Full Terms amp Conditions of access and use can be found athttpswwwtandfonlinecomactionjournalInformationjournalCode=kpsb20

Plant Signaling amp Behavior

ISSN (Print) 1559-2324 (Online) Journal homepage httpswwwtandfonlinecomloikpsb20

Phenolics flavonoids and antioxidant capacities inCitrus species with different degree of tolerance toHuanglongbing

Faraj Hijaz Fuad Al-Rimawi John A Manthey amp Nabil Killiny

To cite this article Faraj Hijaz Fuad Al-Rimawi John A Manthey amp Nabil Killiny (2020)Phenolics flavonoids and antioxidant capacities in Citrus species with different degree of toleranceto Huanglongbing Plant Signaling amp Behavior DOI 1010801559232420201752447

To link to this article httpsdoiorg1010801559232420201752447

Published online 14 Apr 2020

Submit your article to this journal

Article views 88

View related articles

View Crossmark data

RESEARCH PAPER

Phenolics flavonoids and antioxidant capacities in Citrus species with differentdegree of tolerance to HuanglongbingFaraj Hijaza Fuad Al-Rimawiab John A Mantheyc and Nabil Killiny a

aDepartment of Plant Pathology Citrus Research and Education Center IFAS University of Florida Lake Alfred FL USA bChemistry DepartmentFaculty of Science and Technology Al-Quds University Jerusalem Palestine cUS Horticultural Research Laboratory USDA ARS Fort Pierce FL USA

ABSTRACTHuanglongbing (HLB) is a highly destructive disease to the citrus industry in Florida caused by thebacterium Candidatus Liberibacter asiaticus(CLas) and is transmitted by Diaphorina citri It is hypothe-sized that plants with high phenolic contents show higher tolerance to certain plant pathogens In thisregard different citrus varieties and plants of genera related to Citrus were analyzed for their totalphenolic and flavonoids contents and their antioxidant capacities In addition the secondary metabo-lites in the leaves of seven citrus species were analyzed using high performance liquid chromatography-mass spectrometry (HPLC-MS) Colorimetric assays showed that curry leaf contained the highest totalphenolic content and free radical scavenging activity (DPPH) Curry leaf also contained high concentra-tions of an unusual class of carbazole alkaloids Tolerant Citrus species contained high levels of phenolicsand flavonoids and showed high antioxidant capacities Our results suggest that high phenolic andflavonoid leaf contents correlate with increased citrus tolerance to CLas bacterium The results alsosuggest that the high level of carbazole alkaloids known for their strong antimicrobial properties incurry leaf could make it immune to the CLas bacteria Understanding the mechanisms underpinningcitrus tolerance to HLB will contribute to the development of commercially tolerant citrus cultivars

ARTICLE HISTORYReceived 19 March 2020Revised 31 March 2020Accepted 2 April 2020

KEYWORDSCitrus Huanglongbingphenolics flavonoidsantioxidants

Introduction

In North America Huanglongbing (HLB) is caused by thebacterium Candidatus Liberibacter asiaticus (CLas) and istransmitted by the Asian citrus psyllid Diaphorina citriCurrently there is no cure for the HLB disease and itsmanagement mainly depends on insecticides to controlthe insect vector (D citri)1 Several other managementstrategies including enhanced nutritional programs ther-motherapy and removal of infected trees have been testedfor the control of HLB however these strategies haveusually shown poor results in the field2 For these rea-sons the development of CLas-resistant citrus cultivarsmight be the best means for the control of the HLBdisease

Field observations showed thatMurraya paniculata (L)Jack (orange jasmine) and sweet oranges were the most pre-ferred host for D citri while Poncirus trifoliata was a poorhost3 A greenhouse-controlled study showed that grapefruitwas a more preferable host than Citrus jambhiri Lushington(rough lemon) Murraya paniculata and Citrus aurantiumL (sour orange)4 Similarly it was found that D citri pre-ferred Citrus macrophylla Wester (Alemow) more than acces-sions of Poncirus trifoliata x Citroncirus spp5 Fewer eggs werelaid on P trifoliata accessions than C macrophylla5 Theresistance of P trifoliateto D citri was attributed to the effectof antixenosis (deterrents) and antibiosis (antagonisticassociation)5

In a similar manner field observations have shown thatsome citrus cultivars are more tolerant to the HLB pathogenthan others3 Using graft inoculation Folimonova et al (2009)studied the responses of several citrus species to CLas and inagreement with the previous reports certain tested speciesshowed different degrees of tolerance to CLas For examplePoncirus trifoliata Eureka lemon (C limonia Osbeck) andCitrus latipes were tolerant to the CLas bacterium whereassweet oranges and grapefruits were very sensitive6 The sensi-tive species showed severe leaf chlorosis decreased growthand death whereas tolerant cultivars showed little or nosymptoms In a similar study Malaysian citrus species alsoshowed different degrees of tolerance to CLas pathogen7

Unfortunately it is not clear why are some citrus speciesare more tolerant to CLas than others In order to examinepossible mechanisms underpinning the tolerance of somecultivars we investigated the metabolite profiles of severalcultivars of citrus89 Our results showed that tolerant cultivarscontained higher levels of metabolites that possess strongantimicrobial activities and compounds implicated in plantdefense89 In another study we analyzed the metabolite pro-files of lsquoSugar Bellersquo mandarin and four of its parents in orderto understand why lsquoSugar Bellersquo was more tolerant to HLB10

Our metabolomic analysis showed that ldquoSugar Bellerdquo man-darin was high in thymol which has a strong anti-fungal andantimicrobial activity10 lsquoSugar Bellersquo was also high in severalother polar compounds including benzoic acid ferulic acid

CONTACT Nabil Killiny nabilkillinyufledu Department of Plant Pathology Citrus Research and Education Center University of Florida 700 ExperimentStation Rd Lake Alfred FL 33850 USA

PLANT SIGNALING amp BEHAVIORhttpsdoiorg1010801559232420201752447

copy 2020 Taylor amp Francis Group LLC

caffeic acid synephrine and inositol which may protect itfrom biotic and abiotic stress10 We also showed that themetabolomic profile of Australian finger lime a highly toler-ant Citrus relative was different from that of lsquoSugar Bellersquoindicating that they could have different tolerancemechanisms11

Plants produce a wide variety of secondary metabolites thatcan act as antimicrobial substances such as alkaloids flavonoidsand phenols Phenolics are a group of secondary metaboliteswhich are produced via the shikimic acid pathway through thephenylpropanoid pathways12 It is believed that accumulation ofphenolic compounds at the infection site could result in isolationof the attacking pathogens and prevent their spread to otherplantrsquos tissues13 In addition crosslinking of phenylpropanoidesters resultsin the formation of lignin-like polymers whichleads to the stiffening of the plant cell wall13 Previous reportsalso showed that apple resistance toVenturia inaequalis dependson the regulatory genes of phenol synthesis14 The total pheno-lics and shikimic acid contents in Venturia inaequalis-tolerantcultivars are higher than susceptible apple cultivars15

Flavonoids are widely distributed in plants and they aresynthesized in the cytosol through the phenylpropanoid path-way by a set of enzymes1617 Previous reports showed thatflavonoids were implicated in plant resistance to biotic (her-bivory and nematodes) and abiotic stresses including droughtcold UV-radiation and toxic metals such as aluminum16

Flavonoids are also implicated in plant resistance to fungaland microbial attacks Flavonoids could exhibit their resis-tance to pathogens by inhibition and crosslinking of themicrobial enzymes chelation of metals necessary for enzymeactivity and formation of physical barrier16

The total phenolic content is normally measured using theFolin-Ciocalteau method which is based on the reactionbetween Folin-Ciocalteau reagent with reductants such asphenols resulting in blue color formation18 Yet a majorlimitation to this assay is its lack of specificity and the results

of this assay cannot be unequivocally attributed to a singleclass of reductants (ie phenolics) in biological tissuesFlavonoids are generally estimated using aluminum chloridewhich forms acid stable complexes with the C-4 keto groupand either the C-3 or C-5 hydroxyl group of flavones andflavonol19 Yet in complex plant extracts even this methodcan show responses to compounds unrelated to flavones andflavonoids The antioxidant activity is normally estimatedusing the DPPH assay which is based on the ability of anti-oxidants to transfer an electron or a hydrogen to the stablefree radical 22-diphenyl-1-picrylhydrazyl (DPPH)2021

In this study we investigated the total phenolic and flavo-noid contents as well as the antioxidant and reducing activityof 21 citrus species and twoCitrus relatives We hypothesizethat CLas-tolerant cultivars contain higher amounts of phe-nolic and flavonoid compounds and that they will have higherantioxidant andor reducing activity compared to susceptiblecultivars Furthermore we investigated the secondary meta-bolites of a representative subclass of these species coveringa wide range of CLas tolerance This was done with theintention to better understand the mechanisms behind citrustolerance to HLB and to help citrus breeders to developcommercially tolerant citrus cultivars

Materials and methods

Plant materials

Healthy seedlings from 24 citrus cultivars andCitrus relatives wereused in this study (Table 1) Seeds were purchased from LynCitrus Seed Inc (Arvin CA USA) and individually potted insquare plastic pots (30 times 10 times 10 cm) containing Sungro profes-sional growing mix (Sungro Horticulture Agawam MA)Germinated seedlings were kept in a greenhouse (28 plusmn 1ordmC60 plusmn 5 relative humidity L16D8 h photoperiod) at the CitrusResearch and Education Center (CREC) University of Florida

Table 1 Citrus species and citrus relatives used in this study and their degree of tolerance to CLas

Species used in this study Degree of tolerance to CLas

1 Valencia sweet orange (C sinensis (L) Osbeck) 1a

2 Madam Vinous sweet orange (C sinensis (L) Osbeck) 1a

3 Hamlin sweet orange (C sinensis L Osbeck) 1a

4 Duncan grapefruit (C paradisi MacFadyen) 1a

5 Ruby red grapefruit (C paradisi MacFadyen) 1a

6 Sour orange (C aurantium L) 2a

7 C macrophylla Wester (Alemow) 2a

8 Mexican lime (C aurantifolia (Christm) 2a

9 Carrizo citrange (X Citroncirus webberi) 3a

10 Citrus latipes 3a

11 Minneola tangelo 1a

12 Citron (Citrus medica) 2a

13 Cleopatra mandarin 1a

14 Sun Chu Sha Kat mandarin (C reticulata Blanco RUTACEAE) 2b

15 Orange jasmine (Murraya paniculata) 1b

16 Pineapple sweet orange 1b

17 Sugar Belle tangerine 2b

18 Finger lime (Citrus australasica) 2b

19 Curry leaf (Murraya koenigii (L) Spreng) 4b

20 Bearss lime (C latifolia) 2b

21 Frost Marsh grapefruit (Citrus paradisi Macfadyen) 2b

22 Bingo tangerine lsquoLB8-9rsquo 2b

23 Dancy Tangerine 3b

aFolimonova et al6bField and greenhouse observations

e1752447-2 F HIJAZ ET AL

Lake Alfred Florida Seedlings were watered twice weekly Allseedlings were about one-year old Five plants (about one-year-old) were sampled from each variety by collecting three matureleaves from three different locations (top middle and bottom)

Extraction of phenolics and flavonoids from plant tissues

Leaves collected from each variety were pooled and ground inliquid nitrogen using a mortar and pestle and 100 mg of thehomogenous sample was transferred to a 2-mL centrifugetube One ml of methanol was added to each tube and thesample was vortexed at 750 rpm for 15 min at 20ordmC usingEppendorf Thermomixer followed by sonication for 15 minThe samples were centrifuged at 12000 rpm for 10 min at20ordmC The supernatant was transferred into a new tube andkept at minus20ordmC until analysis

Total Phenolic Content (TPC)

Total phenolics were determined using Folin-Ciocalteaureagents according to Singleton amp Rossi (1965)22 Plantextracts or gallic acid standard (20 microl) were mixed with09 mL of Folin-Ciocalteu reagent (10 in water) and then06 mL of sodium carbonate (75 wv) was added to themixture After standing for 60 min at room temperatureabsorbance was measured at 760 nm Aqueous solutions ofknown gallic acid concentrations in the range of 100ndash600 ppmwere used for calibration Results were expressed as mg gallicacid equivalents (GAE) gminus1fresh weight (FW)

Total Flavonoid Content (TFC)

Determination of total flavonoids was performed according tothe colorimetric assay of Aktumsek et al (2013) using alumi-num chloride23 Distilled water (200 microL) was added to 50 microLof the extract in a test tube A 15 microL portion of 5 sodiumnitrite solution was added followed by 15 microL of 10 alumi-num chloride solution Test tubes were incubated at ambienttemperature for 5 min and then 100 microL of 1 M sodiumhydroxide and 12 mL of distilled water were added to themixture The mixture was vortexed and the absorbance of theresulting pink color was measured at 510 nm Aqueous solu-tions of known catechin concentrations in the range of10ndash200 ppm were used for calibration and the results wereexpressed as mg catechin equivalents (CEQ) gminus1FW sample

Free radical scavenging activity (DPPH assay)

DPPH assay was performed according to Brand-Williams et -al24 Briefly a 1000 microL aliquot of a 01 mM of DPPH solutionin methanol was added to 25 microl of each extract or Troloxstandard (prepared in the range of 20ndash200 ppm dissolved inmethanol) The mixture was vortexed for 5ndash10 sec and incu-bated at room temperature in dark for 30 min and then theabsorbance was measured at 515 nm The results wereexpressed as mg Trolox gminus1 FW

Chromatographic conditions

The HPLC-MSanalysis of the extracts were run on eclipse C18column (Eclipse 150 mm 5 μm) The mobile phase wasa mixture of 05 acetic acid solution (A) and acetonitrile(B) and was run in a linear gradient mode The start wasa 95 (A) that descended to 70 (A) in 40 min Then to 40(A) in 20 min and finally to 10 (A) in 2 min and stayedthere for 6 min and then back to the initial conditions in 2min The HPLC system was equilibrated for 5 min with theinitial acidic water mobile phase (95 A) before injecting nextsample All the samples were filtered with a 045 μm PTFEfilter The column temperature was set at 25degC

A 20-microL aliquot of the final leaf extract was analyzed byHPLCndashMS using a Waters Alliance 2695 HPLC coupled witha 996 photodiode array detector and a Waters ZQ single quadmass spectrometer A flow splitter (10-to-1) was used tosimultaneously monitor UV and mass spectra of the elutinganalytes UV spectra were monitored between 600 and240 nm ZQ parameters were as follows MS parameterswere as follows ionization mode ES+ capillary voltage 30kV extractor voltage 5 V source temperature 100degC desolva-tion temperature 225degC desolvation N2 flow 465 Lh cone N2

flow 70 Lh scan range mz 50ndash1000 scan rate 1 scans andcone voltages 20 and 40 V Analysis of the chromatogramswas performed using ZQ calculated mass-extracted total ionchromatograms (TIC) obtained in scanning mode or in thesingle ion response mode To normalize the mass spectro-meter response during sequential runs an internal standardmangiferin was used Data handling was done with theMassLynx software version 41

Phenolics in methanol extract were tentatively identifiedclassified by their UV and mass spectra The numbers ofcompounds in particular classes of phenols can were esti-mated with reasonable accuracy using the high peak resolu-tion afforded by analytical-reversed-phase of the HPLCEstimates of the levels of these compounds were obtained byusing peak area-to-microgram conversion factors of authenticstandards identified in each group The measured conversionfactors for ferulic acid nobiletin hesperidin diosmin vitexinmarmin and mahanimbine were used for hydroxycinnamatespolymethoxylated flavones flavanone glycosides flavone-O-glycosides flavone-C-glycosides coumarins and carbazolealkaloids respectively

Saponification of hydroxycinnamates in citrus leafextracts

Portions (150 uL of each leaf extract) were combined with 50uL 4 N KOH and 200 uL 2 N KOH The samples werevortexed for 3 min and stored at room temperature for 1 hThe saponified samples were then mixed with 50uL glacialacid and concentrated HCL until the resulting solutions wereadjusted to pH 45ndash6 The final volumes of the samples werebrought to 10 mL with deionized water Analytical HPLCusing a Waters XBridge C8 column (150 x 46 mm) usingthe same solvent gradients as described above was used tomeasure the amounts of liberated hydroxycinnamic acids

PLANT SIGNALING amp BEHAVIOR e1752447-3

Statistical analysis

Data were analyzed using JMP 90 software (SAS Cary NC)Analysis of variance (ANOVA) followed by post hoc pairwisecomparisons using TukeyndashKramer honestly significant differ-ent test (Tukey HSD) were used to compare the studiedparameters (TPC TFC DPPH and hydroxycinnamates)among the selected varieties

Results

Total phenolic content

The colorimetricFolin-Ciocalteauassay showed the highest totalphenolic content (1368 plusmn 171mgGAg) in the leaves ofMurrayakoenigii (curry) followed by Dancy tangerine (1255 plusmn 093mgGAgminus1 FW) Carrizo citrange (1115 plusmn 200mgGAgminus1 FW) and lsquoLB8-9ʹ Bingo (1055 plusmn 194mg GA gminus1 FW) (Figure 1(a)) The Tukeyrsquostest showed that the level of total phenolic content in curry leaf wassignificantly higher than all other cultivars (Figure 1(a)) The low-est phenolic content occurred in Duncan grapefruit (411 plusmn057 mg GA gminus1 FW) ruby red grapefruit (462 plusmn 026 mg GAgminus1 FW) and C macrophylla (479 plusmn 053 mg GA gminus1 FW) leaves(Figure 1(a)) The Tukeyrsquos test showed that the level of totalphenolic content in Duncan grapefruit was significantly lowerthan all other cultivars The rest of cultivars contained moderatelevels of phenolic content between 531 and 937 mg GA gminus1 FW(Figure 1(a))

Total flavonoid content

The highest level of flavonoids as measured by the aluminumchloride assay occurred in curry leaf (081 plusmn 010 mg gminus1 FW)followed byBearss lime Sunchu mandarin sour orange Carrizocitrange orange jasmine Bingo Clatipes and Carrizo citrange(049ndash043mggminus1 FW) (Figure 1(b)) The level of total flavonoidsin curry leaf measured by the aluminum chloride assay wassignificantly higher than the rest of other plants (Figure 1(b))The lowest level of flavonoids occurred in Ruby red (023 plusmn0018 mg gminus1 FW) and Minneola tangelo (030 plusmn 0010 mg gminus1

FW) (Figure 1(b)) The rest of the cultivars contained moderatelevels of flavonoids (with the range of 032ndash042 mg gminus1 FW)(Figure 1(b))

Total antioxidant activity

In agreement with the total phenolic and flavonoids contentscurry leaf was the highest free radical scavenging antioxidantactivity DPPH (200 plusmn 027mggminus1 FW) (Figure 1(c)) Bearss limewas the second highest plant in DPPH (152 plusmn 013 mg gminus1 FW)followed by Dancy tangerine (120 plusmn 024 mgg) orange jasmine(092 plusmn 0078mg gminus1 FW) and finger lime (088 plusmn 019mg gminus1 FW)(Figure 1(c)) Ruby red grapefruit had the lowest free radicalscavenging activity (013 plusmn 0051 mg gminus1 FW) (Figure 1(c)) Therest of the cultivars showed moderate free radical scavengingactivity (021ndash08 mg gminus1 FW) (Figure 1(c))

HPLC-MS results

Results of the HPLC-MS analyses of the phenolics in seven of thevarieties in this study are summarized in Table 2 Themain groupsof compounds detected in each variety are shown in Figure 2 Thenumber of compounds detected in each class of plant phenols andthe calculated estimates of the sum total amounts in each class aregiven in Table 2 for leaves of curry Duncan and Ruby Redgrapefruits Bearss lime Carrizo citrange Valencia orange andDancy tangerine leaves The results in Table 2 show that Dancytangerine and Bears lime contained the highest summed values forflavone-O-glycosides and flavone-C-glycosides Dancy tangerinecontained the highest amounts of polymethoxylated flavonoids(PMFs and was also the highest in hydroxycinnamates (Table 2)Unique to curry leaf were the high concentration of carbazolealkaloids Calculations based on the known compoundmahanim-bine show an average carbazole alkaloid concentration of about14 mgg

Accurate measurements of the total concentrations of ferulicsinapinic and p-coumaric acid-bound hydroxycinnamates wereobtained by HPLC analysis of saponified leaf extracts Dancytangerine was shown to contain the highest levels of total boundhydroxycinnamic acids (430mggminus1 FW) followed by Carrizocitrange (36 mgg FW) Bearss lime (190mggminus1 FW) andcurry leaf (150mggminus1 FW) (Table 3) Curry leaf was the highestin P-coumaric acid followed by Bears lime (Table 3) Dancytangerine was the highest in ferulic acid followed by Carrizocitrange (Table 3) The two grapefruit varieties Ruby Red andDuncan contained the lowest levels of bound hydroxycinnamicacids Curry leaves differed from the other citrus varieties byhaving a significantly higher p-coumaric acid content(130mggminus1 FW) than the other leaf sources

Discussion

Although field observations and greenhouse studies showedthat certain citrus genotypes are more tolerant to HLB thanother genotypes the mechanisms behind this toleranceremained largely unknown In the broad view citrus toleranceto HLB could result from resistance to the insect vector or tothe plant pathogen The resistance to the vector insect couldresult from several reasons including antibiosis or antixenosiswhereas as resistance to the CLas bacterium could result frommany different factors primarily which is the presence ofbacteriostatic and bactericidal compounds In our currentinvestigation we measured the total phenolic and flavonoidcontents as well as the DPPH activity of 23 citrus genotypes totest if any of these criteria correlated closely with citrustolerance to the CLas bacterium

Total phenolics total flavonoids and antioxidant activitiescan be measured using several methods with differentmechanisms For example total flavonoids can be measuredby colorimetric methods using aluminum chloride and24-dinitrophenylhydrazine Among the different classes offlavonoids the aluminum chloride assay is specific for fla-vones and flavonols while the 24-dinitrophenylhydrazinemethod is specific for flavanones19 In the same mannerseveral methods with different mechanisms have been devel-oped to determine the antioxidant activities in plants and

e1752447-4 F HIJAZ ET AL

Figure 1 Total phenolics (a) flavonoids (b) contents and antioxidant activity measured as (DPPH) (c) in selected citrus and citrus relative genotypes Error barsrepresent standard deviation (n = 5) Varieties with different letters are statistically different using Tukeyrsquos HSD test (P-value lt 005)

PLANT SIGNALING amp BEHAVIOR e1752447-5

foods25 Consequently it is recommended to use more thanone method to measure the total antioxidant activities ofa biological tissue25 In this investigation we measured thetotal phenolic and flavonoid contents as well as the DPPHactivity in the leaves of a wide range of citrus varieties usingcolorimetric assays and further analyzed the methanol extractof seven of the selected genotypes by HPLC-MS To gain anaccurate measure of the bound hydroxycinnamic acids in theleaf tissues the leaf extracts were also saponified and analyzedby HPLC-MS

The colorimetric assays showed that curry leaf was the high-est in total phenolic total flavonoids and free radical scaven-ging antioxidant activity (DPPH) The HPLC-MS analysisshowed that curry leaf was high in hydroxycinnamatesHowever only four flavonoids were detected and the estimatedtotal flavonoid content in curry leaf was low (060 mg gminus1 FW)These results indicated that phenolic compounds in curry leafwere interfering with the aluminum chloride assay In agree-ment with the HPLC-MS results saponification of the metha-nol extracts showed that curry leaf was the highest inp-coumaric acid which has 14 Trolox equivalent antioxidantcapacity (TEAC)26 Our results were in agreement with pre-vious reports which showed high phenolic contents as well ashigh antioxidant capacities in curry leaves27 In another studycurry leaf was also found to be rich in gallic acid and totalphenolic compounds and showed high DPPH value28

The HPLC-MS analysis also showed that curry leaf was highin carbazole alkaloid contents The alkaloid mahanimbine andthe essential oil of curry leaf showed strong antibacterial activ-ities against several antibiotic-resistant pathogenic bacteria andwere effective against several cancer cell lines29 In a separatestudy the methanol extract of curry leaves showed strong anti-microbial activities against several pathogens includingStaphylococcus Streptococcus and E coli30 The antibacterialactivities of the curry leaf extract were comparable with genta-mycin and amikacin30 Carbazole compounds from curry leafalso showed high oil stability index and a strong radical scaven-ging activity against DPPH31 The DPPH radical scavengingactivities of most carbazoles were stronger than that of α-tocopherol31 The previous results together indicated that carba-zole alkaloids could have a strong antimicrobial activity againstCLas However future research is needed to confirm thissuggestion

Our results showed that Dancy tangerine leaveswere second highest in total phenolic compounds and thethird in antioxidant activity In agreement with the colori-metric assays HPLC-MS also showed that Dancy tangerinewas rich in hydroxycinnamates flavanones and flavones and

polymethoxylated flavones The saponification results showedthat Dancy tangerine was rich in ferulic and sinapic acidHigh levels of hydrolyzable hydroxycinnamic acids were ear-lier found in Dancy tangerine peel32 Levels of polymethoxy-lated flavones in Dancy tangerines were several times higherthan in most other citrus cultivars32 Our earlier researchshowed that several hydroxycinnamates and polymethoxy-lated flavones were induced in CLas-infected sweet orangetrees33 These results suggested that hydroxycinnamates andpolymethoxylated flavones could be involved in citrus defenseagainst CLas Induction of hydroxycinnamates was alsoobserved in different plants upon bacterial and viralinfections33-35 It is believed that hydroxycinnamates couldact as direct antimicrobial agents and signal molecules andcan be deposited into the cell wall to reinforce it againstpathogen attack36 These observations further indicate thatDancy tangerine could be tolerant to the HLB pathogenthrough its high phenolic content

Bearss lime was the second in total flavonoids DPPH andit contained moderate amounts of total phenolic In agree-ment with the colorimetric result the HPLC-MS resultsshowed that Bearss lime was rich in flavonoids (22 com-pounds) In addition the saponification results showed thatBearss lime was the highest in sinapic acid and the fourth intotal hydroxycinnmates These results indicated that it couldshow some tolerance to the HLB disease

Previous studies showed that C citrangewas relatively tolerantto theCLas pathogen6 Our current results showed thatC citrangewas the third highest variety in total phenolic and the fifth in totalflavonoids and the seventh in DPPH In agreement with theseresults the HPLC-MS analysis showed that C citrangewas rich inhydroxycinnamates (15 compounds) and it contained moderateamount of flavonoids (20 compounds) Saponification of themethanol extract also showed that C citrange was rich in ferulicacid Our previous study showed that C citrange was the highestamong 13 different varieties in total volatiles and sesquiterpenesand second in total monoterpenes8 With respect to single vola-tiles C citrange was the highest cultivar in t-caryophyllene γ-elemene d-limonene β-elemene and germacrene D8 In additionC citrange was the second highest variety in quinic acid9

Flavonoids which are also considered as a group of phenoliccompounds have a good antioxidant activity2637 Recently Zuoet al (2019) showed that flavones such as luteolin and apigeninand flavonols (quercetin) had potent inhibitory effects on YbeY ofCLas bacterium38 YbeY is considered part of the 206 genes thatmake up the minimal bacterial genome set which is necessary forthe maturation of RNAs In addition the YbeY portion of thebacteriumrsquos genetic code was found to play an important role in

Table 2 Concentrations (mggminus1 FW) of main classes of metabolites detected in seven citrus genotypes (n = 5)

Species

Chemical group

FN-C-glyc FN-O-glyc Flavanone HCA PMF Psoralen Carbazole

Bearss lime 473 (10) 1109 (7) 035 (1) 019 (2) 0 0095 (2) 0Valencia orange 091 (7) 187 (7) 365 (2) 010 (1) 103 (11) 0 0Ruby red grapefruit 093 (4) 417 (6) 213 (7) 015 (2) 004 (3) 013(2) 0Duncan grapefruit 011 (2) 116 (9) 028 (9) 0031 (2) 010 (5) 005 (1) 0Dancy tangerine 488 (7) 192 (2) 546 (2) 488 (9) 718 (12) 0 0Carrizo citrange 239 (8) 227 (4) 373 (2) 055 (15) 266 (6) 0 0Curry leaf 0 059 (4) 0 075 (15) 0 0 143 (14)

FN-C-glyc Flavone-C-glycosides FN-O-glyc flavone-O-glycosides

e1752447-6 F HIJAZ ET AL

Figure 2 Representative HPLC chromatograms of selected citrus and citrus relative genotypes (a Dancy mandarin b Carrizo citrange c Bearss lime d Valenciasweet orange e Curry leaf f Duncan grapefruit and g Ruby red) showing main peaks at 280 nm

PLANT SIGNALING amp BEHAVIOR e1752447-7

bacterial symbiosis and pathogenicity38 Tested flavones and fla-vonols showed a significant reduction in CLas titer in HLB-infected citrus trees38 On the other hand the flavanones (hesper-etin and naringenin) were found to have little to no inhibitoryeffect38 The previous results suggested that HLB resistant plantscould be generated via genetic or metabolic modification of thebiosynthetic pathways of flavones and flavonols38 Our previousstudy showed that several flavonoids compounds such as 68-di-C-glucosylapigenin 2rdquo-O-xylosylvitexin hesperidin sinensetin andpolymethoxylated flavones were induced in CLas-infected sweetorange trees33 Taken together these previous results suggest thatflavonoids could play a role in citrus defense against CLas

Conclusion

Our results showed that tolerant varieties contained higherlevels of phenolic compounds and flavonoids than sensitivevarieties Curry leaf which is immune toCLas contained highlevel of hyrdoxycinnamates and carbazole alkaloids Ourresults suggest that high levels of flavonoids and phenoliccompounds might enhance citrus tolerance to HLB Furtherstudies into the roles of these compounds in imparting toler-ance in citrus to the CLas bacterium may provide usefulinformation for the control of citrus HLB disease

Acknowledgments

We thank Yasser Nehela for the technical assistance and Lorraine Jonesfor maintaining the trees in greenhouses This work was kindly fundedby Citrus Research and Development Foundation grant number 19-015

Funding

This work was supported by the Citrus Research and DevelopmentFoundation [19-015]

ORCID

Nabil Killiny httporcidorg0000-0002-3895-6943

References

1 Tiwari S Killiny N Stelinski LL Dynamic insecticide susceptibil-ity changes in Florida populations of Diaphorina citri (Hemipterapsyllidae) J Econ Entomol 2013106393ndash399 doi101603EC12281

2 Blaustein RA Lorca GL Teplitski M Challenges for ManagingCandidatus Liberibacter spp (Huanglongbing disease pathogen)current control measures and future directions Phytopathology2018108424ndash435 doi101094PHYTO-07-17-0260-RVW

3 Halbert SE Manjunath KL Asian citrus psyllids(Sternorrhyncha psyllidae) and greening disease of citrusa literature review and assessment of risk in Florida FloridaEntomol 200487330ndash353 doi1016530015-4040(2004)087[0330ACPSPA]20CO2

4 Tsai JH Liu YH Biology of Diaphorina citri (Homoptera psylli-dae) on four host plants J Econ Entomol 2009931721ndash1725doi1016030022-0493-9361721

5 Richardson ML Hall DG Westbrook R Stover EW Duan YPResistance of Poncirus and Citrus x Poncirusgermplasm to theAsian Citrus Psyllid J Citrus Pathol 20141266

6 Folimonova SY Robertson CJ Garnsey SM Gowda SDawson WO Examination of the responses of different genotypesof citrus to Huanglongbing (citrus greening) under differentconditions Phytopathology 2009991346ndash1354 doi101094PHYTO-99-12-1346

7 Shokrollah Differential reaction of citrus species in Malaysia toHuanglongbing (HLB) disease using grafting method Am J AgricBiol Sci 2009432ndash38 doi103844ajabssp20093238

8 Hijaz F Nehela Y Killiny N Possible role of plant volatiles intolerance against huanglongbing in citrus Plant Signal Behav201611e1138193 doi1010801559232420161138193

9 Killiny N Hijaz F Amino acids implicated in plant defense arehigher in Candidatus Liberibacter asiaticus-tolerant citrusvarieties Plant Signal Behav 201611e1171449 doi1010801559232420161171449

10 Killiny N Valim MF Jones SE Omar AA Hijaz F Gmitter FGGrosser JW Metabolically speaking possible reasons behind thetolerance of lsquoSugar Bellersquo mandarin hybrid to HuanglongbingPlant Physiol Biochem 201711636ndash47 doi101016jplaphy201705001

11 Killiny N Jones SE Nehela Y Hijaz F Dutt M Gmitter FGGrosser JW All roads lead to Rome towards understandingdifferent avenues of tolerance to Huanglongbing in citruscultivars Plant Physiol Biochem PPB 20181291ndash10doi101016jplaphy201805005

12 Lin D Xiao M Zhao J Li Z Xing B Li X Kong M Li L Zhang QLiu Y et al An overview of plant phenolic compounds and theirimportance in human nutrition and management of type 2diabetes Molecules 201621(10)pii E1374 doi103390molecules21101374

13 Nicholson RL Hammerschmidt R Phenolic compounds and theirrole in disease resistance Annu Rev Phytopathol199230369ndash389 doi101146annurevpy30090192002101

14 Michalek S Mayr U Treutter D Lux-Endrich A Gutmann MFeucht W Geibel M Role of flavan-3-OLS in resistance of appletrees to venturia inaequalis Acta Hortic 1998484535ndash539doi1017660ActaHortic199848491

15 Arici SE Kafkas E Kaymak S Koc NK Phenolic compounds ofapple cultivars resistant or susceptible to Venturia inaequalisPharm Biol 201452904ndash908 doi103109138802092013872674

16 Treutter D Significance of flavonoids in plant resistance Areview Environ Chem Lett 20064147ndash157 doi101007s10311-006-0068-8

17 Petrussa E Braidot E Zancani M Peresson C Bertolini A Patui SVianello A Plant Flavonoids-Biosynthesis Transport and involve-ment in stress responses Int J Mol Sci 20131414950ndash14973doi103390ijms140714950

Table 3 Concentrations (mg gminus1 FW) of hydroxycinnamic acids existing as hydrolysable hydroxycinnamates in seven citrus leaf extracts Hydroxycinnamic acidsmeasured after leaf extract saponification (n = 5)

Species P-Coumaric Sinapic Ferulic

Bearss lime 062 plusmn 008b 084 plusmn 005a 049 plusmn 004b

Valencia orange 011 plusmn 001d 045 plusmn 004 c 066 plusmn 038b

Ruby Red grapefruit 012 plusmn 001d 006 plusmn 001e 061 plusmn 005b

Duncan grapefruit 011 plusmn 001d 003 plusmn 002e 060 plusmn 041b

Dancy tangerine 028 plusmn 003 c 057 plusmn 002b 344 plusmn 016a

Carrizo citrange 026 plusmn 030 cd 032 plusmn 001d 298 plusmn 266ab

Curry leaf 129 plusmn 030a 003 plusmn 001e 018 plusmn 004 c

Varieties with different superscript letters are statistically different using Tukeyrsquos HSD test (P-value lt 005)

e1752447-8 F HIJAZ ET AL

Lake Alfred Florida Seedlings were watered twice weekly Allseedlings were about one-year old Five plants (about one-year-old) were sampled from each variety by collecting three matureleaves from three different locations (top middle and bottom)

Extraction of phenolics and flavonoids from plant tissues

Leaves collected from each variety were pooled and ground inliquid nitrogen using a mortar and pestle and 100 mg of thehomogenous sample was transferred to a 2-mL centrifugetube One ml of methanol was added to each tube and thesample was vortexed at 750 rpm for 15 min at 20ordmC usingEppendorf Thermomixer followed by sonication for 15 minThe samples were centrifuged at 12000 rpm for 10 min at20ordmC The supernatant was transferred into a new tube andkept at minus20ordmC until analysis

Total Phenolic Content (TPC)

Total phenolics were determined using Folin-Ciocalteaureagents according to Singleton amp Rossi (1965)22 Plantextracts or gallic acid standard (20 microl) were mixed with09 mL of Folin-Ciocalteu reagent (10 in water) and then06 mL of sodium carbonate (75 wv) was added to themixture After standing for 60 min at room temperatureabsorbance was measured at 760 nm Aqueous solutions ofknown gallic acid concentrations in the range of 100ndash600 ppmwere used for calibration Results were expressed as mg gallicacid equivalents (GAE) gminus1fresh weight (FW)

Total Flavonoid Content (TFC)

Determination of total flavonoids was performed according tothe colorimetric assay of Aktumsek et al (2013) using alumi-num chloride23 Distilled water (200 microL) was added to 50 microLof the extract in a test tube A 15 microL portion of 5 sodiumnitrite solution was added followed by 15 microL of 10 alumi-num chloride solution Test tubes were incubated at ambienttemperature for 5 min and then 100 microL of 1 M sodiumhydroxide and 12 mL of distilled water were added to themixture The mixture was vortexed and the absorbance of theresulting pink color was measured at 510 nm Aqueous solu-tions of known catechin concentrations in the range of10ndash200 ppm were used for calibration and the results wereexpressed as mg catechin equivalents (CEQ) gminus1FW sample

Free radical scavenging activity (DPPH assay)

DPPH assay was performed according to Brand-Williams et -al24 Briefly a 1000 microL aliquot of a 01 mM of DPPH solutionin methanol was added to 25 microl of each extract or Troloxstandard (prepared in the range of 20ndash200 ppm dissolved inmethanol) The mixture was vortexed for 5ndash10 sec and incu-bated at room temperature in dark for 30 min and then theabsorbance was measured at 515 nm The results wereexpressed as mg Trolox gminus1 FW

Chromatographic conditions

The HPLC-MSanalysis of the extracts were run on eclipse C18column (Eclipse 150 mm 5 μm) The mobile phase wasa mixture of 05 acetic acid solution (A) and acetonitrile(B) and was run in a linear gradient mode The start wasa 95 (A) that descended to 70 (A) in 40 min Then to 40(A) in 20 min and finally to 10 (A) in 2 min and stayedthere for 6 min and then back to the initial conditions in 2min The HPLC system was equilibrated for 5 min with theinitial acidic water mobile phase (95 A) before injecting nextsample All the samples were filtered with a 045 μm PTFEfilter The column temperature was set at 25degC

A 20-microL aliquot of the final leaf extract was analyzed byHPLCndashMS using a Waters Alliance 2695 HPLC coupled witha 996 photodiode array detector and a Waters ZQ single quadmass spectrometer A flow splitter (10-to-1) was used tosimultaneously monitor UV and mass spectra of the elutinganalytes UV spectra were monitored between 600 and240 nm ZQ parameters were as follows MS parameterswere as follows ionization mode ES+ capillary voltage 30kV extractor voltage 5 V source temperature 100degC desolva-tion temperature 225degC desolvation N2 flow 465 Lh cone N2

flow 70 Lh scan range mz 50ndash1000 scan rate 1 scans andcone voltages 20 and 40 V Analysis of the chromatogramswas performed using ZQ calculated mass-extracted total ionchromatograms (TIC) obtained in scanning mode or in thesingle ion response mode To normalize the mass spectro-meter response during sequential runs an internal standardmangiferin was used Data handling was done with theMassLynx software version 41

Phenolics in methanol extract were tentatively identifiedclassified by their UV and mass spectra The numbers ofcompounds in particular classes of phenols can were esti-mated with reasonable accuracy using the high peak resolu-tion afforded by analytical-reversed-phase of the HPLCEstimates of the levels of these compounds were obtained byusing peak area-to-microgram conversion factors of authenticstandards identified in each group The measured conversionfactors for ferulic acid nobiletin hesperidin diosmin vitexinmarmin and mahanimbine were used for hydroxycinnamatespolymethoxylated flavones flavanone glycosides flavone-O-glycosides flavone-C-glycosides coumarins and carbazolealkaloids respectively

Saponification of hydroxycinnamates in citrus leafextracts

Portions (150 uL of each leaf extract) were combined with 50uL 4 N KOH and 200 uL 2 N KOH The samples werevortexed for 3 min and stored at room temperature for 1 hThe saponified samples were then mixed with 50uL glacialacid and concentrated HCL until the resulting solutions wereadjusted to pH 45ndash6 The final volumes of the samples werebrought to 10 mL with deionized water Analytical HPLCusing a Waters XBridge C8 column (150 x 46 mm) usingthe same solvent gradients as described above was used tomeasure the amounts of liberated hydroxycinnamic acids

PLANT SIGNALING amp BEHAVIOR e1752447-3

Statistical analysis

Data were analyzed using JMP 90 software (SAS Cary NC)Analysis of variance (ANOVA) followed by post hoc pairwisecomparisons using TukeyndashKramer honestly significant differ-ent test (Tukey HSD) were used to compare the studiedparameters (TPC TFC DPPH and hydroxycinnamates)among the selected varieties

Results

Total phenolic content

The colorimetricFolin-Ciocalteauassay showed the highest totalphenolic content (1368 plusmn 171mgGAg) in the leaves ofMurrayakoenigii (curry) followed by Dancy tangerine (1255 plusmn 093mgGAgminus1 FW) Carrizo citrange (1115 plusmn 200mgGAgminus1 FW) and lsquoLB8-9ʹ Bingo (1055 plusmn 194mg GA gminus1 FW) (Figure 1(a)) The Tukeyrsquostest showed that the level of total phenolic content in curry leaf wassignificantly higher than all other cultivars (Figure 1(a)) The low-est phenolic content occurred in Duncan grapefruit (411 plusmn057 mg GA gminus1 FW) ruby red grapefruit (462 plusmn 026 mg GAgminus1 FW) and C macrophylla (479 plusmn 053 mg GA gminus1 FW) leaves(Figure 1(a)) The Tukeyrsquos test showed that the level of totalphenolic content in Duncan grapefruit was significantly lowerthan all other cultivars The rest of cultivars contained moderatelevels of phenolic content between 531 and 937 mg GA gminus1 FW(Figure 1(a))

Total flavonoid content

The highest level of flavonoids as measured by the aluminumchloride assay occurred in curry leaf (081 plusmn 010 mg gminus1 FW)followed byBearss lime Sunchu mandarin sour orange Carrizocitrange orange jasmine Bingo Clatipes and Carrizo citrange(049ndash043mggminus1 FW) (Figure 1(b)) The level of total flavonoidsin curry leaf measured by the aluminum chloride assay wassignificantly higher than the rest of other plants (Figure 1(b))The lowest level of flavonoids occurred in Ruby red (023 plusmn0018 mg gminus1 FW) and Minneola tangelo (030 plusmn 0010 mg gminus1

FW) (Figure 1(b)) The rest of the cultivars contained moderatelevels of flavonoids (with the range of 032ndash042 mg gminus1 FW)(Figure 1(b))

Total antioxidant activity

In agreement with the total phenolic and flavonoids contentscurry leaf was the highest free radical scavenging antioxidantactivity DPPH (200 plusmn 027mggminus1 FW) (Figure 1(c)) Bearss limewas the second highest plant in DPPH (152 plusmn 013 mg gminus1 FW)followed by Dancy tangerine (120 plusmn 024 mgg) orange jasmine(092 plusmn 0078mg gminus1 FW) and finger lime (088 plusmn 019mg gminus1 FW)(Figure 1(c)) Ruby red grapefruit had the lowest free radicalscavenging activity (013 plusmn 0051 mg gminus1 FW) (Figure 1(c)) Therest of the cultivars showed moderate free radical scavengingactivity (021ndash08 mg gminus1 FW) (Figure 1(c))

HPLC-MS results

Results of the HPLC-MS analyses of the phenolics in seven of thevarieties in this study are summarized in Table 2 Themain groupsof compounds detected in each variety are shown in Figure 2 Thenumber of compounds detected in each class of plant phenols andthe calculated estimates of the sum total amounts in each class aregiven in Table 2 for leaves of curry Duncan and Ruby Redgrapefruits Bearss lime Carrizo citrange Valencia orange andDancy tangerine leaves The results in Table 2 show that Dancytangerine and Bears lime contained the highest summed values forflavone-O-glycosides and flavone-C-glycosides Dancy tangerinecontained the highest amounts of polymethoxylated flavonoids(PMFs and was also the highest in hydroxycinnamates (Table 2)Unique to curry leaf were the high concentration of carbazolealkaloids Calculations based on the known compoundmahanim-bine show an average carbazole alkaloid concentration of about14 mgg

Accurate measurements of the total concentrations of ferulicsinapinic and p-coumaric acid-bound hydroxycinnamates wereobtained by HPLC analysis of saponified leaf extracts Dancytangerine was shown to contain the highest levels of total boundhydroxycinnamic acids (430mggminus1 FW) followed by Carrizocitrange (36 mgg FW) Bearss lime (190mggminus1 FW) andcurry leaf (150mggminus1 FW) (Table 3) Curry leaf was the highestin P-coumaric acid followed by Bears lime (Table 3) Dancytangerine was the highest in ferulic acid followed by Carrizocitrange (Table 3) The two grapefruit varieties Ruby Red andDuncan contained the lowest levels of bound hydroxycinnamicacids Curry leaves differed from the other citrus varieties byhaving a significantly higher p-coumaric acid content(130mggminus1 FW) than the other leaf sources

Discussion

Although field observations and greenhouse studies showedthat certain citrus genotypes are more tolerant to HLB thanother genotypes the mechanisms behind this toleranceremained largely unknown In the broad view citrus toleranceto HLB could result from resistance to the insect vector or tothe plant pathogen The resistance to the vector insect couldresult from several reasons including antibiosis or antixenosiswhereas as resistance to the CLas bacterium could result frommany different factors primarily which is the presence ofbacteriostatic and bactericidal compounds In our currentinvestigation we measured the total phenolic and flavonoidcontents as well as the DPPH activity of 23 citrus genotypes totest if any of these criteria correlated closely with citrustolerance to the CLas bacterium

Total phenolics total flavonoids and antioxidant activitiescan be measured using several methods with differentmechanisms For example total flavonoids can be measuredby colorimetric methods using aluminum chloride and24-dinitrophenylhydrazine Among the different classes offlavonoids the aluminum chloride assay is specific for fla-vones and flavonols while the 24-dinitrophenylhydrazinemethod is specific for flavanones19 In the same mannerseveral methods with different mechanisms have been devel-oped to determine the antioxidant activities in plants and

e1752447-4 F HIJAZ ET AL

Figure 1 Total phenolics (a) flavonoids (b) contents and antioxidant activity measured as (DPPH) (c) in selected citrus and citrus relative genotypes Error barsrepresent standard deviation (n = 5) Varieties with different letters are statistically different using Tukeyrsquos HSD test (P-value lt 005)

PLANT SIGNALING amp BEHAVIOR e1752447-5

foods25 Consequently it is recommended to use more thanone method to measure the total antioxidant activities ofa biological tissue25 In this investigation we measured thetotal phenolic and flavonoid contents as well as the DPPHactivity in the leaves of a wide range of citrus varieties usingcolorimetric assays and further analyzed the methanol extractof seven of the selected genotypes by HPLC-MS To gain anaccurate measure of the bound hydroxycinnamic acids in theleaf tissues the leaf extracts were also saponified and analyzedby HPLC-MS

The colorimetric assays showed that curry leaf was the high-est in total phenolic total flavonoids and free radical scaven-ging antioxidant activity (DPPH) The HPLC-MS analysisshowed that curry leaf was high in hydroxycinnamatesHowever only four flavonoids were detected and the estimatedtotal flavonoid content in curry leaf was low (060 mg gminus1 FW)These results indicated that phenolic compounds in curry leafwere interfering with the aluminum chloride assay In agree-ment with the HPLC-MS results saponification of the metha-nol extracts showed that curry leaf was the highest inp-coumaric acid which has 14 Trolox equivalent antioxidantcapacity (TEAC)26 Our results were in agreement with pre-vious reports which showed high phenolic contents as well ashigh antioxidant capacities in curry leaves27 In another studycurry leaf was also found to be rich in gallic acid and totalphenolic compounds and showed high DPPH value28

The HPLC-MS analysis also showed that curry leaf was highin carbazole alkaloid contents The alkaloid mahanimbine andthe essential oil of curry leaf showed strong antibacterial activ-ities against several antibiotic-resistant pathogenic bacteria andwere effective against several cancer cell lines29 In a separatestudy the methanol extract of curry leaves showed strong anti-microbial activities against several pathogens includingStaphylococcus Streptococcus and E coli30 The antibacterialactivities of the curry leaf extract were comparable with genta-mycin and amikacin30 Carbazole compounds from curry leafalso showed high oil stability index and a strong radical scaven-ging activity against DPPH31 The DPPH radical scavengingactivities of most carbazoles were stronger than that of α-tocopherol31 The previous results together indicated that carba-zole alkaloids could have a strong antimicrobial activity againstCLas However future research is needed to confirm thissuggestion

Our results showed that Dancy tangerine leaveswere second highest in total phenolic compounds and thethird in antioxidant activity In agreement with the colori-metric assays HPLC-MS also showed that Dancy tangerinewas rich in hydroxycinnamates flavanones and flavones and

polymethoxylated flavones The saponification results showedthat Dancy tangerine was rich in ferulic and sinapic acidHigh levels of hydrolyzable hydroxycinnamic acids were ear-lier found in Dancy tangerine peel32 Levels of polymethoxy-lated flavones in Dancy tangerines were several times higherthan in most other citrus cultivars32 Our earlier researchshowed that several hydroxycinnamates and polymethoxy-lated flavones were induced in CLas-infected sweet orangetrees33 These results suggested that hydroxycinnamates andpolymethoxylated flavones could be involved in citrus defenseagainst CLas Induction of hydroxycinnamates was alsoobserved in different plants upon bacterial and viralinfections33-35 It is believed that hydroxycinnamates couldact as direct antimicrobial agents and signal molecules andcan be deposited into the cell wall to reinforce it againstpathogen attack36 These observations further indicate thatDancy tangerine could be tolerant to the HLB pathogenthrough its high phenolic content

Bearss lime was the second in total flavonoids DPPH andit contained moderate amounts of total phenolic In agree-ment with the colorimetric result the HPLC-MS resultsshowed that Bearss lime was rich in flavonoids (22 com-pounds) In addition the saponification results showed thatBearss lime was the highest in sinapic acid and the fourth intotal hydroxycinnmates These results indicated that it couldshow some tolerance to the HLB disease

Previous studies showed that C citrangewas relatively tolerantto theCLas pathogen6 Our current results showed thatC citrangewas the third highest variety in total phenolic and the fifth in totalflavonoids and the seventh in DPPH In agreement with theseresults the HPLC-MS analysis showed that C citrangewas rich inhydroxycinnamates (15 compounds) and it contained moderateamount of flavonoids (20 compounds) Saponification of themethanol extract also showed that C citrange was rich in ferulicacid Our previous study showed that C citrange was the highestamong 13 different varieties in total volatiles and sesquiterpenesand second in total monoterpenes8 With respect to single vola-tiles C citrange was the highest cultivar in t-caryophyllene γ-elemene d-limonene β-elemene and germacrene D8 In additionC citrange was the second highest variety in quinic acid9

Flavonoids which are also considered as a group of phenoliccompounds have a good antioxidant activity2637 Recently Zuoet al (2019) showed that flavones such as luteolin and apigeninand flavonols (quercetin) had potent inhibitory effects on YbeY ofCLas bacterium38 YbeY is considered part of the 206 genes thatmake up the minimal bacterial genome set which is necessary forthe maturation of RNAs In addition the YbeY portion of thebacteriumrsquos genetic code was found to play an important role in

Table 2 Concentrations (mggminus1 FW) of main classes of metabolites detected in seven citrus genotypes (n = 5)

Species

Chemical group

FN-C-glyc FN-O-glyc Flavanone HCA PMF Psoralen Carbazole

Bearss lime 473 (10) 1109 (7) 035 (1) 019 (2) 0 0095 (2) 0Valencia orange 091 (7) 187 (7) 365 (2) 010 (1) 103 (11) 0 0Ruby red grapefruit 093 (4) 417 (6) 213 (7) 015 (2) 004 (3) 013(2) 0Duncan grapefruit 011 (2) 116 (9) 028 (9) 0031 (2) 010 (5) 005 (1) 0Dancy tangerine 488 (7) 192 (2) 546 (2) 488 (9) 718 (12) 0 0Carrizo citrange 239 (8) 227 (4) 373 (2) 055 (15) 266 (6) 0 0Curry leaf 0 059 (4) 0 075 (15) 0 0 143 (14)

FN-C-glyc Flavone-C-glycosides FN-O-glyc flavone-O-glycosides

e1752447-6 F HIJAZ ET AL

Figure 2 Representative HPLC chromatograms of selected citrus and citrus relative genotypes (a Dancy mandarin b Carrizo citrange c Bearss lime d Valenciasweet orange e Curry leaf f Duncan grapefruit and g Ruby red) showing main peaks at 280 nm

PLANT SIGNALING amp BEHAVIOR e1752447-7

bacterial symbiosis and pathogenicity38 Tested flavones and fla-vonols showed a significant reduction in CLas titer in HLB-infected citrus trees38 On the other hand the flavanones (hesper-etin and naringenin) were found to have little to no inhibitoryeffect38 The previous results suggested that HLB resistant plantscould be generated via genetic or metabolic modification of thebiosynthetic pathways of flavones and flavonols38 Our previousstudy showed that several flavonoids compounds such as 68-di-C-glucosylapigenin 2rdquo-O-xylosylvitexin hesperidin sinensetin andpolymethoxylated flavones were induced in CLas-infected sweetorange trees33 Taken together these previous results suggest thatflavonoids could play a role in citrus defense against CLas

Conclusion

Our results showed that tolerant varieties contained higherlevels of phenolic compounds and flavonoids than sensitivevarieties Curry leaf which is immune toCLas contained highlevel of hyrdoxycinnamates and carbazole alkaloids Ourresults suggest that high levels of flavonoids and phenoliccompounds might enhance citrus tolerance to HLB Furtherstudies into the roles of these compounds in imparting toler-ance in citrus to the CLas bacterium may provide usefulinformation for the control of citrus HLB disease

Acknowledgments

We thank Yasser Nehela for the technical assistance and Lorraine Jonesfor maintaining the trees in greenhouses This work was kindly fundedby Citrus Research and Development Foundation grant number 19-015

Funding

This work was supported by the Citrus Research and DevelopmentFoundation [19-015]

ORCID

Nabil Killiny httporcidorg0000-0002-3895-6943

References

1 Tiwari S Killiny N Stelinski LL Dynamic insecticide susceptibil-ity changes in Florida populations of Diaphorina citri (Hemipterapsyllidae) J Econ Entomol 2013106393ndash399 doi101603EC12281

2 Blaustein RA Lorca GL Teplitski M Challenges for ManagingCandidatus Liberibacter spp (Huanglongbing disease pathogen)current control measures and future directions Phytopathology2018108424ndash435 doi101094PHYTO-07-17-0260-RVW

3 Halbert SE Manjunath KL Asian citrus psyllids(Sternorrhyncha psyllidae) and greening disease of citrusa literature review and assessment of risk in Florida FloridaEntomol 200487330ndash353 doi1016530015-4040(2004)087[0330ACPSPA]20CO2

4 Tsai JH Liu YH Biology of Diaphorina citri (Homoptera psylli-dae) on four host plants J Econ Entomol 2009931721ndash1725doi1016030022-0493-9361721

5 Richardson ML Hall DG Westbrook R Stover EW Duan YPResistance of Poncirus and Citrus x Poncirusgermplasm to theAsian Citrus Psyllid J Citrus Pathol 20141266

6 Folimonova SY Robertson CJ Garnsey SM Gowda SDawson WO Examination of the responses of different genotypesof citrus to Huanglongbing (citrus greening) under differentconditions Phytopathology 2009991346ndash1354 doi101094PHYTO-99-12-1346

7 Shokrollah Differential reaction of citrus species in Malaysia toHuanglongbing (HLB) disease using grafting method Am J AgricBiol Sci 2009432ndash38 doi103844ajabssp20093238

8 Hijaz F Nehela Y Killiny N Possible role of plant volatiles intolerance against huanglongbing in citrus Plant Signal Behav201611e1138193 doi1010801559232420161138193

9 Killiny N Hijaz F Amino acids implicated in plant defense arehigher in Candidatus Liberibacter asiaticus-tolerant citrusvarieties Plant Signal Behav 201611e1171449 doi1010801559232420161171449

10 Killiny N Valim MF Jones SE Omar AA Hijaz F Gmitter FGGrosser JW Metabolically speaking possible reasons behind thetolerance of lsquoSugar Bellersquo mandarin hybrid to HuanglongbingPlant Physiol Biochem 201711636ndash47 doi101016jplaphy201705001

11 Killiny N Jones SE Nehela Y Hijaz F Dutt M Gmitter FGGrosser JW All roads lead to Rome towards understandingdifferent avenues of tolerance to Huanglongbing in citruscultivars Plant Physiol Biochem PPB 20181291ndash10doi101016jplaphy201805005

12 Lin D Xiao M Zhao J Li Z Xing B Li X Kong M Li L Zhang QLiu Y et al An overview of plant phenolic compounds and theirimportance in human nutrition and management of type 2diabetes Molecules 201621(10)pii E1374 doi103390molecules21101374

13 Nicholson RL Hammerschmidt R Phenolic compounds and theirrole in disease resistance Annu Rev Phytopathol199230369ndash389 doi101146annurevpy30090192002101

14 Michalek S Mayr U Treutter D Lux-Endrich A Gutmann MFeucht W Geibel M Role of flavan-3-OLS in resistance of appletrees to venturia inaequalis Acta Hortic 1998484535ndash539doi1017660ActaHortic199848491

15 Arici SE Kafkas E Kaymak S Koc NK Phenolic compounds ofapple cultivars resistant or susceptible to Venturia inaequalisPharm Biol 201452904ndash908 doi103109138802092013872674

16 Treutter D Significance of flavonoids in plant resistance Areview Environ Chem Lett 20064147ndash157 doi101007s10311-006-0068-8

17 Petrussa E Braidot E Zancani M Peresson C Bertolini A Patui SVianello A Plant Flavonoids-Biosynthesis Transport and involve-ment in stress responses Int J Mol Sci 20131414950ndash14973doi103390ijms140714950

Table 3 Concentrations (mg gminus1 FW) of hydroxycinnamic acids existing as hydrolysable hydroxycinnamates in seven citrus leaf extracts Hydroxycinnamic acidsmeasured after leaf extract saponification (n = 5)

Species P-Coumaric Sinapic Ferulic

Bearss lime 062 plusmn 008b 084 plusmn 005a 049 plusmn 004b

Valencia orange 011 plusmn 001d 045 plusmn 004 c 066 plusmn 038b

Ruby Red grapefruit 012 plusmn 001d 006 plusmn 001e 061 plusmn 005b

Duncan grapefruit 011 plusmn 001d 003 plusmn 002e 060 plusmn 041b

Dancy tangerine 028 plusmn 003 c 057 plusmn 002b 344 plusmn 016a

Carrizo citrange 026 plusmn 030 cd 032 plusmn 001d 298 plusmn 266ab

Curry leaf 129 plusmn 030a 003 plusmn 001e 018 plusmn 004 c

Varieties with different superscript letters are statistically different using Tukeyrsquos HSD test (P-value lt 005)

e1752447-8 F HIJAZ ET AL

Statistical analysis

Data were analyzed using JMP 90 software (SAS Cary NC)Analysis of variance (ANOVA) followed by post hoc pairwisecomparisons using TukeyndashKramer honestly significant differ-ent test (Tukey HSD) were used to compare the studiedparameters (TPC TFC DPPH and hydroxycinnamates)among the selected varieties

Results

Total phenolic content

The colorimetricFolin-Ciocalteauassay showed the highest totalphenolic content (1368 plusmn 171mgGAg) in the leaves ofMurrayakoenigii (curry) followed by Dancy tangerine (1255 plusmn 093mgGAgminus1 FW) Carrizo citrange (1115 plusmn 200mgGAgminus1 FW) and lsquoLB8-9ʹ Bingo (1055 plusmn 194mg GA gminus1 FW) (Figure 1(a)) The Tukeyrsquostest showed that the level of total phenolic content in curry leaf wassignificantly higher than all other cultivars (Figure 1(a)) The low-est phenolic content occurred in Duncan grapefruit (411 plusmn057 mg GA gminus1 FW) ruby red grapefruit (462 plusmn 026 mg GAgminus1 FW) and C macrophylla (479 plusmn 053 mg GA gminus1 FW) leaves(Figure 1(a)) The Tukeyrsquos test showed that the level of totalphenolic content in Duncan grapefruit was significantly lowerthan all other cultivars The rest of cultivars contained moderatelevels of phenolic content between 531 and 937 mg GA gminus1 FW(Figure 1(a))

Total flavonoid content

The highest level of flavonoids as measured by the aluminumchloride assay occurred in curry leaf (081 plusmn 010 mg gminus1 FW)followed byBearss lime Sunchu mandarin sour orange Carrizocitrange orange jasmine Bingo Clatipes and Carrizo citrange(049ndash043mggminus1 FW) (Figure 1(b)) The level of total flavonoidsin curry leaf measured by the aluminum chloride assay wassignificantly higher than the rest of other plants (Figure 1(b))The lowest level of flavonoids occurred in Ruby red (023 plusmn0018 mg gminus1 FW) and Minneola tangelo (030 plusmn 0010 mg gminus1

FW) (Figure 1(b)) The rest of the cultivars contained moderatelevels of flavonoids (with the range of 032ndash042 mg gminus1 FW)(Figure 1(b))

Total antioxidant activity

In agreement with the total phenolic and flavonoids contentscurry leaf was the highest free radical scavenging antioxidantactivity DPPH (200 plusmn 027mggminus1 FW) (Figure 1(c)) Bearss limewas the second highest plant in DPPH (152 plusmn 013 mg gminus1 FW)followed by Dancy tangerine (120 plusmn 024 mgg) orange jasmine(092 plusmn 0078mg gminus1 FW) and finger lime (088 plusmn 019mg gminus1 FW)(Figure 1(c)) Ruby red grapefruit had the lowest free radicalscavenging activity (013 plusmn 0051 mg gminus1 FW) (Figure 1(c)) Therest of the cultivars showed moderate free radical scavengingactivity (021ndash08 mg gminus1 FW) (Figure 1(c))

HPLC-MS results

Results of the HPLC-MS analyses of the phenolics in seven of thevarieties in this study are summarized in Table 2 Themain groupsof compounds detected in each variety are shown in Figure 2 Thenumber of compounds detected in each class of plant phenols andthe calculated estimates of the sum total amounts in each class aregiven in Table 2 for leaves of curry Duncan and Ruby Redgrapefruits Bearss lime Carrizo citrange Valencia orange andDancy tangerine leaves The results in Table 2 show that Dancytangerine and Bears lime contained the highest summed values forflavone-O-glycosides and flavone-C-glycosides Dancy tangerinecontained the highest amounts of polymethoxylated flavonoids(PMFs and was also the highest in hydroxycinnamates (Table 2)Unique to curry leaf were the high concentration of carbazolealkaloids Calculations based on the known compoundmahanim-bine show an average carbazole alkaloid concentration of about14 mgg

Accurate measurements of the total concentrations of ferulicsinapinic and p-coumaric acid-bound hydroxycinnamates wereobtained by HPLC analysis of saponified leaf extracts Dancytangerine was shown to contain the highest levels of total boundhydroxycinnamic acids (430mggminus1 FW) followed by Carrizocitrange (36 mgg FW) Bearss lime (190mggminus1 FW) andcurry leaf (150mggminus1 FW) (Table 3) Curry leaf was the highestin P-coumaric acid followed by Bears lime (Table 3) Dancytangerine was the highest in ferulic acid followed by Carrizocitrange (Table 3) The two grapefruit varieties Ruby Red andDuncan contained the lowest levels of bound hydroxycinnamicacids Curry leaves differed from the other citrus varieties byhaving a significantly higher p-coumaric acid content(130mggminus1 FW) than the other leaf sources

Discussion

Although field observations and greenhouse studies showedthat certain citrus genotypes are more tolerant to HLB thanother genotypes the mechanisms behind this toleranceremained largely unknown In the broad view citrus toleranceto HLB could result from resistance to the insect vector or tothe plant pathogen The resistance to the vector insect couldresult from several reasons including antibiosis or antixenosiswhereas as resistance to the CLas bacterium could result frommany different factors primarily which is the presence ofbacteriostatic and bactericidal compounds In our currentinvestigation we measured the total phenolic and flavonoidcontents as well as the DPPH activity of 23 citrus genotypes totest if any of these criteria correlated closely with citrustolerance to the CLas bacterium

Total phenolics total flavonoids and antioxidant activitiescan be measured using several methods with differentmechanisms For example total flavonoids can be measuredby colorimetric methods using aluminum chloride and24-dinitrophenylhydrazine Among the different classes offlavonoids the aluminum chloride assay is specific for fla-vones and flavonols while the 24-dinitrophenylhydrazinemethod is specific for flavanones19 In the same mannerseveral methods with different mechanisms have been devel-oped to determine the antioxidant activities in plants and

e1752447-4 F HIJAZ ET AL

Figure 1 Total phenolics (a) flavonoids (b) contents and antioxidant activity measured as (DPPH) (c) in selected citrus and citrus relative genotypes Error barsrepresent standard deviation (n = 5) Varieties with different letters are statistically different using Tukeyrsquos HSD test (P-value lt 005)

PLANT SIGNALING amp BEHAVIOR e1752447-5

foods25 Consequently it is recommended to use more thanone method to measure the total antioxidant activities ofa biological tissue25 In this investigation we measured thetotal phenolic and flavonoid contents as well as the DPPHactivity in the leaves of a wide range of citrus varieties usingcolorimetric assays and further analyzed the methanol extractof seven of the selected genotypes by HPLC-MS To gain anaccurate measure of the bound hydroxycinnamic acids in theleaf tissues the leaf extracts were also saponified and analyzedby HPLC-MS

The colorimetric assays showed that curry leaf was the high-est in total phenolic total flavonoids and free radical scaven-ging antioxidant activity (DPPH) The HPLC-MS analysisshowed that curry leaf was high in hydroxycinnamatesHowever only four flavonoids were detected and the estimatedtotal flavonoid content in curry leaf was low (060 mg gminus1 FW)These results indicated that phenolic compounds in curry leafwere interfering with the aluminum chloride assay In agree-ment with the HPLC-MS results saponification of the metha-nol extracts showed that curry leaf was the highest inp-coumaric acid which has 14 Trolox equivalent antioxidantcapacity (TEAC)26 Our results were in agreement with pre-vious reports which showed high phenolic contents as well ashigh antioxidant capacities in curry leaves27 In another studycurry leaf was also found to be rich in gallic acid and totalphenolic compounds and showed high DPPH value28

The HPLC-MS analysis also showed that curry leaf was highin carbazole alkaloid contents The alkaloid mahanimbine andthe essential oil of curry leaf showed strong antibacterial activ-ities against several antibiotic-resistant pathogenic bacteria andwere effective against several cancer cell lines29 In a separatestudy the methanol extract of curry leaves showed strong anti-microbial activities against several pathogens includingStaphylococcus Streptococcus and E coli30 The antibacterialactivities of the curry leaf extract were comparable with genta-mycin and amikacin30 Carbazole compounds from curry leafalso showed high oil stability index and a strong radical scaven-ging activity against DPPH31 The DPPH radical scavengingactivities of most carbazoles were stronger than that of α-tocopherol31 The previous results together indicated that carba-zole alkaloids could have a strong antimicrobial activity againstCLas However future research is needed to confirm thissuggestion

Our results showed that Dancy tangerine leaveswere second highest in total phenolic compounds and thethird in antioxidant activity In agreement with the colori-metric assays HPLC-MS also showed that Dancy tangerinewas rich in hydroxycinnamates flavanones and flavones and

polymethoxylated flavones The saponification results showedthat Dancy tangerine was rich in ferulic and sinapic acidHigh levels of hydrolyzable hydroxycinnamic acids were ear-lier found in Dancy tangerine peel32 Levels of polymethoxy-lated flavones in Dancy tangerines were several times higherthan in most other citrus cultivars32 Our earlier researchshowed that several hydroxycinnamates and polymethoxy-lated flavones were induced in CLas-infected sweet orangetrees33 These results suggested that hydroxycinnamates andpolymethoxylated flavones could be involved in citrus defenseagainst CLas Induction of hydroxycinnamates was alsoobserved in different plants upon bacterial and viralinfections33-35 It is believed that hydroxycinnamates couldact as direct antimicrobial agents and signal molecules andcan be deposited into the cell wall to reinforce it againstpathogen attack36 These observations further indicate thatDancy tangerine could be tolerant to the HLB pathogenthrough its high phenolic content

Bearss lime was the second in total flavonoids DPPH andit contained moderate amounts of total phenolic In agree-ment with the colorimetric result the HPLC-MS resultsshowed that Bearss lime was rich in flavonoids (22 com-pounds) In addition the saponification results showed thatBearss lime was the highest in sinapic acid and the fourth intotal hydroxycinnmates These results indicated that it couldshow some tolerance to the HLB disease

Previous studies showed that C citrangewas relatively tolerantto theCLas pathogen6 Our current results showed thatC citrangewas the third highest variety in total phenolic and the fifth in totalflavonoids and the seventh in DPPH In agreement with theseresults the HPLC-MS analysis showed that C citrangewas rich inhydroxycinnamates (15 compounds) and it contained moderateamount of flavonoids (20 compounds) Saponification of themethanol extract also showed that C citrange was rich in ferulicacid Our previous study showed that C citrange was the highestamong 13 different varieties in total volatiles and sesquiterpenesand second in total monoterpenes8 With respect to single vola-tiles C citrange was the highest cultivar in t-caryophyllene γ-elemene d-limonene β-elemene and germacrene D8 In additionC citrange was the second highest variety in quinic acid9

Flavonoids which are also considered as a group of phenoliccompounds have a good antioxidant activity2637 Recently Zuoet al (2019) showed that flavones such as luteolin and apigeninand flavonols (quercetin) had potent inhibitory effects on YbeY ofCLas bacterium38 YbeY is considered part of the 206 genes thatmake up the minimal bacterial genome set which is necessary forthe maturation of RNAs In addition the YbeY portion of thebacteriumrsquos genetic code was found to play an important role in

Table 2 Concentrations (mggminus1 FW) of main classes of metabolites detected in seven citrus genotypes (n = 5)

Species

Chemical group

FN-C-glyc FN-O-glyc Flavanone HCA PMF Psoralen Carbazole

Bearss lime 473 (10) 1109 (7) 035 (1) 019 (2) 0 0095 (2) 0Valencia orange 091 (7) 187 (7) 365 (2) 010 (1) 103 (11) 0 0Ruby red grapefruit 093 (4) 417 (6) 213 (7) 015 (2) 004 (3) 013(2) 0Duncan grapefruit 011 (2) 116 (9) 028 (9) 0031 (2) 010 (5) 005 (1) 0Dancy tangerine 488 (7) 192 (2) 546 (2) 488 (9) 718 (12) 0 0Carrizo citrange 239 (8) 227 (4) 373 (2) 055 (15) 266 (6) 0 0Curry leaf 0 059 (4) 0 075 (15) 0 0 143 (14)

FN-C-glyc Flavone-C-glycosides FN-O-glyc flavone-O-glycosides

e1752447-6 F HIJAZ ET AL

Figure 2 Representative HPLC chromatograms of selected citrus and citrus relative genotypes (a Dancy mandarin b Carrizo citrange c Bearss lime d Valenciasweet orange e Curry leaf f Duncan grapefruit and g Ruby red) showing main peaks at 280 nm

PLANT SIGNALING amp BEHAVIOR e1752447-7

bacterial symbiosis and pathogenicity38 Tested flavones and fla-vonols showed a significant reduction in CLas titer in HLB-infected citrus trees38 On the other hand the flavanones (hesper-etin and naringenin) were found to have little to no inhibitoryeffect38 The previous results suggested that HLB resistant plantscould be generated via genetic or metabolic modification of thebiosynthetic pathways of flavones and flavonols38 Our previousstudy showed that several flavonoids compounds such as 68-di-C-glucosylapigenin 2rdquo-O-xylosylvitexin hesperidin sinensetin andpolymethoxylated flavones were induced in CLas-infected sweetorange trees33 Taken together these previous results suggest thatflavonoids could play a role in citrus defense against CLas

Conclusion

Our results showed that tolerant varieties contained higherlevels of phenolic compounds and flavonoids than sensitivevarieties Curry leaf which is immune toCLas contained highlevel of hyrdoxycinnamates and carbazole alkaloids Ourresults suggest that high levels of flavonoids and phenoliccompounds might enhance citrus tolerance to HLB Furtherstudies into the roles of these compounds in imparting toler-ance in citrus to the CLas bacterium may provide usefulinformation for the control of citrus HLB disease

Acknowledgments

We thank Yasser Nehela for the technical assistance and Lorraine Jonesfor maintaining the trees in greenhouses This work was kindly fundedby Citrus Research and Development Foundation grant number 19-015

Funding

This work was supported by the Citrus Research and DevelopmentFoundation [19-015]

ORCID

Nabil Killiny httporcidorg0000-0002-3895-6943

References

1 Tiwari S Killiny N Stelinski LL Dynamic insecticide susceptibil-ity changes in Florida populations of Diaphorina citri (Hemipterapsyllidae) J Econ Entomol 2013106393ndash399 doi101603EC12281

2 Blaustein RA Lorca GL Teplitski M Challenges for ManagingCandidatus Liberibacter spp (Huanglongbing disease pathogen)current control measures and future directions Phytopathology2018108424ndash435 doi101094PHYTO-07-17-0260-RVW

3 Halbert SE Manjunath KL Asian citrus psyllids(Sternorrhyncha psyllidae) and greening disease of citrusa literature review and assessment of risk in Florida FloridaEntomol 200487330ndash353 doi1016530015-4040(2004)087[0330ACPSPA]20CO2

4 Tsai JH Liu YH Biology of Diaphorina citri (Homoptera psylli-dae) on four host plants J Econ Entomol 2009931721ndash1725doi1016030022-0493-9361721

5 Richardson ML Hall DG Westbrook R Stover EW Duan YPResistance of Poncirus and Citrus x Poncirusgermplasm to theAsian Citrus Psyllid J Citrus Pathol 20141266

6 Folimonova SY Robertson CJ Garnsey SM Gowda SDawson WO Examination of the responses of different genotypesof citrus to Huanglongbing (citrus greening) under differentconditions Phytopathology 2009991346ndash1354 doi101094PHYTO-99-12-1346

7 Shokrollah Differential reaction of citrus species in Malaysia toHuanglongbing (HLB) disease using grafting method Am J AgricBiol Sci 2009432ndash38 doi103844ajabssp20093238

8 Hijaz F Nehela Y Killiny N Possible role of plant volatiles intolerance against huanglongbing in citrus Plant Signal Behav201611e1138193 doi1010801559232420161138193

9 Killiny N Hijaz F Amino acids implicated in plant defense arehigher in Candidatus Liberibacter asiaticus-tolerant citrusvarieties Plant Signal Behav 201611e1171449 doi1010801559232420161171449

10 Killiny N Valim MF Jones SE Omar AA Hijaz F Gmitter FGGrosser JW Metabolically speaking possible reasons behind thetolerance of lsquoSugar Bellersquo mandarin hybrid to HuanglongbingPlant Physiol Biochem 201711636ndash47 doi101016jplaphy201705001

11 Killiny N Jones SE Nehela Y Hijaz F Dutt M Gmitter FGGrosser JW All roads lead to Rome towards understandingdifferent avenues of tolerance to Huanglongbing in citruscultivars Plant Physiol Biochem PPB 20181291ndash10doi101016jplaphy201805005

12 Lin D Xiao M Zhao J Li Z Xing B Li X Kong M Li L Zhang QLiu Y et al An overview of plant phenolic compounds and theirimportance in human nutrition and management of type 2diabetes Molecules 201621(10)pii E1374 doi103390molecules21101374

13 Nicholson RL Hammerschmidt R Phenolic compounds and theirrole in disease resistance Annu Rev Phytopathol199230369ndash389 doi101146annurevpy30090192002101

14 Michalek S Mayr U Treutter D Lux-Endrich A Gutmann MFeucht W Geibel M Role of flavan-3-OLS in resistance of appletrees to venturia inaequalis Acta Hortic 1998484535ndash539doi1017660ActaHortic199848491

15 Arici SE Kafkas E Kaymak S Koc NK Phenolic compounds ofapple cultivars resistant or susceptible to Venturia inaequalisPharm Biol 201452904ndash908 doi103109138802092013872674

16 Treutter D Significance of flavonoids in plant resistance Areview Environ Chem Lett 20064147ndash157 doi101007s10311-006-0068-8

17 Petrussa E Braidot E Zancani M Peresson C Bertolini A Patui SVianello A Plant Flavonoids-Biosynthesis Transport and involve-ment in stress responses Int J Mol Sci 20131414950ndash14973doi103390ijms140714950

Table 3 Concentrations (mg gminus1 FW) of hydroxycinnamic acids existing as hydrolysable hydroxycinnamates in seven citrus leaf extracts Hydroxycinnamic acidsmeasured after leaf extract saponification (n = 5)

Species P-Coumaric Sinapic Ferulic

Bearss lime 062 plusmn 008b 084 plusmn 005a 049 plusmn 004b

Valencia orange 011 plusmn 001d 045 plusmn 004 c 066 plusmn 038b

Ruby Red grapefruit 012 plusmn 001d 006 plusmn 001e 061 plusmn 005b

Duncan grapefruit 011 plusmn 001d 003 plusmn 002e 060 plusmn 041b

Dancy tangerine 028 plusmn 003 c 057 plusmn 002b 344 plusmn 016a

Carrizo citrange 026 plusmn 030 cd 032 plusmn 001d 298 plusmn 266ab

Curry leaf 129 plusmn 030a 003 plusmn 001e 018 plusmn 004 c

Varieties with different superscript letters are statistically different using Tukeyrsquos HSD test (P-value lt 005)

e1752447-8 F HIJAZ ET AL

Figure 1 Total phenolics (a) flavonoids (b) contents and antioxidant activity measured as (DPPH) (c) in selected citrus and citrus relative genotypes Error barsrepresent standard deviation (n = 5) Varieties with different letters are statistically different using Tukeyrsquos HSD test (P-value lt 005)

PLANT SIGNALING amp BEHAVIOR e1752447-5

foods25 Consequently it is recommended to use more thanone method to measure the total antioxidant activities ofa biological tissue25 In this investigation we measured thetotal phenolic and flavonoid contents as well as the DPPHactivity in the leaves of a wide range of citrus varieties usingcolorimetric assays and further analyzed the methanol extractof seven of the selected genotypes by HPLC-MS To gain anaccurate measure of the bound hydroxycinnamic acids in theleaf tissues the leaf extracts were also saponified and analyzedby HPLC-MS

The colorimetric assays showed that curry leaf was the high-est in total phenolic total flavonoids and free radical scaven-ging antioxidant activity (DPPH) The HPLC-MS analysisshowed that curry leaf was high in hydroxycinnamatesHowever only four flavonoids were detected and the estimatedtotal flavonoid content in curry leaf was low (060 mg gminus1 FW)These results indicated that phenolic compounds in curry leafwere interfering with the aluminum chloride assay In agree-ment with the HPLC-MS results saponification of the metha-nol extracts showed that curry leaf was the highest inp-coumaric acid which has 14 Trolox equivalent antioxidantcapacity (TEAC)26 Our results were in agreement with pre-vious reports which showed high phenolic contents as well ashigh antioxidant capacities in curry leaves27 In another studycurry leaf was also found to be rich in gallic acid and totalphenolic compounds and showed high DPPH value28

The HPLC-MS analysis also showed that curry leaf was highin carbazole alkaloid contents The alkaloid mahanimbine andthe essential oil of curry leaf showed strong antibacterial activ-ities against several antibiotic-resistant pathogenic bacteria andwere effective against several cancer cell lines29 In a separatestudy the methanol extract of curry leaves showed strong anti-microbial activities against several pathogens includingStaphylococcus Streptococcus and E coli30 The antibacterialactivities of the curry leaf extract were comparable with genta-mycin and amikacin30 Carbazole compounds from curry leafalso showed high oil stability index and a strong radical scaven-ging activity against DPPH31 The DPPH radical scavengingactivities of most carbazoles were stronger than that of α-tocopherol31 The previous results together indicated that carba-zole alkaloids could have a strong antimicrobial activity againstCLas However future research is needed to confirm thissuggestion

Our results showed that Dancy tangerine leaveswere second highest in total phenolic compounds and thethird in antioxidant activity In agreement with the colori-metric assays HPLC-MS also showed that Dancy tangerinewas rich in hydroxycinnamates flavanones and flavones and

polymethoxylated flavones The saponification results showedthat Dancy tangerine was rich in ferulic and sinapic acidHigh levels of hydrolyzable hydroxycinnamic acids were ear-lier found in Dancy tangerine peel32 Levels of polymethoxy-lated flavones in Dancy tangerines were several times higherthan in most other citrus cultivars32 Our earlier researchshowed that several hydroxycinnamates and polymethoxy-lated flavones were induced in CLas-infected sweet orangetrees33 These results suggested that hydroxycinnamates andpolymethoxylated flavones could be involved in citrus defenseagainst CLas Induction of hydroxycinnamates was alsoobserved in different plants upon bacterial and viralinfections33-35 It is believed that hydroxycinnamates couldact as direct antimicrobial agents and signal molecules andcan be deposited into the cell wall to reinforce it againstpathogen attack36 These observations further indicate thatDancy tangerine could be tolerant to the HLB pathogenthrough its high phenolic content

Bearss lime was the second in total flavonoids DPPH andit contained moderate amounts of total phenolic In agree-ment with the colorimetric result the HPLC-MS resultsshowed that Bearss lime was rich in flavonoids (22 com-pounds) In addition the saponification results showed thatBearss lime was the highest in sinapic acid and the fourth intotal hydroxycinnmates These results indicated that it couldshow some tolerance to the HLB disease

Previous studies showed that C citrangewas relatively tolerantto theCLas pathogen6 Our current results showed thatC citrangewas the third highest variety in total phenolic and the fifth in totalflavonoids and the seventh in DPPH In agreement with theseresults the HPLC-MS analysis showed that C citrangewas rich inhydroxycinnamates (15 compounds) and it contained moderateamount of flavonoids (20 compounds) Saponification of themethanol extract also showed that C citrange was rich in ferulicacid Our previous study showed that C citrange was the highestamong 13 different varieties in total volatiles and sesquiterpenesand second in total monoterpenes8 With respect to single vola-tiles C citrange was the highest cultivar in t-caryophyllene γ-elemene d-limonene β-elemene and germacrene D8 In additionC citrange was the second highest variety in quinic acid9

Flavonoids which are also considered as a group of phenoliccompounds have a good antioxidant activity2637 Recently Zuoet al (2019) showed that flavones such as luteolin and apigeninand flavonols (quercetin) had potent inhibitory effects on YbeY ofCLas bacterium38 YbeY is considered part of the 206 genes thatmake up the minimal bacterial genome set which is necessary forthe maturation of RNAs In addition the YbeY portion of thebacteriumrsquos genetic code was found to play an important role in

Table 2 Concentrations (mggminus1 FW) of main classes of metabolites detected in seven citrus genotypes (n = 5)

Species

Chemical group

FN-C-glyc FN-O-glyc Flavanone HCA PMF Psoralen Carbazole

Bearss lime 473 (10) 1109 (7) 035 (1) 019 (2) 0 0095 (2) 0Valencia orange 091 (7) 187 (7) 365 (2) 010 (1) 103 (11) 0 0Ruby red grapefruit 093 (4) 417 (6) 213 (7) 015 (2) 004 (3) 013(2) 0Duncan grapefruit 011 (2) 116 (9) 028 (9) 0031 (2) 010 (5) 005 (1) 0Dancy tangerine 488 (7) 192 (2) 546 (2) 488 (9) 718 (12) 0 0Carrizo citrange 239 (8) 227 (4) 373 (2) 055 (15) 266 (6) 0 0Curry leaf 0 059 (4) 0 075 (15) 0 0 143 (14)

FN-C-glyc Flavone-C-glycosides FN-O-glyc flavone-O-glycosides

e1752447-6 F HIJAZ ET AL

Figure 2 Representative HPLC chromatograms of selected citrus and citrus relative genotypes (a Dancy mandarin b Carrizo citrange c Bearss lime d Valenciasweet orange e Curry leaf f Duncan grapefruit and g Ruby red) showing main peaks at 280 nm

PLANT SIGNALING amp BEHAVIOR e1752447-7

bacterial symbiosis and pathogenicity38 Tested flavones and fla-vonols showed a significant reduction in CLas titer in HLB-infected citrus trees38 On the other hand the flavanones (hesper-etin and naringenin) were found to have little to no inhibitoryeffect38 The previous results suggested that HLB resistant plantscould be generated via genetic or metabolic modification of thebiosynthetic pathways of flavones and flavonols38 Our previousstudy showed that several flavonoids compounds such as 68-di-C-glucosylapigenin 2rdquo-O-xylosylvitexin hesperidin sinensetin andpolymethoxylated flavones were induced in CLas-infected sweetorange trees33 Taken together these previous results suggest thatflavonoids could play a role in citrus defense against CLas

Conclusion

Our results showed that tolerant varieties contained higherlevels of phenolic compounds and flavonoids than sensitivevarieties Curry leaf which is immune toCLas contained highlevel of hyrdoxycinnamates and carbazole alkaloids Ourresults suggest that high levels of flavonoids and phenoliccompounds might enhance citrus tolerance to HLB Furtherstudies into the roles of these compounds in imparting toler-ance in citrus to the CLas bacterium may provide usefulinformation for the control of citrus HLB disease

Acknowledgments

We thank Yasser Nehela for the technical assistance and Lorraine Jonesfor maintaining the trees in greenhouses This work was kindly fundedby Citrus Research and Development Foundation grant number 19-015

Funding

This work was supported by the Citrus Research and DevelopmentFoundation [19-015]

ORCID

Nabil Killiny httporcidorg0000-0002-3895-6943

References

1 Tiwari S Killiny N Stelinski LL Dynamic insecticide susceptibil-ity changes in Florida populations of Diaphorina citri (Hemipterapsyllidae) J Econ Entomol 2013106393ndash399 doi101603EC12281

2 Blaustein RA Lorca GL Teplitski M Challenges for ManagingCandidatus Liberibacter spp (Huanglongbing disease pathogen)current control measures and future directions Phytopathology2018108424ndash435 doi101094PHYTO-07-17-0260-RVW

3 Halbert SE Manjunath KL Asian citrus psyllids(Sternorrhyncha psyllidae) and greening disease of citrusa literature review and assessment of risk in Florida FloridaEntomol 200487330ndash353 doi1016530015-4040(2004)087[0330ACPSPA]20CO2

4 Tsai JH Liu YH Biology of Diaphorina citri (Homoptera psylli-dae) on four host plants J Econ Entomol 2009931721ndash1725doi1016030022-0493-9361721

5 Richardson ML Hall DG Westbrook R Stover EW Duan YPResistance of Poncirus and Citrus x Poncirusgermplasm to theAsian Citrus Psyllid J Citrus Pathol 20141266

6 Folimonova SY Robertson CJ Garnsey SM Gowda SDawson WO Examination of the responses of different genotypesof citrus to Huanglongbing (citrus greening) under differentconditions Phytopathology 2009991346ndash1354 doi101094PHYTO-99-12-1346

7 Shokrollah Differential reaction of citrus species in Malaysia toHuanglongbing (HLB) disease using grafting method Am J AgricBiol Sci 2009432ndash38 doi103844ajabssp20093238

8 Hijaz F Nehela Y Killiny N Possible role of plant volatiles intolerance against huanglongbing in citrus Plant Signal Behav201611e1138193 doi1010801559232420161138193

9 Killiny N Hijaz F Amino acids implicated in plant defense arehigher in Candidatus Liberibacter asiaticus-tolerant citrusvarieties Plant Signal Behav 201611e1171449 doi1010801559232420161171449

10 Killiny N Valim MF Jones SE Omar AA Hijaz F Gmitter FGGrosser JW Metabolically speaking possible reasons behind thetolerance of lsquoSugar Bellersquo mandarin hybrid to HuanglongbingPlant Physiol Biochem 201711636ndash47 doi101016jplaphy201705001

11 Killiny N Jones SE Nehela Y Hijaz F Dutt M Gmitter FGGrosser JW All roads lead to Rome towards understandingdifferent avenues of tolerance to Huanglongbing in citruscultivars Plant Physiol Biochem PPB 20181291ndash10doi101016jplaphy201805005

12 Lin D Xiao M Zhao J Li Z Xing B Li X Kong M Li L Zhang QLiu Y et al An overview of plant phenolic compounds and theirimportance in human nutrition and management of type 2diabetes Molecules 201621(10)pii E1374 doi103390molecules21101374

13 Nicholson RL Hammerschmidt R Phenolic compounds and theirrole in disease resistance Annu Rev Phytopathol199230369ndash389 doi101146annurevpy30090192002101

14 Michalek S Mayr U Treutter D Lux-Endrich A Gutmann MFeucht W Geibel M Role of flavan-3-OLS in resistance of appletrees to venturia inaequalis Acta Hortic 1998484535ndash539doi1017660ActaHortic199848491

15 Arici SE Kafkas E Kaymak S Koc NK Phenolic compounds ofapple cultivars resistant or susceptible to Venturia inaequalisPharm Biol 201452904ndash908 doi103109138802092013872674

16 Treutter D Significance of flavonoids in plant resistance Areview Environ Chem Lett 20064147ndash157 doi101007s10311-006-0068-8

17 Petrussa E Braidot E Zancani M Peresson C Bertolini A Patui SVianello A Plant Flavonoids-Biosynthesis Transport and involve-ment in stress responses Int J Mol Sci 20131414950ndash14973doi103390ijms140714950

Table 3 Concentrations (mg gminus1 FW) of hydroxycinnamic acids existing as hydrolysable hydroxycinnamates in seven citrus leaf extracts Hydroxycinnamic acidsmeasured after leaf extract saponification (n = 5)

Species P-Coumaric Sinapic Ferulic

Bearss lime 062 plusmn 008b 084 plusmn 005a 049 plusmn 004b

Valencia orange 011 plusmn 001d 045 plusmn 004 c 066 plusmn 038b

Ruby Red grapefruit 012 plusmn 001d 006 plusmn 001e 061 plusmn 005b

Duncan grapefruit 011 plusmn 001d 003 plusmn 002e 060 plusmn 041b

Dancy tangerine 028 plusmn 003 c 057 plusmn 002b 344 plusmn 016a

Carrizo citrange 026 plusmn 030 cd 032 plusmn 001d 298 plusmn 266ab

Curry leaf 129 plusmn 030a 003 plusmn 001e 018 plusmn 004 c

Varieties with different superscript letters are statistically different using Tukeyrsquos HSD test (P-value lt 005)

e1752447-8 F HIJAZ ET AL

foods25 Consequently it is recommended to use more thanone method to measure the total antioxidant activities ofa biological tissue25 In this investigation we measured thetotal phenolic and flavonoid contents as well as the DPPHactivity in the leaves of a wide range of citrus varieties usingcolorimetric assays and further analyzed the methanol extractof seven of the selected genotypes by HPLC-MS To gain anaccurate measure of the bound hydroxycinnamic acids in theleaf tissues the leaf extracts were also saponified and analyzedby HPLC-MS

The colorimetric assays showed that curry leaf was the high-est in total phenolic total flavonoids and free radical scaven-ging antioxidant activity (DPPH) The HPLC-MS analysisshowed that curry leaf was high in hydroxycinnamatesHowever only four flavonoids were detected and the estimatedtotal flavonoid content in curry leaf was low (060 mg gminus1 FW)These results indicated that phenolic compounds in curry leafwere interfering with the aluminum chloride assay In agree-ment with the HPLC-MS results saponification of the metha-nol extracts showed that curry leaf was the highest inp-coumaric acid which has 14 Trolox equivalent antioxidantcapacity (TEAC)26 Our results were in agreement with pre-vious reports which showed high phenolic contents as well ashigh antioxidant capacities in curry leaves27 In another studycurry leaf was also found to be rich in gallic acid and totalphenolic compounds and showed high DPPH value28

The HPLC-MS analysis also showed that curry leaf was highin carbazole alkaloid contents The alkaloid mahanimbine andthe essential oil of curry leaf showed strong antibacterial activ-ities against several antibiotic-resistant pathogenic bacteria andwere effective against several cancer cell lines29 In a separatestudy the methanol extract of curry leaves showed strong anti-microbial activities against several pathogens includingStaphylococcus Streptococcus and E coli30 The antibacterialactivities of the curry leaf extract were comparable with genta-mycin and amikacin30 Carbazole compounds from curry leafalso showed high oil stability index and a strong radical scaven-ging activity against DPPH31 The DPPH radical scavengingactivities of most carbazoles were stronger than that of α-tocopherol31 The previous results together indicated that carba-zole alkaloids could have a strong antimicrobial activity againstCLas However future research is needed to confirm thissuggestion

Our results showed that Dancy tangerine leaveswere second highest in total phenolic compounds and thethird in antioxidant activity In agreement with the colori-metric assays HPLC-MS also showed that Dancy tangerinewas rich in hydroxycinnamates flavanones and flavones and

polymethoxylated flavones The saponification results showedthat Dancy tangerine was rich in ferulic and sinapic acidHigh levels of hydrolyzable hydroxycinnamic acids were ear-lier found in Dancy tangerine peel32 Levels of polymethoxy-lated flavones in Dancy tangerines were several times higherthan in most other citrus cultivars32 Our earlier researchshowed that several hydroxycinnamates and polymethoxy-lated flavones were induced in CLas-infected sweet orangetrees33 These results suggested that hydroxycinnamates andpolymethoxylated flavones could be involved in citrus defenseagainst CLas Induction of hydroxycinnamates was alsoobserved in different plants upon bacterial and viralinfections33-35 It is believed that hydroxycinnamates couldact as direct antimicrobial agents and signal molecules andcan be deposited into the cell wall to reinforce it againstpathogen attack36 These observations further indicate thatDancy tangerine could be tolerant to the HLB pathogenthrough its high phenolic content

Bearss lime was the second in total flavonoids DPPH andit contained moderate amounts of total phenolic In agree-ment with the colorimetric result the HPLC-MS resultsshowed that Bearss lime was rich in flavonoids (22 com-pounds) In addition the saponification results showed thatBearss lime was the highest in sinapic acid and the fourth intotal hydroxycinnmates These results indicated that it couldshow some tolerance to the HLB disease

Previous studies showed that C citrangewas relatively tolerantto theCLas pathogen6 Our current results showed thatC citrangewas the third highest variety in total phenolic and the fifth in totalflavonoids and the seventh in DPPH In agreement with theseresults the HPLC-MS analysis showed that C citrangewas rich inhydroxycinnamates (15 compounds) and it contained moderateamount of flavonoids (20 compounds) Saponification of themethanol extract also showed that C citrange was rich in ferulicacid Our previous study showed that C citrange was the highestamong 13 different varieties in total volatiles and sesquiterpenesand second in total monoterpenes8 With respect to single vola-tiles C citrange was the highest cultivar in t-caryophyllene γ-elemene d-limonene β-elemene and germacrene D8 In additionC citrange was the second highest variety in quinic acid9

Flavonoids which are also considered as a group of phenoliccompounds have a good antioxidant activity2637 Recently Zuoet al (2019) showed that flavones such as luteolin and apigeninand flavonols (quercetin) had potent inhibitory effects on YbeY ofCLas bacterium38 YbeY is considered part of the 206 genes thatmake up the minimal bacterial genome set which is necessary forthe maturation of RNAs In addition the YbeY portion of thebacteriumrsquos genetic code was found to play an important role in

Table 2 Concentrations (mggminus1 FW) of main classes of metabolites detected in seven citrus genotypes (n = 5)

Species

Chemical group

FN-C-glyc FN-O-glyc Flavanone HCA PMF Psoralen Carbazole

Bearss lime 473 (10) 1109 (7) 035 (1) 019 (2) 0 0095 (2) 0Valencia orange 091 (7) 187 (7) 365 (2) 010 (1) 103 (11) 0 0Ruby red grapefruit 093 (4) 417 (6) 213 (7) 015 (2) 004 (3) 013(2) 0Duncan grapefruit 011 (2) 116 (9) 028 (9) 0031 (2) 010 (5) 005 (1) 0Dancy tangerine 488 (7) 192 (2) 546 (2) 488 (9) 718 (12) 0 0Carrizo citrange 239 (8) 227 (4) 373 (2) 055 (15) 266 (6) 0 0Curry leaf 0 059 (4) 0 075 (15) 0 0 143 (14)

FN-C-glyc Flavone-C-glycosides FN-O-glyc flavone-O-glycosides

e1752447-6 F HIJAZ ET AL

Figure 2 Representative HPLC chromatograms of selected citrus and citrus relative genotypes (a Dancy mandarin b Carrizo citrange c Bearss lime d Valenciasweet orange e Curry leaf f Duncan grapefruit and g Ruby red) showing main peaks at 280 nm

PLANT SIGNALING amp BEHAVIOR e1752447-7

bacterial symbiosis and pathogenicity38 Tested flavones and fla-vonols showed a significant reduction in CLas titer in HLB-infected citrus trees38 On the other hand the flavanones (hesper-etin and naringenin) were found to have little to no inhibitoryeffect38 The previous results suggested that HLB resistant plantscould be generated via genetic or metabolic modification of thebiosynthetic pathways of flavones and flavonols38 Our previousstudy showed that several flavonoids compounds such as 68-di-C-glucosylapigenin 2rdquo-O-xylosylvitexin hesperidin sinensetin andpolymethoxylated flavones were induced in CLas-infected sweetorange trees33 Taken together these previous results suggest thatflavonoids could play a role in citrus defense against CLas

Conclusion

Our results showed that tolerant varieties contained higherlevels of phenolic compounds and flavonoids than sensitivevarieties Curry leaf which is immune toCLas contained highlevel of hyrdoxycinnamates and carbazole alkaloids Ourresults suggest that high levels of flavonoids and phenoliccompounds might enhance citrus tolerance to HLB Furtherstudies into the roles of these compounds in imparting toler-ance in citrus to the CLas bacterium may provide usefulinformation for the control of citrus HLB disease

Acknowledgments

We thank Yasser Nehela for the technical assistance and Lorraine Jonesfor maintaining the trees in greenhouses This work was kindly fundedby Citrus Research and Development Foundation grant number 19-015

Funding

This work was supported by the Citrus Research and DevelopmentFoundation [19-015]

ORCID

Nabil Killiny httporcidorg0000-0002-3895-6943

References

1 Tiwari S Killiny N Stelinski LL Dynamic insecticide susceptibil-ity changes in Florida populations of Diaphorina citri (Hemipterapsyllidae) J Econ Entomol 2013106393ndash399 doi101603EC12281

2 Blaustein RA Lorca GL Teplitski M Challenges for ManagingCandidatus Liberibacter spp (Huanglongbing disease pathogen)current control measures and future directions Phytopathology2018108424ndash435 doi101094PHYTO-07-17-0260-RVW

3 Halbert SE Manjunath KL Asian citrus psyllids(Sternorrhyncha psyllidae) and greening disease of citrusa literature review and assessment of risk in Florida FloridaEntomol 200487330ndash353 doi1016530015-4040(2004)087[0330ACPSPA]20CO2

4 Tsai JH Liu YH Biology of Diaphorina citri (Homoptera psylli-dae) on four host plants J Econ Entomol 2009931721ndash1725doi1016030022-0493-9361721

5 Richardson ML Hall DG Westbrook R Stover EW Duan YPResistance of Poncirus and Citrus x Poncirusgermplasm to theAsian Citrus Psyllid J Citrus Pathol 20141266

6 Folimonova SY Robertson CJ Garnsey SM Gowda SDawson WO Examination of the responses of different genotypesof citrus to Huanglongbing (citrus greening) under differentconditions Phytopathology 2009991346ndash1354 doi101094PHYTO-99-12-1346

7 Shokrollah Differential reaction of citrus species in Malaysia toHuanglongbing (HLB) disease using grafting method Am J AgricBiol Sci 2009432ndash38 doi103844ajabssp20093238

8 Hijaz F Nehela Y Killiny N Possible role of plant volatiles intolerance against huanglongbing in citrus Plant Signal Behav201611e1138193 doi1010801559232420161138193

9 Killiny N Hijaz F Amino acids implicated in plant defense arehigher in Candidatus Liberibacter asiaticus-tolerant citrusvarieties Plant Signal Behav 201611e1171449 doi1010801559232420161171449

10 Killiny N Valim MF Jones SE Omar AA Hijaz F Gmitter FGGrosser JW Metabolically speaking possible reasons behind thetolerance of lsquoSugar Bellersquo mandarin hybrid to HuanglongbingPlant Physiol Biochem 201711636ndash47 doi101016jplaphy201705001

11 Killiny N Jones SE Nehela Y Hijaz F Dutt M Gmitter FGGrosser JW All roads lead to Rome towards understandingdifferent avenues of tolerance to Huanglongbing in citruscultivars Plant Physiol Biochem PPB 20181291ndash10doi101016jplaphy201805005

12 Lin D Xiao M Zhao J Li Z Xing B Li X Kong M Li L Zhang QLiu Y et al An overview of plant phenolic compounds and theirimportance in human nutrition and management of type 2diabetes Molecules 201621(10)pii E1374 doi103390molecules21101374

13 Nicholson RL Hammerschmidt R Phenolic compounds and theirrole in disease resistance Annu Rev Phytopathol199230369ndash389 doi101146annurevpy30090192002101

14 Michalek S Mayr U Treutter D Lux-Endrich A Gutmann MFeucht W Geibel M Role of flavan-3-OLS in resistance of appletrees to venturia inaequalis Acta Hortic 1998484535ndash539doi1017660ActaHortic199848491

15 Arici SE Kafkas E Kaymak S Koc NK Phenolic compounds ofapple cultivars resistant or susceptible to Venturia inaequalisPharm Biol 201452904ndash908 doi103109138802092013872674

16 Treutter D Significance of flavonoids in plant resistance Areview Environ Chem Lett 20064147ndash157 doi101007s10311-006-0068-8

17 Petrussa E Braidot E Zancani M Peresson C Bertolini A Patui SVianello A Plant Flavonoids-Biosynthesis Transport and involve-ment in stress responses Int J Mol Sci 20131414950ndash14973doi103390ijms140714950

Table 3 Concentrations (mg gminus1 FW) of hydroxycinnamic acids existing as hydrolysable hydroxycinnamates in seven citrus leaf extracts Hydroxycinnamic acidsmeasured after leaf extract saponification (n = 5)

Species P-Coumaric Sinapic Ferulic

Bearss lime 062 plusmn 008b 084 plusmn 005a 049 plusmn 004b

Valencia orange 011 plusmn 001d 045 plusmn 004 c 066 plusmn 038b

Ruby Red grapefruit 012 plusmn 001d 006 plusmn 001e 061 plusmn 005b

Duncan grapefruit 011 plusmn 001d 003 plusmn 002e 060 plusmn 041b

Dancy tangerine 028 plusmn 003 c 057 plusmn 002b 344 plusmn 016a

Carrizo citrange 026 plusmn 030 cd 032 plusmn 001d 298 plusmn 266ab

Curry leaf 129 plusmn 030a 003 plusmn 001e 018 plusmn 004 c

Varieties with different superscript letters are statistically different using Tukeyrsquos HSD test (P-value lt 005)

e1752447-8 F HIJAZ ET AL

Figure 2 Representative HPLC chromatograms of selected citrus and citrus relative genotypes (a Dancy mandarin b Carrizo citrange c Bearss lime d Valenciasweet orange e Curry leaf f Duncan grapefruit and g Ruby red) showing main peaks at 280 nm

PLANT SIGNALING amp BEHAVIOR e1752447-7

bacterial symbiosis and pathogenicity38 Tested flavones and fla-vonols showed a significant reduction in CLas titer in HLB-infected citrus trees38 On the other hand the flavanones (hesper-etin and naringenin) were found to have little to no inhibitoryeffect38 The previous results suggested that HLB resistant plantscould be generated via genetic or metabolic modification of thebiosynthetic pathways of flavones and flavonols38 Our previousstudy showed that several flavonoids compounds such as 68-di-C-glucosylapigenin 2rdquo-O-xylosylvitexin hesperidin sinensetin andpolymethoxylated flavones were induced in CLas-infected sweetorange trees33 Taken together these previous results suggest thatflavonoids could play a role in citrus defense against CLas

Conclusion

Our results showed that tolerant varieties contained higherlevels of phenolic compounds and flavonoids than sensitivevarieties Curry leaf which is immune toCLas contained highlevel of hyrdoxycinnamates and carbazole alkaloids Ourresults suggest that high levels of flavonoids and phenoliccompounds might enhance citrus tolerance to HLB Furtherstudies into the roles of these compounds in imparting toler-ance in citrus to the CLas bacterium may provide usefulinformation for the control of citrus HLB disease

Acknowledgments

We thank Yasser Nehela for the technical assistance and Lorraine Jonesfor maintaining the trees in greenhouses This work was kindly fundedby Citrus Research and Development Foundation grant number 19-015

Funding

This work was supported by the Citrus Research and DevelopmentFoundation [19-015]

ORCID

Nabil Killiny httporcidorg0000-0002-3895-6943

References

1 Tiwari S Killiny N Stelinski LL Dynamic insecticide susceptibil-ity changes in Florida populations of Diaphorina citri (Hemipterapsyllidae) J Econ Entomol 2013106393ndash399 doi101603EC12281

2 Blaustein RA Lorca GL Teplitski M Challenges for ManagingCandidatus Liberibacter spp (Huanglongbing disease pathogen)current control measures and future directions Phytopathology2018108424ndash435 doi101094PHYTO-07-17-0260-RVW

3 Halbert SE Manjunath KL Asian citrus psyllids(Sternorrhyncha psyllidae) and greening disease of citrusa literature review and assessment of risk in Florida FloridaEntomol 200487330ndash353 doi1016530015-4040(2004)087[0330ACPSPA]20CO2

4 Tsai JH Liu YH Biology of Diaphorina citri (Homoptera psylli-dae) on four host plants J Econ Entomol 2009931721ndash1725doi1016030022-0493-9361721

5 Richardson ML Hall DG Westbrook R Stover EW Duan YPResistance of Poncirus and Citrus x Poncirusgermplasm to theAsian Citrus Psyllid J Citrus Pathol 20141266

6 Folimonova SY Robertson CJ Garnsey SM Gowda SDawson WO Examination of the responses of different genotypesof citrus to Huanglongbing (citrus greening) under differentconditions Phytopathology 2009991346ndash1354 doi101094PHYTO-99-12-1346

7 Shokrollah Differential reaction of citrus species in Malaysia toHuanglongbing (HLB) disease using grafting method Am J AgricBiol Sci 2009432ndash38 doi103844ajabssp20093238

8 Hijaz F Nehela Y Killiny N Possible role of plant volatiles intolerance against huanglongbing in citrus Plant Signal Behav201611e1138193 doi1010801559232420161138193

9 Killiny N Hijaz F Amino acids implicated in plant defense arehigher in Candidatus Liberibacter asiaticus-tolerant citrusvarieties Plant Signal Behav 201611e1171449 doi1010801559232420161171449

10 Killiny N Valim MF Jones SE Omar AA Hijaz F Gmitter FGGrosser JW Metabolically speaking possible reasons behind thetolerance of lsquoSugar Bellersquo mandarin hybrid to HuanglongbingPlant Physiol Biochem 201711636ndash47 doi101016jplaphy201705001

11 Killiny N Jones SE Nehela Y Hijaz F Dutt M Gmitter FGGrosser JW All roads lead to Rome towards understandingdifferent avenues of tolerance to Huanglongbing in citruscultivars Plant Physiol Biochem PPB 20181291ndash10doi101016jplaphy201805005

12 Lin D Xiao M Zhao J Li Z Xing B Li X Kong M Li L Zhang QLiu Y et al An overview of plant phenolic compounds and theirimportance in human nutrition and management of type 2diabetes Molecules 201621(10)pii E1374 doi103390molecules21101374

13 Nicholson RL Hammerschmidt R Phenolic compounds and theirrole in disease resistance Annu Rev Phytopathol199230369ndash389 doi101146annurevpy30090192002101

14 Michalek S Mayr U Treutter D Lux-Endrich A Gutmann MFeucht W Geibel M Role of flavan-3-OLS in resistance of appletrees to venturia inaequalis Acta Hortic 1998484535ndash539doi1017660ActaHortic199848491

15 Arici SE Kafkas E Kaymak S Koc NK Phenolic compounds ofapple cultivars resistant or susceptible to Venturia inaequalisPharm Biol 201452904ndash908 doi103109138802092013872674

16 Treutter D Significance of flavonoids in plant resistance Areview Environ Chem Lett 20064147ndash157 doi101007s10311-006-0068-8

17 Petrussa E Braidot E Zancani M Peresson C Bertolini A Patui SVianello A Plant Flavonoids-Biosynthesis Transport and involve-ment in stress responses Int J Mol Sci 20131414950ndash14973doi103390ijms140714950

Table 3 Concentrations (mg gminus1 FW) of hydroxycinnamic acids existing as hydrolysable hydroxycinnamates in seven citrus leaf extracts Hydroxycinnamic acidsmeasured after leaf extract saponification (n = 5)

Species P-Coumaric Sinapic Ferulic

Bearss lime 062 plusmn 008b 084 plusmn 005a 049 plusmn 004b

Valencia orange 011 plusmn 001d 045 plusmn 004 c 066 plusmn 038b

Ruby Red grapefruit 012 plusmn 001d 006 plusmn 001e 061 plusmn 005b

Duncan grapefruit 011 plusmn 001d 003 plusmn 002e 060 plusmn 041b

Dancy tangerine 028 plusmn 003 c 057 plusmn 002b 344 plusmn 016a

Carrizo citrange 026 plusmn 030 cd 032 plusmn 001d 298 plusmn 266ab

Curry leaf 129 plusmn 030a 003 plusmn 001e 018 plusmn 004 c

Varieties with different superscript letters are statistically different using Tukeyrsquos HSD test (P-value lt 005)

e1752447-8 F HIJAZ ET AL

bacterial symbiosis and pathogenicity38 Tested flavones and fla-vonols showed a significant reduction in CLas titer in HLB-infected citrus trees38 On the other hand the flavanones (hesper-etin and naringenin) were found to have little to no inhibitoryeffect38 The previous results suggested that HLB resistant plantscould be generated via genetic or metabolic modification of thebiosynthetic pathways of flavones and flavonols38 Our previousstudy showed that several flavonoids compounds such as 68-di-C-glucosylapigenin 2rdquo-O-xylosylvitexin hesperidin sinensetin andpolymethoxylated flavones were induced in CLas-infected sweetorange trees33 Taken together these previous results suggest thatflavonoids could play a role in citrus defense against CLas

Conclusion

Our results showed that tolerant varieties contained higherlevels of phenolic compounds and flavonoids than sensitivevarieties Curry leaf which is immune toCLas contained highlevel of hyrdoxycinnamates and carbazole alkaloids Ourresults suggest that high levels of flavonoids and phenoliccompounds might enhance citrus tolerance to HLB Furtherstudies into the roles of these compounds in imparting toler-ance in citrus to the CLas bacterium may provide usefulinformation for the control of citrus HLB disease

Acknowledgments

We thank Yasser Nehela for the technical assistance and Lorraine Jonesfor maintaining the trees in greenhouses This work was kindly fundedby Citrus Research and Development Foundation grant number 19-015

Funding

This work was supported by the Citrus Research and DevelopmentFoundation [19-015]

ORCID

Nabil Killiny httporcidorg0000-0002-3895-6943

References

1 Tiwari S Killiny N Stelinski LL Dynamic insecticide susceptibil-ity changes in Florida populations of Diaphorina citri (Hemipterapsyllidae) J Econ Entomol 2013106393ndash399 doi101603EC12281

2 Blaustein RA Lorca GL Teplitski M Challenges for ManagingCandidatus Liberibacter spp (Huanglongbing disease pathogen)current control measures and future directions Phytopathology2018108424ndash435 doi101094PHYTO-07-17-0260-RVW

3 Halbert SE Manjunath KL Asian citrus psyllids(Sternorrhyncha psyllidae) and greening disease of citrusa literature review and assessment of risk in Florida FloridaEntomol 200487330ndash353 doi1016530015-4040(2004)087[0330ACPSPA]20CO2

4 Tsai JH Liu YH Biology of Diaphorina citri (Homoptera psylli-dae) on four host plants J Econ Entomol 2009931721ndash1725doi1016030022-0493-9361721

5 Richardson ML Hall DG Westbrook R Stover EW Duan YPResistance of Poncirus and Citrus x Poncirusgermplasm to theAsian Citrus Psyllid J Citrus Pathol 20141266

6 Folimonova SY Robertson CJ Garnsey SM Gowda SDawson WO Examination of the responses of different genotypesof citrus to Huanglongbing (citrus greening) under differentconditions Phytopathology 2009991346ndash1354 doi101094PHYTO-99-12-1346

7 Shokrollah Differential reaction of citrus species in Malaysia toHuanglongbing (HLB) disease using grafting method Am J AgricBiol Sci 2009432ndash38 doi103844ajabssp20093238

8 Hijaz F Nehela Y Killiny N Possible role of plant volatiles intolerance against huanglongbing in citrus Plant Signal Behav201611e1138193 doi1010801559232420161138193

9 Killiny N Hijaz F Amino acids implicated in plant defense arehigher in Candidatus Liberibacter asiaticus-tolerant citrusvarieties Plant Signal Behav 201611e1171449 doi1010801559232420161171449

10 Killiny N Valim MF Jones SE Omar AA Hijaz F Gmitter FGGrosser JW Metabolically speaking possible reasons behind thetolerance of lsquoSugar Bellersquo mandarin hybrid to HuanglongbingPlant Physiol Biochem 201711636ndash47 doi101016jplaphy201705001

11 Killiny N Jones SE Nehela Y Hijaz F Dutt M Gmitter FGGrosser JW All roads lead to Rome towards understandingdifferent avenues of tolerance to Huanglongbing in citruscultivars Plant Physiol Biochem PPB 20181291ndash10doi101016jplaphy201805005

12 Lin D Xiao M Zhao J Li Z Xing B Li X Kong M Li L Zhang QLiu Y et al An overview of plant phenolic compounds and theirimportance in human nutrition and management of type 2diabetes Molecules 201621(10)pii E1374 doi103390molecules21101374

13 Nicholson RL Hammerschmidt R Phenolic compounds and theirrole in disease resistance Annu Rev Phytopathol199230369ndash389 doi101146annurevpy30090192002101

14 Michalek S Mayr U Treutter D Lux-Endrich A Gutmann MFeucht W Geibel M Role of flavan-3-OLS in resistance of appletrees to venturia inaequalis Acta Hortic 1998484535ndash539doi1017660ActaHortic199848491

15 Arici SE Kafkas E Kaymak S Koc NK Phenolic compounds ofapple cultivars resistant or susceptible to Venturia inaequalisPharm Biol 201452904ndash908 doi103109138802092013872674

16 Treutter D Significance of flavonoids in plant resistance Areview Environ Chem Lett 20064147ndash157 doi101007s10311-006-0068-8

17 Petrussa E Braidot E Zancani M Peresson C Bertolini A Patui SVianello A Plant Flavonoids-Biosynthesis Transport and involve-ment in stress responses Int J Mol Sci 20131414950ndash14973doi103390ijms140714950

Table 3 Concentrations (mg gminus1 FW) of hydroxycinnamic acids existing as hydrolysable hydroxycinnamates in seven citrus leaf extracts Hydroxycinnamic acidsmeasured after leaf extract saponification (n = 5)

Species P-Coumaric Sinapic Ferulic

Bearss lime 062 plusmn 008b 084 plusmn 005a 049 plusmn 004b

Valencia orange 011 plusmn 001d 045 plusmn 004 c 066 plusmn 038b

Ruby Red grapefruit 012 plusmn 001d 006 plusmn 001e 061 plusmn 005b

Duncan grapefruit 011 plusmn 001d 003 plusmn 002e 060 plusmn 041b

Dancy tangerine 028 plusmn 003 c 057 plusmn 002b 344 plusmn 016a

Carrizo citrange 026 plusmn 030 cd 032 plusmn 001d 298 plusmn 266ab

Curry leaf 129 plusmn 030a 003 plusmn 001e 018 plusmn 004 c

Varieties with different superscript letters are statistically different using Tukeyrsquos HSD test (P-value lt 005)

e1752447-8 F HIJAZ ET AL

18 Saacutenchez-Rangel JC Benavides J Heredia JB Cisneros-Zevallos LJacobo-Velaacutezquez DA The Folin-Ciocalteu assay revisited improve-ment of its specificity for total phenolic content determination AnalMethods 201355990ndash5999 doi101039c3ay41125g

19 Chang CC Yang MH Wen HM Chern JC Estimation of totalflavonoid content in propolis by two complementary colorimetricmethods J Food Drug Anal 200210178ndash182

20 Sakanaka S Tachibana Y Okada Y Preparation and antioxidantproperties of extracts of Japanese persimmon leaf tea(kakinoha-cha) Food Chem 200589569ndash575 doi101016jfoodchem200403013

21 Naik GH Priyadarsini KI Satav JG Banavalikar MM Sohoni DPBiyani MK Mohana H Comparative antioxidant activity of indi-vidual herbal components used in ayurvedic medicinePhytochemistry 20036397ndash104 doi101016S0031-9422(02)00754-9

22 Singleton V Rossi JA Colorimetry of total phenolics withphosphomolybdic-phosphotungstic acid reagents Am J EnolVitic 196516144ndash158

23 Aktumsek A Zengin G Guler GO Cakmak YS Duran AAntioxidant potentials and anticholinesterase activities of methano-lic and aqueous extracts of three endemic Centaurea L species FoodChem Toxicol 201355290ndash296 doi101016jfct201301018

24 Brand-Williams W Cuvelier ME Berset C Use of a free radicalmethod to evaluate antioxidant activity LWT - Food Sci Technol19952825ndash30 doi101016S0023-6438(95)80008-5

25 Moon JK Shibamoto T Antioxidant assays for plant and foodcomponents J Agric Food Chem 2009571655ndash1666doi101021jf803537k

26 Baderschneider B Winterhalter P Isolation and characterizationof novel benzoates cinnamates flavonoids and lignans fromRiesling wine and screening for antioxidant activity J AgricFood Chem 2001492788ndash2798 doi101021jf010396d

27 Singh AP Wilson T Luthria D Freeman MR Scott RMBilenker D Shah S Somasundaram S Vorsa N LC-MS-MS char-acterisation of curry leaf flavonols and antioxidant activity FoodChem 201112780ndash85 doi101016jfoodchem201012091

28 Ghasemzadeh A Jaafar HZE Rahmat A Devarajan T Evaluationof bioactive compounds pharmaceutical quality and anticanceractivity of curry leaf (Murraya koenigii L) Evidence-basedComplement Altern Med 201420141ndash8 doi1011552014873803

29 Nagappan T Ramasamy P Wahid MEA Segaran TCVairappan CS Biological activity of carbazole alkaloids and essen-tial oil of murraya koenigii against antibiotic resistant microbesand cancer cell lines Molecules 2011169651ndash9664 doi103390molecules16119651

30 Al Harbi H Irfan UM Ali S The antibacterial effect of curryleaves (Murraya Koenigii) EJPMR 20163382ndash387

31 Yukari T Hiroe K Lajis NH Nakatani N Comparison of anti-oxidative properties of carbazole alkaloids from Murraya koenigiiLeaves J Agric Food Chem 2003 doi101021JF034700+

32 Manthey JA Grohmann K Phenols in citrus peel by productsConcentrations of hydroxycinnamates and polymethoxylated fla-vones in citrus peel molasses J Agric Food Chem2001493268ndash3273 doi101021jf010011r

33 Hijaz FM Manthey JA Folimonova SY Davis CL Jones SEReyes-De-Corcuera JI An HPLC-MS Characterization of thechanges in sweet orange leaf metabolite profile following infectionby the bacterial pathogenCandidatus Liberibacter asiaticus PLoSOne 20138(11)e79485 doi101371journalpone0079485

34 Loacutepez-Gresa MP Torres C Campos L Lisoacuten P Rodrigo IBelleacutes JM Conejero V Identification of defence metabolites intomato plants infected by the bacterial pathogen Pseudomonassyringae Environ Exp Bot 201174216ndash228 doi101016jenvexpbot201106003

35 Belleacutes JM Loacutepez-Gresa MP Fayos J Pallaacutes V Rodrigo IConejero V Induction of cinnamate 4-hydroxylase and phenyl-propanoids in virus-infected cucumber and melon plants PlantSci 2008174524ndash533 doi101016jplantsci200802008

36 Von Roepenack-Lahaye E Newman MA Schornack SHammond-Kosack KE Lahaye T Jones JDG Daniels MJDow JM p-Coumaroylnoradrenaline a novel plant metaboliteimplicated in tomato defense against pathogens J Biol Chem200327843373ndash43383 doi101074jbcM305084200

37 Chen GL Fan MX Wu JL Li N Guo MQ Antioxidant andanti-inflammatory properties of flavonoids from lotus plumuleFood Chem 2019277706ndash712 doi101016jfoodchem201811040

38 Zuo R Oliveira A Bullita E Torino MI Padgett-Pagliai KAGardner CL Harrison NA da Silva D Merli ML Gonzalez CFet al Identification of flavonoids as regulators of YbeY activity inLiberibacter asiaticus Environ Microbiol 201921(12)4822ndash4835doi1011111462-292014831

PLANT SIGNALING amp BEHAVIOR e1752447-9