the calix (Fig. 1). The juice

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from the different fruit types for early and late season ‘Hamlin’ as well as ‘Valencia’ were also

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tested for CLas titer using qPCR 20, where lower Ct values indicate greater infection by CLas.

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This was done to determine how infected the actual fruit (and associated vascular system for that

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part of the tree) were. ‘Hamlin’ healthy samples had Ct values generally close to or above what

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is considered the “healthy” range, for which there is no consensus on Ct cut-off, but the literature

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shows anywhere above Ct 32-37 for the Li primer (Fig. 2A). It appears that the H, and to a

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lesser extent H-R, samples came from trees, that although were symptomless for HLB, may have

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had some infection in some part of the tree, which increased between December and January.

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Meanwhile, the HLB samples were within what is considered the CLas “infected” range which is

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below Ct 32-35 23, 24. Identified ranges for “healthy” or “infected” have not been reported for the

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LJ primer (Fig. 2B), however, below Ct 32 seems reasonable in accordance with symptoms. For

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both Li and LJ primers the HLB juices had generally lower Ct values, and HLB-D appeared to be

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lower than HLB-R, whereas there was no obvious difference between H-R and H-D. To

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determine if the extent of the abscission zone formation (looseness of the fruit attachment to the

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tree) affects fruit and fruit juice quality, the fruit in this study were tested for fruit/juice physical,

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chemical and sensorial characteristics related to quality.

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Fruit color and size. Size distribution, as determined by the processor’s sizing

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equipment, showed that the HLB fruit types had the most fruit less than 6.35 cm and the least

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fruit in the 6.35 cm to 7.94 cm or the greater than 7.94 cm ranges (Fig. 3A). The HLB fruit

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were smaller than the healthy fruit, but there was no difference between HLB-R and HLB-D.


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For fruit diameter, HLB ‘Hamlin’ fruit from the December, 2014 harvest (early season ‘Hamlin’)

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had smaller diameters than all but H fruit, for which there was no significant difference (Fig.

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3B). For ‘Hamlin’ fruit harvested in January, 2015 (late season ‘Hamlin’), and the ‘Valencia’

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April, 2015 fruit, both HLB fruit types were smaller in diameter than the healthy types.

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For fruit peel color (Table 1), early season ‘Hamlin’ HLB-R and HLB-D samples had the

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lowest chromameter a* values (higher values indicate red color), HLB-R lowest chromameter b*

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values (higher value indicates less yellow, more green color) and both HLB samples exhibited

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lowest a*/b* (higher value roughly indicates orange color) and highest hue values (lower value

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indicates more red color), indicating they were more yellow/green and less orange than all the

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healthy samples (Table 1). For late season ‘Hamlin’ and ‘Valencia’ fruit, the results were similar

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with HLB fruit generally having lower chromameter a*, b*, a*/b* and higher hue values than the

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healthy samples (especially HLB-D for late season ‘Hamlin’ and HLB-R for ’Valencia’), again

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indicating that the HLB fruit were more yellow/green and less orange than the healthy samples.

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So overall the HLB fruit were greener/less orange than the healthy fruit, but there was no

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difference between HLB-R and HLB-D except for Valencia where HLB-R exhibited lower a*,

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b*, a*/b* and higher hue than HLB-D, indicating that it was more green/less orange than HLB-

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D, as indicated in Fig. 1. The dropped HLB fruit were shown to produce ethylene gas 12, which

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degreens fruit by breaking down chlorophyll 25, 26.

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Sugars and acids. For early season ‘Hamlin’ orange juice, the HLB-R fruit juice was

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lowest in pH, with H-D being highest (Table 2). The H-R fruit were lowest in TA compared to

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the other samples. There were no differences for citric acid but HLB-R was highest in malic acid

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with H-R being lowest. H-R, H-D and HLB-D were highest in TAA compared to H and HLB-R.

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For sugars, H-R was lowest in SS compared to H and HLB-D; There were no differences in 13 ACS Paragon Plus Environment


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sugar acid ratio (SS/TA) but H was highest in sucrose, while the HLB-D was lowest, HLB-R was

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highest in glucose and H highest in fructose (not always significant), while H was highest in TS

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with HLB-D being lowest (not always significant). For late season ‘Hamlin’ orange juice acid

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measurements, HLB-D had the lowest pH, with H and H-D having the highest among the

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samples. HLB-D had the highest TA with H and H-R having the lowest. There were no

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differences for citrate, but the HLB samples had the lowest malate and highest TAA along with

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H-D, with H having the lowest TAA. For sugars, the H fruit juice had the lowest SS; the H-R

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juice the highest SS/TA and HLB-D the lowest. H-D had the highest sucrose, TS and SE, while

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the HLB samples had the highest glucose and fructose levels. For the ‘Valencia’ fruit acid

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measurements, the HLB fruit had the lowest pH and highest TA, with no difference in citric or

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TAA, but H was highest in malic acid compared to the other samples. For sugars H-D was

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lowest in SS and the HLB samples lowest in SS/TA and sucrose and highest in glucose and

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fructose (H being lowest in fructose). HLB-D was highest in TS and SE. So overall for all the

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seasons, the HLB samples exhibited lower pH and higher TA, no difference in citric but

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sometimes lower malic and no pattern for TAA. Sugars showed no particular pattern, except for

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sucrose and SS/TA generally being lower in HLB fruit. Sugars, especially sucrose has been

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found previously to be lower in HLB fruit 4, 6.

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Limonoids and Flavonoids. For early ‘Hamlin’ juice, the H fruit juice was highest in

314

many flavonoids including hesperidin, narirutin, vicenin2, didymin, diosmin, sinensetin,

315

nobiletin, tangeretin and heptamethoxyflavone (HMF) as well as some non-bitter limonoids

316

(Table 3). The HLB-D juice was also high in sinensetin, nobiletin and tangeretin, but not

317

significantly different from the H juice. The HLB-D juice was highest overall in the two bitter

318

limonoids, limonin and nomilin. H-R and H-D were intermediate or lowest in many of the


Page 15 of 46


319

flavonoids and/or bitter limonoids. For late season ‘Hamlin’, there were no differences for

320

vicenin2, diosmin or tangeretin, meanwhile HLB-R and/or HLB-D were generally higher in

321

flavonoids and the two bitter limonoids (only H-D for nomilin) compared to healthy samples.

322

This was unlike the early season where H was very high in many of these compounds except for

323

the bitter limonoids. For ‘Valencia’, there was no difference for vicenin2, but HLB-D was

324

highest in all but nomilinic acid glucoside and limonin glucoside, although not different from

325

HLB-R for narirutin and hesperidin (along with H), and not different from healthy samples for

326

diosmin or H for tangeretin. HLB-R and HLB-D were highest for limonin and nomilin with

327

HLB-D the highest of the two (Table 3). So overall, the HLB-D had the highest levels of the

328

bitter limonoids, limonin and nomilin, often followed by HLB-R. For flavonoids, H and HLB-D

329

were generally highest among the treatments for early season ‘Hamlin’, HLB-R and HLB-D for

330

late season ‘Hamlin’ and HLB-D for the ‘Valencia’ harvest.

331

Aroma Volatiles. For early ‘Hamlin’, there was no particular pattern except that H was

332

lowest for acetaldehyde (solvent-like, fruity, fresh) 27, HLB-D was highest in hexanal but HLB-R

333

low in octanal (green, citrus-like, geranium, floral) among the samples for aldehydes (Table 4).

334

The HLB samples were highest in hexanol and cis-3-hexenol (not different from H) for C-6

335

aldehydes, which along with hexanal contribute green, grassy aromas 27. HLB-D was highest in

336

methanol, but lowest in ethanol (fermented off-odor). Many of the terpenoid type volatiles were

337

highest in HLB-D including tepinen-4-ol (metallic), α-terpineol (considered a thermal processing

338

off-flavor) 28, α-pinene (resin, pine tree), sabinene (warm, spicy), β-myrcene (mossy, geranium,

339

musty), limonene (minty, lemon, citrus like) and γ-terpinene (citrus-like aroma), although not

340

always significantly different from one or another healthy samples (not different from H-D for α-

341

terpineol and H-R for sabinene). The H samples were generally low in many of these



Page 16 of 46

342

compounds, except for valencene. For the fruity esters, HLB-D was highest among the samples

343

for methyl butanoate (fruity, strawberry), but along with HLB-R, lowest in ethyl butanoate

344

(fruity, pineapple) 27, 29, 30. H-D was highest in ethyl butanoate and ethyl hexanoate, and along

345

with H and H-R, ethyl-3-hydroxyhexanoate. HLB-D was also lowest in acetone among the

346

samples.

347

For late season ‘Hamlin’, HLB-D and H-D were highest for acetaldehyde and 2-

348

methylpropanol, HLB-D for octanal, methanol, hexanol (along with HLB-R), terpinene-4-ol, α-

349

pinene, β-myrcene, limonene, γ-terpinene, valencene, ethyl acetate (fruity, solvent-like), and

350

ethyl buanoate (Table 4), often not different from HLB-R, or H-D. H was low in many volatiles

351

(with the exception of decanal and α-terpineol). HLB-R or HLB-D were low in decanal, cis-3-

352

hexenal and linalool. For Valencia, it was a similar story with HLB-D being highest in hexanal,

353

methanol, ethanol, hexanol, octanol, linalool, terpinen-4-ol, α-terpineol, α-pinene, sabinene, β-

354

myrcene, limonene, ethyl acetate, methylbutanoate and acetone as well as lowest in valencene

355

and ethyl-3-hydroxyhexanoate (Table 4). So, similarities are for the C-6 aldehydes (hexanal,

356

hexanol and sometimes cis-3-hexenol), methanol and sometimes ethanol, octanol, and many

357

terpenoid volatiles (terpinen-4-ol, α-terpineol, α-pinene, sabinene, β-myrcene, limonene, γ-

358

terpinene) as well as some of the esters (ethyl acetate and methyl butanoate) and acetone, being

359

relatively high in HLB samples, especially HLB-D. The HLB samples were always lower in

360

valencene except for late season ‘Hamlin’ (weak citrus-like aroma) 30.

361

Putting all the chemical and physical measurements into a PCA makes it easier to make

362

sense of large datasets. The first two components of the PCAs (PC1 and PC2) for the two

363

‘Hamlin’ and one ‘Valencia’ harvest explained 74-80% percent of the variation (Fig. 4). For

364

early season ‘Hamlin’, the healthy fruit types clearly separated from HLB fruit types, but also H 16 ACS Paragon Plus Environment

Page 17 of 46


365

from H-D and H-R, which overlapped. Early ‘Hamlin’ H samples had positive scores on PC1

366

corresponding to flavonoids and sugars as well as decanal and linalool, two volatiles indicative

367

of orange flavor, while HLB-D had positive scores on PC2 (44.1% and 30.3%, respectively, for

368

PC1 and 2), corresponding to the bitter limonoids (limonin and nomilin), C-6 aldehydes and

369

alcohols (hexanal, hexanol and cis-3-hexenol) as well as terpenoid volatiles in general (Fig 4A).

370

H-R and H-D had negative scores on PC2 and intermediate on PC1, corresponding to some

371

esters and aldehydes. For late season ‘Hamlin’, the HLB samples are well separated from the

372

healthy, with HLB-R and HLB-D clustering together and H-R and H-D closely associated (Fig.

373

4B). HLB samples generally had positive scores on PC1 and negative on PC2 (55.3% and

374

21.7% of the variation, respectively), associated with flavonoids, limonoids (including bitter

375

limonoids) and acids, while healthy samples were intermediate (H-D) to very negative (H) on

376

PC1, associated with SS/TA and color. The H samples were very negative on PC-1 and PC-2,

377

associated with the flavonoid tangeretin and volatile aldehyde decanal (Fig. 4B). For ‘Valencia’

378

(Fig. 4C), similar to late season ‘Hamlin’, HLB-D and HLB-R had positive scores on PC1

379

(60.6% of the variation), but HLB-D had positive scores on PC-2 (18.9% of the variation) while

380

HLB-R had negative PC2 scores. HLB-D was associated with flavonoids, terpenoids, C-6

381

volatiles, bitter limonoids, acids and sugars, but H-D and H were associated with SS/TA. All

382

healthy samples were negative on PC1, but separated on PC2, with H-D and H clustering

383

together this time, separating from H-R. All this would predict that the HLB samples and

384

especially HLB-D, would have flavor problems due to low sugar or SS/TA, high acid, high

385

astringency from flavonoids and high bitterness from bitter limonoids. Therefore sensory

386

analyses were done to confirm the chemical prediction and to see if HLB-D samples were

387

sensorially different from HLB-R.



388

Page 18 of 46

Sensory. For the consumer panel simple difference test, panelists were asked to compare

389

two samples: retained/dropped, retained/retained or dropped/dropped for both healthy (H-R and

390

H-D) and HLB-affected (HLB-R and HLB-D) fruit juice, and asked if the juice was the same of

391

different and then to add comments. For early season ‘Hamlin’ fruit juice, panelists could not

392

detect the difference between juices for H-R and H-D for any of the 3 replications. However,

393

they could detect a difference between juice samples from HLB-R and HLB-D at P = 0.017, P =

394

0.044 and P = 0.000, for replications 1, 2 and 3, respectively. Comments for HLB-D compared

395

to HLB-R were that HLB-D tasted more bitter and sour than HLB-R. For late season ‘Hamlin’,

396

the results were similar in that panelists could not distinguish between H-R and H-D in any of

397

the replications, and could distinguish between HLB-R and HLB-D in two out of the three

398

replicates (P = 0.666, P = 0.001 and P = 0.080 for replication 1, 2 and 3, respectively), however

399

with inconsistent comments. This reflects the chemical analyses, where differences were greater

400

for early season than for later season ‘Hamlin’. For ‘Valencia’, the results were similar to late

401

season ‘Hamlin’, with panelists not being able to distinguish H-R from H-D, but being able to

402

distinguish HLB-R from HLB-D in two out of the three replicates (P = 0.688, P = 0.002 and P =

403

0.005 for replication 1, 2 and 3, respectively).

404

For the trained panels all 20 descriptors had differences for H, H-R, H-D, HLB-R and

405

HLB-D (Fig. 5). For early season ‘Hamlin’ (Fig. 5A), all healthy samples had higher ratings for

406

the favorable descriptors ‘orange’ and ‘fruity-non-citrus’ (H-D not significantly different from

407

HLB samples) flavors and ‘sweet’ taste. All HLB samples, but especially HLB-D, had higher

408

perceived intensities for the less than desirable descriptors including ‘grapefruit’, ‘orange peel’,

409

‘green’, ‘stale’, oxidized oil’ and ‘typical HLB’ flavors as well as ‘sour’, ‘umami’, ‘bitter’, and

410

metallic’ tastes. In addition, HLB samples, and especially HLB-D had more ‘tingling’,


Page 19 of 46


411

‘astringent’ and ‘burning’ mouthfeel as well as ‘after-bitter’, ‘after-astringent’ and ‘after-

412

burning’ aftertastes. Fruit type H was perceived to have the most ‘body’, which is somewhat

413

related to juice viscosity. For late season ‘Hamlin’ (Fig. 5B), the results are similar but less

414

extreme and with differences mostly for HLB-D compared to healthy samples and less for HLB-

415

R. There were no statistical differences for ‘fruity-non-citrus, ‘sour’, ‘body’ or ‘burning’

416

aftertaste. For the ‘Valencia samples (Fig. 5C), the results are similar to early season ‘Hamlin’

417

with HLB, and especially HLB-D, generally being different from the Healthy samples in that the

418

HLB samples were lower in the desirable ‘orange’, ‘fruity-non-citrus’ and ‘sweet’ descriptors

419

and higher in all the rest of the undesirable descriptors for flavor, taste, mouthfeel and after-taste,

420

as well as being lowest in ‘body’, but not significantly different from H-R and HLB-R for this

421

descriptor. So overall, the sensory tests were very consistent, with the HLB, and especially

422

HLB-D samples indicated to have flavor problems.

423

Again, using PCA to look at the complex sensory data (Fig. 6), 88 to 98 percent of the

424

variation is explained by the first to PCs, mostly PC1. For early season ‘Hamlin’, the three

425

healthy samples had negative scores on PC1 (90% of the variation), explained by “positive”

426

descriptors (orange, fruity-non-citrus and sweet). They were separated from each other along

427

PC2 (7.9% of the variation), and from HLB samples which had positive scores on PC1. Positive

428

scores on PC1 were associated with the “negative” descriptors (bitter, sour, metallic, burning

429

etc.). HLB-R and HLB-D were also separated from each other on PC2 (Fig. 6A) very similarly

430

to the chemical PCA (Fig. 4A). For late season ‘Hamlin’, the separations were less clear (as was

431

the chemical PCA, Fig 4B), although all Healthy samples clumped together, with negative scores

432

on PC1, separating from HLB samples which had more positive scores on PC1 along, especially

433

HLB-D associated with the “negative” descriptors. HLB-R, was between the healthy samples



434

and HLB-D on PC1 and separated from all the healthy and HLB-D samples on PC2 as well

435

(14.1% of the variation). For ‘Valencia’, only the HLB-D was clearly associated with the

436

“negative” descriptors, and to a lesser extent HLB-R on PC1 (88.7% out of 96.3% of the

437

variation), while H and H-D were associated with the “positive” descriptors more than H-R on

438

PC2.

Page 20 of 46

In conclusion, HLB fruit from shaken trees that fell on the ground abscised below the

439 440

calix, while fruit that were retained on the tree separated above the calix when harvested,

441

indicating that the abscission zone had as least partially formed for the HLB-D fruit. Juice from

442

HLB fruit had generally lower Ct values than for healthy fruit and were in what is considered the

443

“infected” range, while healthy fruit Ct values were generally in the “healthy” range for CLas 23,

444

24

445

healthy trees and positive for HLB trees. Of the HLB juice samples for both varieties, HLB-D

446

had the lowest Ct values (highest in CLas levels) by both Li and LJ primers. This is similar to

447

what was found in a previous study with these fruit for the abscission zones, which had lower

448

Clas and Diplodia Ct values for HLB-D compared to HLB-R fruit 12. The HLB fruit in general

449

were smaller and greener/less orange than the healthy fruit. The sensory data reflects the

450

chemical data in that the HLB, and especially HLB-D samples tasted more bitter, sour and

451

astringent as well as less sweet than healthy samples. This was explained by higher levels of the

452

bitter compounds limonin and nomilin, generally higher TA and often lower SS/TA and sucrose,

453

higher levels of astringent flavonoids (except for H samples in early season ‘Hamlin’) and higher

454

levels of volatile terpenoid aroma compounds compared to healthy samples. The difference in

455

color (HLB fruit being greener) and size (HLB fruit being smaller) is relevant for the fresh fruit

456

market, and indicative of fruit more symptomatic for HLB and thus, more likely to have flavor

. Trees from which these fruit were harvested had tested negative by qPCR for CLas for the


Page 21 of 46


457

issues 4-6. It appears from this data that the HLB fruit loosely attached to the tree (HLB-D) had

458

the most flavor issues, and efforts to retain these fruit on the tree by preventing the abscission

459

zone formation (by perhaps using fungicide to reduce Diplodia infection or an anti-ethylene

460

agent like 1-methylcyclopropene), may help maintain the fruit and their flavor. However, if the

461

tree is shedding fruit it cannot support, then perhaps it is better to let the fruit drop. An earlier

462

transcriptomics study showed up-regulation of ethylene and jasmonic acid-related genes, as well

463

as genes that respond to fungi in the abscission zones of these fruit. Meanwhile genes for

464

abscisic acid that are related to abiotic stress were down-regulated 13. This suggests that fungal

465

infection was exacerbating abscission zone formation in this case, and thus could be controlled

466

by use of fungicides 12 which would hopefully help maintain the quality of retained fruit.

467 468

ABBREVIATIONS USED

469

HLB – huanglongbing

470

H – healthy

471

R – retained

472

D – dropped

473

CLas – Candidatus liberibacter asiaticus

474

Ct – cycle time

475

qPCR – quantitative polymerase chain reaction

476

heptamethoxyflavone (HMF)



Page 22 of 46

477 478

ACKNOWLEDGMENT

479

The study was supported by a grant from Southern Gardens Citrus Nursery

480


Page 23 of 46


481

REFERENCES

482

1.

J. Plant Pathol. 2006, 88, 7-37.

483 484

2.

Bové, J. M., Huanglongbing or yellow shoot, a disease of Gondwanan origin: Will it destroy citrus worldwide? Phytoparasitica 2014, 42, 579-583.

485 486

Bové, J. M., Huanglongbing: A destructive, newly-emerging, century-old disease of citrus.

3.

Gottwald, T. R., Citrus canker and citrus Huanglongbing, two exotic bacterial diseases

487

threatening the citrus industries of the Western Hemisphere. Outlooks on Pest Management

488

2007, 18, 274-279.

489

4.

Baldwin, E.; Plotto, A.; Manthey, J.; McCollum, G.; Bai, J.; Irey, M.; Cameron, R.; Luzio,

490

G., Effect of Liberibacter infection (Huanglongbing disease) of citrus on orange fruit

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physiology and fruit/fruit juice quality: chemical and physical analyses. J. Agric. Food

492

Chem. 2010, 58, 1247-1262.

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5.

orange cultivars in Brazil. http://dx.doi.org/10.1007/s10658-009-9506-3 (2009),

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Bassanezi, R.; Montesino, L.; Stuchi, E. Effects of huanglongbing on fruit quality of sweet

6.

Dagulo, L.; Danyluk, M. D.; Spann, T. M.; Valim, M. F.; Goodrich-Schneider, R.; Sims, C.;

496

Rouseff, R., Chemical characterization of orange juice from trees infected with citrus

497

greening (Huanglongbing). J. Food Sci. 2010, 75, C199-C207.

498

7.

Plotto, A.; Baldwin, E.; McCollum, G.; Manthey, J.; Narciso, J.; Irey, M., Effect of

499

Liberibacter infection (Huanglongbing or “Greening” disease) of citrus on orange juice

500

flavor quality by sensory evaluation. J. Food Sci. 2010, 75, S220-S230.

501

8.

Phytophylactica 1970, 2, 177-194.

502 503

McClean, A. P. D.; Schwarz, R. E., Greening or blotchy-mottle disease of citrus.

9.

Da Graca, J., Citrus greening disease. Annu. Rev. Phytopathol. 1991, 29, 109-136.



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10. USDA-NASS. Citrus Forecast. USDA, National Agricultural Statistics Service

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(2016).https://www.nass.usda.gov/Statistics_by_State/Florida/Publications/ Citrus/cit/2015-

506

16/cit0516.pdf Acessed 03.13.2017.

507

11. Zhao, W.; Bai, J.; McCollum, G.; Baldwin, E., High incidence of preharvest colonization of

508

huanglongbing-symptomatic Citrus sinensis fruit by Lasiodiplodia theobromae (Diplodia

509

natalensis) and its exacerbation of postharvest fruit decay by that fungus. Appl. Environ.

510

Microbiol. 2015, 81, 364-372.

511

12. Zhao, W.; Gottwald, T.; Bai, J.; McCollum, G.; Irey, M.; Plotto, A.; Baldwin, E.,

512

Correlation of Diplodia (Lasiodiplodia theobromae) infection, huanglongbing, ethylene

513

production, fruit removal force and pre-harvest fruit drop. Scientia Horticulturae 2016, 212,

514

162-170.

515

13.

Zhao, W.; Baldwin, E.; Bai, J.; Plotto, A.; Irey, M., HLB-associated pre-harvest fruit

516

abscission is mediated by jasmonate/ethylene signaling triggered by secondary fungal

517

infection. Proceedings of Florida State Horticultural Society 2016, 129, 198–202.

518 519

14. Li, W.; Hartung, J. S.; Levy, L., Quantitative real-time PCR for detection and identification

520

of Candidatus Liberibacter species associated with citrus huanglongbing. J. Microbiol.

521

Methods 2006, 66, 104-115.

522

15. Baldwin, E. A.; Scott, J. W.; Bai, J., Sensory and chemical flavor analyses of tomato

523

genotypes grown in Florida during three different growing seasons in multiple years. J.

524

Amer. Soc. Hort. Sci. 2015, 140, 490-503.

525 526

16. Baldwin, E. A.; Bai, J.; Plotto, A.; Cameron, R.; Luzio, G.; Narciso, J.; Manthey, J.; Widmer, W.; Ford, B. L., Effect of extraction method on quality of orange juice: hand24 ACS Paragon Plus Environment

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squeezed, commercial-fresh squeezed and processed. J. Sci. Food Agric. 2012, 92, 2029-

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2042.

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17. Koehler, P. E.; Kays, S. J., Sweet potato flavor: Quantitative and qualitative assessment of optimum sweetness. J. Food Qual. 1991, 14, 241-249.

531

18. Dea, S.; Plotto, A.; Manthey, J. A.; Raithore, S.; Irey, M.; Baldwin, E., Interactions and

532

thresholds of limonin and nomilin in bitterness perception in orange juice and other

533

matrices. J. Sens. Stud. 2013, 28, 311-323.

534

19. Wang, L.; Baldwin, E. A.; Plotto, A.; Luo, W.; Raithore, S.; Yu, Z.; Bai, J., Effect of methyl

535

salicylate and methyl jasmonate pre-treatment on the volatile profile in tomato fruit

536

subjected to chilling temperature. Postharvest Biol. Technol. 2015, 108, 28-38.

537

20. Zhao, W.; Baldwin, E. A.; Bai, J.; Plotto, A.; Irey, M. S., Method for assessing juice/cider

538

quality and/or safety. U.S. Patent Application Publication 2015, US 20150093755 A1.

539

21. Morgan, J. K.; Zhou, L.; Li, W.; Shatters, R. G.; Keremane, M.; Duan, Y. P., Improved real-

540

time PCR detection of 'Candidatus Liberibacter asiaticus' from citrus and psyllid hosts by

541

targeting the intragenic tandem-repeats of its prophage genes. Mol. Cell. Probes 2012, 26,

542

90-98.

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22. Plotto, A.; Baldwin, E. A.; Bai, J.; Manthey, J.; Raithore, S.; Deterre, S.; Zhao, W.; Stansly,

544

P.; Tansey, J., Effect of vector control and foliar nutrition on quality of orange juice affected

545

by Huanglongbing (HLB): sensory evaluation. HortScience 2017, 52, 1-8.

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23. Stover, E.; McCollum, G., Incidence and severity of huanglongbing and candidatus

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liberibacter asiaticus titer among field-infected citrus cultivars. HortScience 2011, 46, 1344-

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1348.



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24. Gottwald, T. R.; Graham, J. H.; Irey, M. S.; McCollum, T. G.; Wood, B. W.,

550

Inconsequential effect of nutritional treatments on huanglongbing control, fruit quality,

551

bacterial titer and disease progress. Crop Protect. 2012, 36, 73-82.

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25. Hall, D. J., The color-add process as applied in Florida. Proceedings of the Florida State Horticultural Society 2013, 126, 220-224. 26. Manera, F.; Brotons, J.; Conesa, A.; Porras, I., Relation between temperature and the

555

beginning of peel color change in grapefruit (Citrus paradisi Macf.). Scientia Horticulturae

556

2013, 160, 292-299.

557 558 559

27. Perez-Cacho, P. R.; Rouseff, R. L., Fresh squeezed orange juice odor: a review. Crit. Rev. Food Sci. Nutr. 2008, 48, 681-695. 28. Nagy, S.; Rouseff, R. L.; Lee, H. S., Thermally Degraded Flavors in Citrus Juice Products.

560

In Thermal Generation of Aromas, American Chemical Society, Washington, DC: 1989;

561

Vol. 409, pp 331-345.

562 563 564 565

29. Perez-Cacho, P. R.; Rouseff, R., Processing and storage effects on orange juice aroma: a review. J. Agric. Food Chem. 2008, 56, 9785-9796. 30. Nisperos-Carriedo, M. O.; Shaw, P. E., Comparison of volatile flavor components in fresh and processed orange juices. J. Agric. Food Chem. 1990, 38, 1048-1052.

566 567


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568

Figure Legends.

569

Fig. 1. Fruit from Huanglongbing (HLB) affected tree. Left fruit with calix was retained on the

570

tree (HLB-R) and the right fruit without the calix dropped to the ground (HLB-D) when the tree

571

was shaken

572 573

Fig. 2 Cycle time (Ct) values from qPCR of orange juice from ‘Hamlin’ fruit harvested

574

December, 2014 (Early Hamlin), ‘Hamlin’ fruit harvested January 7, 2015 (Late Hamlin) and

575

Valencia harvested January, 2015 (A) using Li primers and (B) using LJ primers from fruit

576

harvested from a non-shaken healthy tree (H), from a shaken healthy trees (H-R) and recovered

577

from the ground after shaking the tree (H-D), harvested from a shaken huanglongbing (HLB)

578

affected tree (HLB-R) and recovered from the ground after shaking the tree (HLB-D).

579 580

Fig.3. Size distribution of orange fruit by sizer at processing plant (A) and fruit diameter

581

measured at the fruit equator for fruit harvested from healthy not shaken trees (Healthy), healthy

582

shaken trees (Healthy Retain) and healthy fruit that dropped to the ground upon shaking the trees

583

(Healthy Drop), fruit retained on shaken Huanglongbing (HLB)-affected trees (HLB Retain) and

584

HLB fruit that dropped (HLB Drop).

585 586

Fig. 4. PCA plots of chemical and physical measurements for juice from fruit harvested from

587

healthy not shaken trees (Healthy), healthy shaken trees (Healthy retain) and healthy fruit that

588

dropped to the ground upon shaking the trees (Healthy drop), fruit retained on shaken

589

Huanglongbing-affected trees (HLB retain) and HLB fruit that dropped (HLB drop) for 27 ACS Paragon Plus Environment


Page 28 of 46

590

December 2014 ‘Hamlin’ (A), January 2014 ‘Hamlin’ (B) and April 2015 ‘Valencia’ (C).

591

TA=titratable acidity, SS=soluble solids, TS= Total sugar, SE-sucrose equvalents, LG=limonin

592

glucoside

593 594

Fig. 5. Trained panel descriptors intensity in juice from fruit harvested from healthy not shaken

595

trees (Healthy), healthy shaken trees (Healthy retain) and healthy fruit that dropped to the ground

596

upon shaking the trees (Healthy drop), fruit retained on shaken Huanglongbing-affected trees

597

(HLB retain) and HLB fruit that dropped (HLB drop) for December 2014 ‘Hamlin’ (A), January

598

2014 ‘Hamlin’ (B) and April 2015 ‘Valencia’ (C).

599 600

Fig. 6. PCA plots of trained panel sensory attributes for juice from fruit harvested from healthy

601

not shaken trees (Healthy), healthy shaken trees (Healthy retain) and healthy fruit that dropped to

602

the ground upon shaking the trees (Healthy drop), fruit retained on shaken Huanglongbing-

603

affected trees (HLB retain) and HLB fruit that dropped (HLB drop) for December 2014 ‘Hamlin’

604

(A), January 2014 ‘Hamlin’ (B) and April 2015 ‘Valencia’ (C).

605 606 607


Page 29 of 46


HLB drop

HLB retain

Healthy drop

Parameter

Healthy retain

Healthy

Table 1. Surface color values for oranges (20 fruit/replicate) harvested from a non-shaken healthy (Healthy), a shaken healthy trees (Healthy retained) and recovered from the ground after shaking the tree (Healthy drop), harvested from a shaken huanglongbing (HLB) affected tree (HLB retain) and recovered from the ground (HLB drop) for ‘Hamlin’ in December 2014 and January 2015, and 'Valencia' in April 2015.

'Hamlin', December 2014 z

a* b* a*/b* hue

0.20 63.53 0.00 89.97

a a a b

a* b* a*/b* hue

12.11 63.76 0.19 79.15

a ab ab b

a* b* a*/b* hue

12.6 60.1 0.2 78.3

a ab a c

-0.26 a -1.92 59.43 ab 60.14 -0.01 a -0.04 90.44 b 92.16 'Hamlin', January 2015 12.72 a 11.37 62.41 ab 64.88 0.21 a 0.18 78.35 b 79.94 'Valencia', April 2015 9.6 ab 11.2 60.0 bc 62.8 0.2 b 0.2 81.4 b 80.1

a ab a b

-5.20 58.68 -0.11 96.07

b b b a

-6.28 63.20 -0.11 96.15

b ab b a

a a ab ab

10.18 58.96 0.17 80.18

ab bc ab ab

7.72 56.17 0.14 82.25

b c b a

ab a ab bc

2.8 53.1 0.0 87.7

c d c a

8.0 57.4 0.1 82.8

b c b b

z

Values (n=3) that are not followed by the same letter within a row show significant difference at the 0.05 level using Tukey's test. Each replicate = 20 fruit. 608 609 610



Page 30 of 46

Table 2. Sugars and acid measurements of juice from oranges harvested from non-shaken healthy (Healthy), shaken healthy (Healthy retained) and shaken huanglongbing (HLB retain) trees, as well as recovered from the ground after shaking the trees (Healthy drop and HLB drop).

'Hamlin', December 2014 pH Titratable acidity (TA) Soluble solids (SS) SS/TA Sucrose Glucose Fructose Total sugar (TS) Sucrose equivalence (SE) Citric acid Malic acid Total ascorbic acid (TAA) 'Hamlin', January 2015 pH Titratable acidity (TA) Soluble solids (SS) SS/TA Sucrose Glucose Fructose Total sugar (TS) Sucrose equivalence (SE) Citric acid Malic acid Total ascorbic acid (TAA) 'Valencia', April 2015 pH

4.302 0.441 10.3 23.39 5.301 1.751 1.902 8.954 9.887

bcz a a a ab a a a

4.389 0.358 8.1 22.57 3.985 1.369 1.382 6.736 7.388

ab b c c c c cd cd

4.41 0.43 9.2 21.45 4.622 1.474 1.57 7.667 8.43

a a abc b c c bc bc

4.289 0.452 8.133 20.92 4.305 1.78 1.865 7.95 8.849

HLB drop

HLB retain

Healthy drop

Healthy retain

Healthy

Compound/attribute

c a bc bc a ab ab ab

4.352 0.425 9.533 22.83 3.302 1.553 1.604 6.459 7.226

abc a ab

a b

0.61 0.213 38.09

bc a

d bc bc d d

0.686 0.219 ab 33.33 b

0.549 0.193 c 38.01 a

0.638 0.232 38.01

ab a

0.672 0.24 31.2

4.379 0.408 10.63 26.17 5.196 2.13 2.15 9.475 10.49

4.35 0.415 11.3 27.33 5.587 2.18 2.223 9.99 11.05

ab b a a ab b bc ab ab

4.375 0.435 11.57 26.67 5.676 2.238 2.208 10.12 11.15

a ab a ab a ab c a a

4.277 0.443 11.67 26.58 5.249 2.429 2.35 10.03 11.11

ab ab a ab c a a ab ab

4.267 0.465 11.5 24.75 5.296 2.402 2.344 10.04 11.13

b a a b bc a ab ab ab

0.615 0.239 a 28.11 b

0.664 0.227 ab 31.21 ab

0.68 0.234 31.55

a a

0.718 0.203 33.24

c a

0.735 0.212 34.41

bc a

4.181 ab

4.197 ab

4.23

a

4.093

bc

4.033

c

a b b ab c b c b b


Page 31 of 46


Titratable acidity (TA) 0.684 bc 0.703 b 0.651 c 0.794 a 0.803 a Soluble solids (SS) 11.75 a 11.68 ab 11.3 b 12.03 a 12.05 a SS/TA 17.19 ab 16.62 b 17.36 a 15.16 c 15.01 c Sucrose 5.488 a 5.341 ab 5.311 abc 5.155 bc 5.118 c Glucose 2.061 bc 2.028 bc 1.959 c 2.162 ab 2.223 a Fructose 2.093 d 2.282 bc 2.178 cd 2.388 b 2.522 a Total sugar (TS) 9.641 ab 9.651 ab 9.448 b 9.704 ab 9.863 a Sucrose equivalence 10.63 b 10.79 ab 10.53 b 10.88 ab 11.13 a (SE) Citric acid 0.744 0.76 0.729 0.811 0.794 Malic acid 0.225 a 0.185 b 0.19 b 0.195 b 0.181 b Total ascorbic acid 32.7 34.96 34.73 33.8 35.34 (TAA) z Values (n=3) that are not followed by the same letter within a row show significant difference at the 0.05 level using Tukey's test. Units for all compounds are g/100g. Each juice replicate = 89-199 fruit from 3 trees 611

Table 3. Flavonid and limonoid measurements of juice from oranges harvested from nonshaken healthy (Healthy), shaken healthy (healthy retained) and shaken huanglongbing (HLB retain) trees, as well as recovered from the ground after shaking the trees (Healthy drop and HLB drop), from ‘Hamlin’ in December 2014, 'Hamlin' in January 2015, and 'Valencia' in April 2015.

'Hamlin', December 2014 Hesperidin Narirutin Vicenin2 Didymin Diosmin Sinensetin Nobiletin Heptamethoxyflavone Tangeretin Nomilinic acid glucoside Limonin glucoside

203.9 26.15 15.51 7.108 0.769 0.157 0.371 0.29 0.065 92.57 43.56

az a a a a a a a a a a

180.6 20.59 12.79 6.284 0.647 0.118 0.307 0.23 0.055 75.19 31.74

ab b b bc bc b ab b a b bc

158.2 16.31 10.11 5.623 0.563 0.096 0.236 0.166 0.034 56.37 25.88

bc b c bc c b b c b c c

148.7 15.38 9.256 5.18 0.615 0.101 0.237 0.164 0.036 60.54 30.53

HLB drop

HLB retain

Healthy drop

Healthy retain

Healthy

Compound mg /L

c c c d c b b c b bc bc

162.4 18.88 10.85 6.647 0.734 0.156 0.342 0.21 0.053 68.87 39.32

bc bc bc ab ab a a bc a bc ab 31

ACS Paragon Plus Environment


Page 32 of 46

Limonin 1.663 b 1.124 c 0.988 c 1.741 b 2.756 a Nomilin 0.149 b 0.092 c 0.082 c 0.178 b 0.359 a 'Hamlin', January 2015 Hesperidin 194.4 b 262.8 a 259.9 a 240.3 ab 256.5 a Narirutin 24.88 b 31.9 a 32.36 a 31.33 a 32.7 a Vicenin2 16.41 17.97 18.71 18.25 18.96 Isosakuranetin 7.905 b 10.91 a 10.74 a 10.34 ab 10.82 a Diosmin 0.955 0.892 0.97 1.09 0.943 Sinensetin 0.194 ab 0.174 b 0.174 b 0.205 a 0.209 a Nobiletin 0.357 d 0.374 cd 0.401 bc 0.434 a 0.42 ab Heptamethoxyflavone 0.366 a 0.282 b 0.282 b 0.363 a 0.315 ab Tangeretin 0.09 0.05 0.07 0.049 0.049 Nomilinic acid glucoside 99.39 b 98.18 b 114.3 a 117.3 a 120.8 a Limonin glucoside 45.11 b 45.53 b 50.82 ab 54.38 a 57.73 a Limonin 0.843 b 0.744 b 0.898 b 0.999 ab 1.288 a Nomilin 0.065 b 0.086 b 0.067 b 0.088 b 0.226 a 'Valencia', April 2015 Hesperidin 287.2 ab 233.3 bc 262.3 b 295.6 ab 313.5 a Narirutin 44.39 c 46 c 50.3 bc 54.84 ab 57.21 a Vicenin2 28.41 28.39 30.26 27.19 29.37 Isosakuranetin 18.09 cd 15.9 d 19.18 c 22.3 b 25.77 a Diosmin 1.37 ab 1.337 ab 1.537 ab 1.226 b 1.601 a Sinensetin 1.171 b 1.128 b 1.23 b 1.274 b 1.771 a Nobiletin 1.561 b 1.406 b 1.494 b 1.479 b 2.032 a Heptamethoxyflavone 0.807 b 0.738 b 0.79 b 0.783 b 1.008 a Tangeretin 0.265 ab 0.257 b 0.246 b 0.227 b 0.307 a Nomilinic acid glucoside 297.6 a 290.7 ab 309 a 256.8 bc 248.5 c Limonin glucoside 154.3 a 135.3 b 153.7 a 127.4 b 126.4 b Limonin 1.554 c 1.017 c 1.681 c 3.034 b 5.374 a Nomilin 0.137 c 0.092 c 0.21 c 0.839 b 1.91 a z Values (n=3) that are not followed by the same letter within a row show significant difference at the 0.05 level using Tukey's test. Each juice replicate = 89-199 fruit from 3 trees. 612


Page 33 of 46


Table 4. Aroma volatile measurements for juice from oranges harvested from non-shaken healthy (Healthy), shaken healthy (healthy retained) and shaken huanglongbing (HLB retain) trees, as well as recovered from the ground after shaking the trees (Healthy drop and HLB drop), from ‘Hamlin’ in December 2014, 'Hamlin' in January 2015, and 'Valencia' in April 2015.

Hexanal

Octanal

Decanal

Methanol

Ethanol

2Methypropanol Hexanol

4. 0 3 0. 2 7 0. 4 5 0. 3 7 1 4. 2 3 2 1 0. 0 7 0. 0 5

b 5. 4 2 b 0. 1 6 c 0. 6 2 a 0. 2

a 3 b 3 3 b 0. 1

a 5. 6 5 d 0. 2 2 a 0. 5 1 b 0. 2 2 c 2 1. 5 a 3 0 5 a 0. 1

c

c

d 1 9

0. 0 5

0. 0 6

a

5. 4 1 c 0. 2 6 b 0. 4 b 0. 2 2 b 2 1. 7 b 2 c 9 4 a 0. 0 8 c 0. 1 1

a

5. 2 6 b 0. 3 4 d 0. 4 6 b 0. 2 b 2 6. 5 c 2 5 0 a 0. b 0 7 b 0. 1 8

a

a

c

4. 6 4 0. 4 1 0. 4

b 0. 2 7 a 1 4. 1 d 3 4 0 b 0. 1 2 a 0. 0 4

b 4. 7 8 b 0. c 4 2 b 0. 5 3 a 0. 2 2 b 1 2. 5 b 3 3 9 b 0. 1 2 c 0. 0 4

b 5. 7 4 b 0. c 3 5 b 0. 5 4 b 0. 1 8 b 1 2 b 4 5 2 b 0. 2 2 c 0. 0 5

a

5. 0 1 c 0. 7 9 b 0. 5 2 b 0. c 1 8 b 1 3. 2 a 2 9 9 a 0. 1 3 c 0. 0 7

b 5. 5 8 a 0. 4 9 b 0. 8 1 c 0. 1 7 b 1 7. 2 b 3 4 1 b 0. 2 a

0. 0 6

a

8. 2 8 b 0. 2 7 a 0. 0 9 c 1. 9 8 a 2 7. 7 b 7 4 4 a 0. 1 6 b 0. 1 2

a

6. 8 7 c 0. 1 8 c 0. 1 4 a 1. 7 7 b 3 4. 1 a 6 b 8 7 a 0. 0 9 d 0. 1 3

b 9. 0 2 d 0. 3 2 a 0. 0 6 b 1. 8 8 b 3 4. 4 b 7 1 0 c 0. 1 3 d 0. 1 8

a

b c b

a b b

b

b

c

9. 1 4 0. 3 4 0. 1 6 1. 3 7 3 5. 9 7 1 7 0. 1 3 0. 2 3

HLB drop

HLB retain

Healthy drop

Healthy

Healthy retain

'Valencia', April 2015

HLB drop

HLB retain

Healthy drop

Healthy

HLB drop

HLB retain

Healthy drop

Healthy Acetaldehyde

'Hamlin', January 2015 Healthy retain

'Hamlin', December 2014 Healthy retain

Compound μL/L

a

b

a

c

b

a b b

b

9. 3 6 0. 4 2 0. 1 5 1. 4 6 5 3. 8 7 9 6 0. 1 2 0. 2 8

a

a

a

c

a

a

b c a



cis-3-Hexenol

trans-2-Hexenol

Octanol

Linalool

Terpinen-4-ol

α-Terpineol

α-Pinene

Sabinene

β-Myrcene

Limonene

γ-Terpinene

Valencene

0. 3 2 0. 0 0 3. 6 4 0. 2 1 0. 1 3 0. 1 1 0. 6 5 0. 2 1 2. 5 1 1 2 5 0. 0 2 3.

a

0. 2 8 0. 0 0 a 3. 2 3 a 0. 1 7 c 0. 1 4 d 0. 1 5 c 1. 2 5 c 0. 6

b 0. 3 3 0. 0 0 c 2. 6

a

a

b

4. 1

b 0. 2 7 0. 0 0 b 2. 8 1 b 0. 2 1 b 0. c 1 5 c 0. d 1 9 b 1. 1 7 a 0. 4 3 b 3. 8

1 8 0 d 0. 0 5 a 3.

b 1 7 3 b 0. 0 4 b 2.

b 1 8 3 c 0. 0 5 b 2.

c

c

b

a

b

b

b

0. 1 7 0. 1 5 0. 1 7 1. 1 3 0. 2 9 3. 8

c

b

b c b

c

0. 3 4 0. 0 0 2. 8 9 0. 1 7 0. 1 7 0. 1 8 1. 5

0. 5 4 b 4. 7 5 b 2 0 5 b 0. c 0 7 c 1.

a

0. 3 5 0. 0 0 c 2. 4 4 b 0. 1 3 a 0. 0 8 a 0. b 1 4 a 0. 8 1 a 0. 2 3 a 2. 6 4 a 1 3 3 a 0. 0 1 d 6.

a 0. 3 5 0. 0 0 b 2. 3 3 b 0. 1 4 c 0. 0 8 a 0. 0 9 c 0. 9 3 b 0. 3 4 b 3. 0 3 b 1 5 1 b 0. 0 2 b 6.

a

b

a

c

b

c

a

a b a b a b b

0. 3 5 0. 0 0 2. 2 3 0. 1 4 0. 0 9 0. 1 2 1. 0 9 0. 2 7 3. 0 2 1 4 9 0. 0 2 7.

a

Page 34 of 46

0. 3

b 0. 3

0. 0 0 b 2. 7

0. 0 0 2. 3 9 0. 1 3 0. 1 2 0. 1 4 1. 2 3 0. 2 1 3. 4 3 1 5 9 0. 0 2 8.

a

a 0. b 1

c

b 0. c 0 9 a 0. b 1 1 b 1. 1 5 a 0. b 3

b

a 3. b 3 5 a 1 b 5 9 a 0. 0 2 b 8.

a b a b a b a

a

a

a

b 1. 0 8 0. 0 0 b 1. 9 1 a 0. b 8 9 a 0. 2 3 a 0. b 1 2 a 1. 7 1 b 0. 5 1 a 5. 2 2 a 2 2 3 a 0. 0 5 a 8.

a

1. 1

a

0. 0 0 1. 2 7 0. 9 2 0. 1 9 0. 1 1 1. 4 5 0. 6 9 4. 7 5 2 1 8 0. 0 4 7.

b

c

a

b

b c c

b c b

a

a

1. 0 4 a 0. b 0 0 d 1. 7 1 c 1. 0 4 c 0. 2 b 0. 1 2 c 1. 6 9 b 0. 6 8 c 5. 1 7 b 2 2 5 0. 0 5 b 7.

a

b 0. 9 2 b 0. 0 0 c 2. 4

b c

a

b 5. c 4

b 1. 2 8 a 0. b 2 5 b 0. 2 4 a 2. b 2 2 b 1. 1 5 a 6 b

b 2 3 0 0. 0 5 a 5.

b 2 4 8 0. 0 5 c 4.

a

a b b c b

c d b

c

b

0. 8 4 0. 0 0 1. 6 2 1. 0 3 0. 2 2 0. 1 3 1. 9 8 0. 7

a b a

a

a

a

a

a

d 34

ACS Paragon Plus Environment

Page 35 of 46


8 0 9 4 7 0 5 2 7 5 2 1 7 b 8 3 5 9 5 4 7 8 9 5 9 8 6 6 6 Ethyl acetate 0. c 0. a 0. a 0. b 0. c 0. c 0. c 0. a 0. b 0. a 0. a 0. c 0. b 0. a 0. a 0 1 0 0 0 1 1 2 1 2 1 1 1 c 1 b 1 7 9 8 7 5 7 5 8 2 7 2 5 5 Methyl 0. d 0. c 0. c 0. b 0. a 0. b 0. b 0. b 0. a 0. b 0. b 0. b 0. a 0. a 0. a butanoate 0 0 d 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 2 2 3 5 5 5 6 5 1 1 2 2 2 Ethyl butanoate 0. c 0. b 0. a 0. d 0. d 0. c 0. b 0. a 0. b 0. a 0. b 0. c 0. a 0. c 0. c 2 2 2 2 1 4 5 c 7 5 7 6 4 7 5 4 2 7 9 9 4 1 1 5 2 1 6 2 9 Ethyl hexanoate 0. c 0. b 0. a 0. b 0. b 0. b 0. b 0. a 0. a 0. a 0. b 0. b 0. a 0. c 0. b 0 0 0 0 0 0 0 1 0 b 1 b 0 c 0 0 0 0 c 1 2 3 2 2 8 8 2 9 1 4 5 8 3 4 Ethyl 32 a 2 a 2 a 1 b 1 b 3 5 4 5 5 5 a 4 b 4 b 4 c 2 d hydroxyhexanoat 3. 1. 2. 8. 8. 8. 8 5. 2. 0 2. 5. c 7. 2. 9 e 3 6 3 8 2 3 8 1 1 8 7 7 Acetone 0. a 0. a 0. a 0. a 0. b 0. b 0. a 0. a 0. a 0. a 0. c 0. a 0. b 0. b 0. a 1 1 1 1 1 3 4 4 b 4 4 b 1 6 2 c 3 6 3 3 4 3 1 8 7 4 6 2 3 5 7 6 z Values (n=4) that are not followed by the same letter within a row, in the same harvest time, show significant difference at the 0.05 level using Tukey's test. Each juice replicate = 89-199 fruits from 3 trees. 613



614

Page 36 of 46

Fig. 1. Fruit from Huanglongbing (HLB) affected tree. Left fruit with calix was retained on the tree (HLB-R) and the right fruit without the calix dropped to the ground (HLB-D) when the tree was shaken

HLB-R

HLB-D


Page 37 of 46


615 616 617 618 619 620 621

Fig. 2. Ct values from qPCR of orange juice from ‘Hamlin’ fruit harvested December, 2014 (Early Hamlin), ‘Hamlin’ fruit harvested January 7, 2015 (Late Hamlin) and Valencia harvested January, 2015 (A) using Li primers and (B) using LJ primers from fruit harvested from a nonshaken healthy tree (H), from a shaken healthy trees (H-R) and recovered from the ground after shaking the tree (H-D), harvested from a shaken huanglongbing (HLB) affected tree (HLB-R) and recovered from the ground after shaking the tree (HLB-D). Each juice replicate = 89-199 fruit from 3 trees.

622

A.)

Ct value

Ct vlaues for Hamlin and Valencia juice 45 40 35 30 25 20 15 10 5 0 H

H-R

H-D

HLB-R

HLB-D

Fruit type Early Hamlin

Late Hamlin

Valencia

623 624

B.)

Ct vlaues for Hamlin and Valencia juice, LJ primers 50

Ct value

40 30 20 10 0 H

H-R

H-D

HLB-R

HLB-D

Fruit type Early Hamlin

Late Hamlin

Valencia

625 626



627 628 629 630 631 632 633

Page 38 of 46

Fig. 3. Size distribution of orange fruit by sizer at processing plant (A) fruit diameter mean of 3 replicates of 89+ fruit each (B) measured at the fruit equator, mean of three replicates of 20 fruit each, for fruit harvested from healthy not shaken trees (Healthy), healthy shaken trees (Healthy Retain) and healthy fruit that dropped to the ground upon shaking the trees (Healthy Drop), fruit retained on shaken Huanglongbing (HLB)-affected trees (HLB Retain) and HLB fruit that dropped (HLB Drop). A)

B.)

634


Page 39 of 46


635 636 637 638 639 640 641

Fig. 4. PCA plots of chemical and physical measurements for juice from fruit harvested from healthy not shaken trees (Healthy), healthy shaken trees (Healthy retain) and healthy fruit that dropped to the ground upon shaking the trees (Healthy drop), fruit retained on shaken Huanglongbing-affected trees (HLB retain) and HLB fruit that dropped (HLB drop) for December 2014 ‘Hamlin’ (A), January 2014 ‘Hamlin’ (B) and April 2015 ‘Valencia’ (C). TA=titratable acidity, SS=soluble solids, TS= Total sugar, SE-sucrose equivalents, LG=limonin glucoside

642

A) Hamlin Dec. 2014 1

Limonin Nomilin Hexanal

cis-3-Hexenol

Hexanol Methyl butanoate hue

C* Sinensetin Diosmin LG b*

HLB-D

Terpinen-4-ol g-Terpinene

TA

PC 2 (30.3%)

Methanol Limonene Myrcene Malic

a-Pinene

HLB-R

0

a-Terpineol Sabinene

Nobiletin SS Isosakuranetin Fructose Tangeretin SS/TA Citric NAG HMF H Narirutin Decanal Glucose

0.5

0 SE

Ascorbic

TS

Acetaldehyde

H-R

Linalool Sucrose

H-D -0.5

pH Ethyl hexanoate

Valencene Ethyl 3hydroxyhexanoate

Octanal

a* Ethanol Acetone

2-Methypropanol Ethyl acetate

-1 -1

-0.5

Vicenin2 Octanol Hesperidin

Ethyl butanoate 0

0.5

1

PC 1 (44.1%)

643 644

B)



Page 40 of 46

Hamlin Jan. 2015 1

Sucrose Hesperidin Linalool

cis-3-Hexenol

H-D

SS/TA

C* b* pH a* a*/b* Malic

Isosakuranetin TS Narirutin Ethyl acetate SE Vicenin2 Ethyl 3hydroxyhexanoate Ethyl butanoateSS 2-Methypropanol g-Terpinene Acetaldehyde Ethyl hexanoate Limonene Acetone

Sabinene

H-R

PC 2 (21.7%)

0.5

Ethanol

0

0

Tangeretin Decanal -0.5

Ascorbic Octanal a-Pinene Citric Methyl butanoate Myrcene Nobiletin NAG TA Valencene HLB-D LG Fructose Nomilin Glucose Terpinen-4-ol HLB-R hue Limonin Hexanol Diosmin Hexanal

H a-Terpineol

Methanol

Octanol Sinensetin HMF -1 -1

-0.5

0

0.5

1

PC 1 (55.3%)

645 646

C)


Page 41 of 46


Valencia Apr. 2015 1

Diosmin Vicenin2

LG 0.5

Decanal

Ethyl hexanoate

SS/TA cis-3-Hexenol NAG Sucrose

PC 2 (18.9%)

Octanol g-Terpinene Tangeretin Ethanol

a*/b* a* Ethyl butanoate C* b*

H-D

HMF Nobiletin Myrcene Hexanal Acetaldehyde HLB-D Sinensetin a-Terpineol Ethyl acetate Limonene Terpinen-4-ol Linalool Hesperidin a-Pinene Methanol Limonin Sabinene Isosakuranetin Methyl butanoate Nomilin

2-Methypropanol

H

Malic

Valencene 0

pH

0

Ascorbic TS

Ethyl 3hydroxyhexanoate

Hexanol Narirutin Glucose SE Fructose

Acetone

SS

TA

H-R

-0.5

Citric

HLB-R

Octanal hue

-1 -1

-0.5

0

0.5

1

PC 1 (60.6%)

647 648



Page 42 of 46

649 650 651 652 653

Fig. 5. Trained panel descriptor intensity juice from fruit harvested from healthy not shaken trees (Healthy), healthy shaken trees (Healthy retain) and healthy fruit that dropped to the ground upon shaking the trees (Healthy drop), fruit retained on shaken Huanglongbing-affected trees (HLB retain) and HLB fruit that dropped (HLB drop) for December 2014 ‘Hamlin’ (A), January 2014 ‘Hamlin’ (B) and April 2015 ‘Valencia’ (C).

654

A) Hamlin December 2014 Healthy Unshaken

Healthy Retain

12

Healthy Drop

HLB Retain

HLB Drop

a

10

a b

8

b a

6

a aa

4

b

a b

2

b

c cc a ab bc cc

ab b bb

a

ab a bc bc c

ab bc cc

b

ccc

a a

a

a ab ab b b

a

a b bb b

c d

a

b bb

a

c a ab b bb

b

c

c

a

a

b a

bc cc

b bb

a

ab

b b b

a

a c cc

bc c c

a ab a c cbc

bbb

0

655 656

B) Hamlin January 2015 Healthy Unshaken

Healthy Retain

Healthy Drop

HLB Retain

HLB Drop

10

8

6

aa a ab b

ab a abc bc c a

4

a a

2

ab b b b

a a a ab b b ab b b bb b b bb

bb b a b bb b

b

a bb b b

b

a

b

a

bb bb

bb

a a b bb ab bb

b

a

a ab b bb

b b bb

a bb b

b

0

657 658 659 660 42 ACS Paragon Plus Environment

Page 43 of 46

661


C) Valencia April 2015 Healthy Unshaken

Healthy Retain

Healthy Drop

HLB Retain

HLB Drop

10

8

aa 6

aaa a a ab

a bc c a b c cc

bc c

a

b

aa ababa

a a ab ab b

a

b

4

2

b bb

b

b c cc

aa

b bb b bb

b

a bbab

bc cbc aa ab bb

b a b c cbc

a bbb

a

a

c bcc b bb

a

a

a b

b b b

b

bb b

a

a

bb b

0

662



Page 44 of 46

663 664 665 666 667

Fig. 6. PCA plots of trained panel sensory attributes for juice from fruit harvested from healthy not shaken trees (Healthy), healthy shaken trees (Healthy retain) and healthy fruit that dropped to the ground upon shaking the trees (Healthy drop), fruit retained on shaken Huanglongbingaffected trees (HLB retain) and HLB fruit that dropped (HLB drop) for December 2014 ‘Hamlin’ (A), January 2014 ‘Hamlin’ (B) and April 2015 ‘Valencia’ (C).

668

A) 1

Body

H

Fruity

0.5

Sweet

PC 2 (7.9%)

Orange 0

0

H-R

-0.5

H-D

Sour Orange peel Metallic HLB-D Grapefruit Umami Green Bitter Tingling Burning Oxidized HLB-R Typical HLB AfterBitter AfterAstringent Astringent Stale AfterBurning

-1 -1

-0.5

0

669 670

0.5

1

PC 1 (90.0%)

B) 1

HLB-R

Orange

Body 0.5

PC 2 (14.1%)

Tingling Sour AfterBurning AfterAstringent AfterBitter Grapefruit Astringent Orange peel Green Burning Bitter HLB-D Umami Oxidized Typical HLB Stale

Sweet

H-D 0

0

Fruity

H-R H

Metallic

-0.5

-1 -1

671

-0.5

0

0.5

1

PC 1 (73.8%)


Page 45 of 46

672


C) 1

H-R Stale

0.5

PC 2 (7.6%)

HLB-DAfterBitter

Oxidized Astringent Bitter Grapefruit Typical HLB Metallic Burning Green Umami Tingling

Sweet 0

0

H-D

Fruity Orange

H

Orange peel AfterBurning Sour

-0.5

Body

HLB-R

-1 -1

673

-0.5

0

0.5

1

PC 1 (88.7%)

674 675



676

Page 46 of 46

TOC Graphic

677

678


Effect of Abscission Zone Formation on Orange (Citrus sinensis) Fruit

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