ORIGINAL RESEARCH

Relation between plant nutrition, hormones, insecticideapplications, bacterial endophytes, and CandidatusLiberibacter Ct values in citrus trees infectedwith Huanglongbing

Weishou Shen & Juan M. Cevallos-Cevallos & Ulisses Nunes da Rocha &

Hector A. Arevalo & Philip A. Stansly & Pamela D. Roberts &

Ariena H. C. van Bruggen

Accepted: 19 August 2013 /Published online: 7 September 2013# KNPV 2013

Abstract Intensive insecticide and nutrient manage-ment have been attempted worldwide to reduce citrushuanglongbing (HLB) symptom development andyield loss. However, effects of insecticide and nutrientapplications on HLB have been poorly understood.Leaf nutrients, jasmonic and salicylic acid contents,

cycle threshold (Ct) values of Ca. Liberibacterasiaticus (Las), and community structure of endophyticα-proteobacteriawere evaluated after insecticide treat-ment, ‘nutrition’ treatment (including systemic resis-tance inducing agents), or both in comparison with acontrol in a two-factor field experiment in 2008–2012.Leaf N,Mn, Zn and B significantly increased whilst Cudecreased after nutrient applications. Salicylic acidsignificantly increased in old leaves treated with insec-ticides, nutrients or both, and in young leaves treatedwith nutrients only. The jasmonic acid concentrationwas highest after the nutrition treatment in both old andyoung leaves. Ct values of Las and leaf area and weightsignificantly increased after long-term nutrient appli-cations in 2011 and/or 2012. Redundancy analysis ofthe endophytic α-proteobacteria community structureindicated that the communities were mainly separatedaccording to nutrient applications, which were posi-tively associated with Ct values of Las and Ca, Mn, Zn,B, Mg, and Fe contents in leaf samples collected in2012. Thus, effects of insecticides on HLB were sig-nificant in the early 2-year period whilst nutrients hadsignificant effects on Las content and leaf size andweight after at least 3 years of application.

Keywords Boyd’s nutritional program .Ca.Liberibacter asiaticus . Cycle threshold (Ct) value .

Induced systemic resistance (ISR) . Huanglongbing(HLB) . Systemic acquired resistance (SAR)

Eur J Plant Pathol (2013) 137:727–742DOI 10.1007/s10658-013-0283-7

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10658-013-0283-7) containssupplementary material, which is available to authorized users.

W. Shen : J. M. Cevallos-Cevallos :U. Nunes da Rocha :A. H. C. van Bruggen (*)Emerging Pathogens Institute and Department of PlantPathology, University of Florida, Gainesville, FL 32611,USAe-mail: ahcvanbruggen@ufl.edu

W. ShenDepartment of Environmental Science and Engineering,Nanjing Normal University, Nanjing 210023, China

J. M. Cevallos-CevallosCentro de Investigaciones Biotecnológicas del Ecuador(CIBE), Escuela Superior Politécnica del Litoral (ESPOL),Guayaquil 090112, Ecuador

U. Nunes da RochaLawrence Berkeley National Laboratory, Earth SciencesDivision, Berkeley, CA 94720, USA

H. A. Arevalo : P. A. Stansly : P. D. RobertsSouthwest Florida Research and Education Center,University of Florida, Immokalee, FL 34142, USA

Introduction

Citrus greening disease or huanglongbing (HLB) isone of the most important citrus diseases worldwide(Bové 2006; Gottwald 2010), causing such severelosses that this disease is threatening commercial citrusproduction wherever the disease occurs (Gottwald2010). This disease has long been known in Asia andAfrica, but was found in South America only in 2004.In Florida, HLB was first detected in 2005 during aroutine survey by regulatory agents, although the vec-tor of the disease, the Asian citrus psyllid (Diaphorinacitri Kuwayama, Sternorrhyncha: Psyllidae) was dis-covered as early as 1998 (Halbert andManjunath 2004;Halbert 2005). Since then, the disease has spreadthrough most of Florida, where the majority of theU.S. commercial citrus groves are located. In recentyears, the vector and pathogen have spread to otherSouth Eastern States in the U.S., and the pathogen wasdetected in California in 2012 (Stokstad 2012). HLBhas not been found yet in Europe or the Middle East.

Citrus greening is consistently associated with a bacte-rium that has not been cultured reliably yet. The putativecausative agent is named Candidatus Liberibacter sp.,belonging to the α-proteobacteria. Three species of Ca.Liberibacter are known to colonize citrus trees: Ca.Liberibacter asiaticus in Asian countries and since recentlyalso in Eastern Africa and in the Americas, Ca.Liberibacter africanus in Southern Africa and some EastAfrican countries, and Ca. Liberibacter americanus inBrazil (Bové 2006; Gottwald et al. 2007; Mangomereet al. 2009; Teixeira et al. 2008). In the USA, the onlyspecies that has been found so far is Ca. Liberibacterasiaticus or Las (Gottwald et al. 2007).

Ca. Liberibacter spp. are transmitted from tree totree by psyllids, Diaphorina citri, in most citrus grow-ing regions and Trioza eritreae in Africa. Both psyllidspecies can transmit all species of Ca. Liberibacter(Bové 2006). Adult psyllids are attracted to volatilesemitted from flushing shoots (Patt and Sétamou 2010),females lay eggs on young twigs, and nymphs developon young twigs and leaves (flush). Ca. Liberibactercells are taken up from the phloem through the mouth-parts of nymphs and adults (Inoue et al. 2009). Thebacteria can cross the gut membranes and move to thesalivary glands from which they are injected into thephloem (Bonani et al. 2010). The psyllids remain in-fectious for life and can transmit the pathogen to theiroffspring (Pelz-Stelinski et al. 2010).

In Florida, the psyllid populations are highest in spring,but they can occur at any time of the year when treesproduce new flush (Rogers and Ebert 2009). ManagementofD. citri populations and consequently HLB is currentlybased on intensive use of insecticides (commonly every2 weeks). Even in winter, application of insecticides isrecommended to minimize psyllid infestations on flushlater on (Qureshi and Stansly 2010). An intensive area-wide insecticide program is now in place in Florida, butHLB continues to be a major problem. Many differentinsecticides are available for managing the Asian citruspsyllid, and insecticides are rotated to limit the develop-ment of resistance in the psyllids. Nevertheless, insecticideresistance, in particular to imidacloprid, has already devel-oped in field populations ofD. citri in Florida (Tiwari et al.2011), and negative side-effects on the parasitoidTamarixia radiata, introduced into Florida for biologicalcontrol of the psyllid, has also been observed (Hall andNguyen 2010). Moreover, secondary pest outbreaks ofvarious citrus scales and mealy bugs can be a real threatresulting from intensive insecticide use for psyllid control(Wakgari andGiliomee 2003). In addition, the insecticidescommonly used for the control of the Asian citrus psyllidhave not been as effective as expected (Ichinose et al.2010). Finally, insecticides may be helpful in reducingthe spread of Ca. Liberibacter asiaticus in the beginningof the epidemic, but may not reduce the pathogen load inthe trees once most trees in a grove are infected (Chiyakaet al. 2012).

After transmission of Ca. Liberibacter asiaticus tocitrus, infected trees respond to the bacteria with a widearray of physiological reactions resulting in a sequenceof symptoms, from mild chlorosis to distinct mottling,associated with an increase in bacterial concentrations(Coletta-Filho et al. 2010). A hormonal imbalance maybe responsible for the development of lopsided fruitswith uneven colouring (Kim et al. 2009). Starch isaccumulated in leaves and stems as the phloem getsblocked (Etxeberria et al. 2009; Kim et al. 2009). As aconsequence, roots starve and can become infected bysecondary pathogens and mineral nutrition is out ofbalance (Pereira and Pereira Milori 2009). Within 2–3 years after inoculation a tree may die. Despite thisgeneral trend in disease development, variation in theextent of plant response to infection can be found. Thiscan be due to differences in genotype (Folimonovaet al. 2009), but some trees with the same genotypecan also differ in disease reaction, suggesting that someother factors may be responsible for relative resistance

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to HLB. One factor that may influence the resistance todisease can be the nutritional condition, which caninfluence the microbial community in the rhizosphereand endosphere of plants (Dordas 2008; Gu et al.2013). In turn, bacterial and fungal endophytes canchange the phloem composition and induce systemicresistance (Musetti et al. 2007).

A reduction in HLB symptom development and yieldloss has been attempted by intensive nutrient manage-ment. In particular, micronutrients combined withsalicylic acid and/or phosphite have been applied, oftenas foliar sprays, to maintain the productive capacity ofHLB infected trees (Ahmad et al. 2011; Masaoka et al.2011; Razi et al. 2011). The effectiveness of these treat-ments has been controversial (Gottwald et al. 2012), butbalanced nutrition likely slows down tree decline due toHLB, considering the occurrence of healthy looking treesmany years after Las infection was demonstrated (fieldobservations by the authors). Asymptomatic infectionhas been noticed in several organically managed groves,despite infestation by Las-positive psyllids (Halbert,unpublished; Shen et al. 2013).

The nutritional programs employed vary tremen-dously and mostly have not been tested in replicatedexperiments (including controls) over many years. Oneof these programs (Table 1), developed by citrus grow-er Maury Boyd, has been instituted since the start of theepidemic in 2005. Trees treated according to the Boydprogram look healthier than untreated trees and haveadequate fruit production, despite being 100 %

infected with Las. The Boyd program has been testedin a replicated experiment in combination with insec-ticide treatments and a negative control since 2008(Stansly et al. 2011). In the first 3 years no effects weredetected of the nutrient plus salicylic acid treatmentson the Las content in the trees or on yield (Stansly et al.2011). In another replicated (but shorter-term) experi-ment no differences were observed in tree health, fruitquality and production, or Las titre between trees treat-ed with a nutritional program (plus or minus phosphite)and a control treatment (Gottwald et al. 2012).Additionally, application of micro nutrients, phosphiteand salicylic acid was compared to regular nutrientmanagement in several commercial blocks, but nodifferences in disease development or yield were ob-served. Both of these experiments lasted 2 years(Gottwald et al. 2012). Thus, it has not been shownso far if nutritional programs can lower the Las contentand reverse its effects on citrus trees in the long run.

The Boyd nutritional program includes agents thatinduce systemic resistance, either systemic acquiredresistance (SAR) or induced systemic resistance(ISR), but individual effects of these agents on HLBhave not been published in scientific journals.Nevertheless, soil-applied imidacloprid, isonicotinicacid and acibenzolar-s-methyl did elicit SAR in citrustrees in greenhouse tests, and reduced the severity ofcitrus canker (Francis et al. 2009).

Boyd’s nutritional program is commonly combinedwith the application of insecticides. The individual

Table 1 Composition of the foliar spray known as ‘Boyd nutrient solution’

Product Unitper acre

Impact on microbes or plants Reference

13-0-44 fertilizer 8.5 lb N-K fertilizer Diamond R

Techmangan (Mn sulfate) 8.5 lb Mn fertilizer Diamond R

Zinc sulfate 2.8 lb Zn fertilizer Diamond R

Epsom salts 8.5 lb Mg fertilizer Diamond R

Sodium molybdate 0.85 oz Mo fertilizer, bactericide Diamond R

Di-oxy solv organic (hydrogen dioxide) 2 qt Oxidizer (fungicide/bactericide) Flo-Tec Inc.

3-18-20 with K-phite (potassium phosphate) 8 gal Oomycte inhibitor, inducedsystemic resistance (ISR)

Plant Food Systems

Serenade Max WP (Bacillus subtilis) 2.25 lb Biological fungicide/bactericide, Systemicacquired resistance (SAR) inducer

AgraQuest, Inc.

SAver (salicylic acid) 1 qt SAR inducer Plant Food Systems

435 oil 5 gal carrier PetroCanada

Citrus grower Maury Boyd developed a program with multiple applications of various nutrients (as listed above plus Borate and Ca-nitrate), hydrogen dioxide, potassium phosphite, Bacillus subtillis, ammonium and potassium salicylate, and 435 spray oil

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effects of insecticide treatments and a nutritional programhave not been studied in detail. In 2008, Stansly andcolleagues started a long-term replicated field experimenttesting effects of insecticides, Boyd’s nutrient program andboth of these in comparison with a non-treated controltreatment. HLB had just been detected in some trees in theexperimental grove at that time. In the first few years(2008–2010), effects of insecticide treatments on HLBdevelopment and yield were significant, but there wereno effects of the nutrient treatment (Stansly et al. 2013).The current study was carried out with the goal to inves-tigate if prolonged treatment with nutrients (including ISRand SAR inducing agents) would have an effect on endo-phytic bacterial communities, Las titres, plant hormoneconcentrations and leaf sizes. Our hypotheses were: (1) inthe long run, insecticides have a different effect onmicrobial communities than ‘Boyd nutrient solution’compared to the control treatment; (2) insecticides donot affect Las titres once most of the trees are infected;(3) ‘Boyd nutrient solution’ enhances the diversity ofendophytic bacteria, induced systemic resistance and/orsystemic acquired resistance, and foliar growth; (4) acombination of both insecticides and ‘Boyd nutrientsolution’ is the best agricultural practice in areas wherea limited number of the trees are infected with Las. Totest these hypotheses, soil and leaf samples were collect-ed from the experimental grove mentioned above(Stansly et al. 2013), and were analyzed for nutrients,endophytic microbial communities, Las titers, plant hor-mones and leaf sizes.

Material and methods

Experimental field and design

The experiment was conducted in block B9 in SilverStrand’s North Grove, a 5.2 ha block of ‘Valencia’ orangeon ‘Swingle’ citromelo rootstock planted in June 2001.The site is located in Collier Co. (26° 29′N 81° 21′W,Florida, USA). The soil type is primarily Immokalee finesand, with a slightly different soil type, Basinger finesand, mostly confined to block 2 (Fig. 1). The annualprecipitation is 117–137 cm, and the average annual airtemperature is 21–25 °C. In February 2008, 2 years afterthe detection of HLB, the block was divided into 16 plotsto fit a randomized complete block design with fourreplicates and four treatments (Fig. 1). Treatment selec-tion corresponded to a 2×2 factorial experiment using the

two main components: foliar nutrient solution (N1) anduntreated (N0), and insecticide (I1) and untreated (I0). Thecombination of these twomain factors determined the fourtreatments: control (N0I0), nutritional of Moury Boyd orMB (N1I0), insecticide or INS (N0I1), and MB + INS(N1I1).

Insecticide applications

Chemical applications were timed by Integrated PestManagement (IPM) guidelines, and applied on an “asneeded” basis according to observations made duringweekly scouting. Five foliar insecticide applicationswere administered during each growing season in theinsecticide and ‘nutritionals plus insecticide’ plots:(1) Danitol® 4 E.C. (fenpropathrin) [Valent USACorp. Walnut Creek, CA] at 0.9–1.2 l/ha, (2)Delegate™ WG (spinetoram) [Dow Agrosciences.Indianapolis, IN] at 0.3–0.4 l/ha, (3) Mustang ® (ze-ta-cypermethrin) [FMC. Philadelphia, PA] at 0.3 l/ha,(4) Movento® (spirotetramat) [Bayer CropSciences.Research Triangle Park, NC] at 0.7–1.2 l/ha, and (5)Lorsban 4E (chlorpyrifos) [Dow Agrosciences.Indianapolis, IN] at 3.5 l/ha. The products were selectedbased on the time of the year and current managementguidelines for Asian citrus psyllids. All of the insecticidetreatments included 2 % of 435 horticultural oil. Allapplications were made using an air blast sprayer at 10bars, averaging 982 l/ha. In addition, Temik 15G(aldicarb) [Bayer CropSciences, Research TrianglePark, NC] was applied at 22–34 kg/ha each winteraccording to the guidelines followed by commercialgrowers.

Nutrient applications

Fertilizers (NPK or as listed) were applied to soil asfollows: 13-0-21 at 336 kg/ha in September 2008; 12-4-16 at 448 kg/ha in January 2009; 8-0-24 at 448 kg/hain May 2009; K-Mag® (22 % K2O, 11 % Mg and22 % S) at 224 kg/ha in October 2009 and August2010; UN-32 (45 % NH4NO3, 35 % urea and 20 %water) at 186 l/ha in October 2009, January and April2010; 0-0-42 at 224 kg/ha on March 2010, May andAugust 2011; 9-0-0 liquid at 93 l/ha in March 2010;Granulite (heat dried biosolids) at 1,120 kg/ha in May2010; 14-0-22 at 336 kg/ha in September 2010; 16-4-16 at 336 kg/ha in January 2011; 20-0-0 plus 5 % Caliquid at 96 l/ha in May and August 2011.

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‘Boyd nutrient solution’ (Table 1) was sprayed onthe foliage in designated plots (nutrition only and in-secticide plus nutrition treatments) three times a yearwhen major flushes were fully expanded but not hard-ened. Applications were performed with an Air-O-Fanairblast sprayer equipped with Albuz® ATR hollowcone nozzles providing an 80o spray pattern with fiveblue and one green nozzle (2.5 and 3.4 l/min,

respectively) at 10 bars and 5.2 km/h, delivering a total39 l/min or 982 l/ha (105 gal/ac).

Sampling

In 2010, soil samples were collected under one ran-domly selected tree from each of the 16 plots. Eachsample consisted of four subsamples around the

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Main road

MB = NutritionalCtrl = ControlIns = InsecticideIns+MB = Combined insecticide + Nutritional

Bk: 1Trt: MB

Bk: 2Trt: Ins

Bk: 1Trt: Ctrl

Bk: 2Trt: Ctrl

Bk: 3Trt: Ctrl

Bk: 4Trt: Ctrl

Bk: 2Trt: MB

Bk: 3Trt: MBBk: 4

Trt: MB

Bk: 1Trt: Ins

Bk: 4Trt: Ins + MB

Bk: 3Trt: Ins

Bk: 4Trt: Ins

Bk: 3Trt: Ins + MB

Bk: 2Trt: Ins + MB

Bk: 1Trt: Ins + MB

Fig. 1 Field layout of an experiment testing the effects ofinsecticide applications, nutritional management (Maury Boyd,MB) or both on ‘Valencia’ orange on ‘Swingle’ citromelo

rootstock compared to non-treated controls. The experimentwas conducted in Silver Strand’s North Grove just North ofImmokalee in South Florida from 2008 to 2012

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selected tree pooled into plastic bags. The soil wastransported from the field to the laboratory inGainesville in an ice cooled container.

All leaf samples were collected from trees of thesame size and age; young, replanted trees wereavoided. In 2011, pooled leaf samples were collectedfrom one tree per plot and transported in liquid nitro-gen to the laboratory in Gainesville for plant hormoneanalysis. In October 2011, leaf samples were collectedfrom six to 17 random trees in each of the 16 plots,totaling 148 samples. The samples were placed in acool box with ice and brought to the HLB laboratory atthe SWFREC for DNA extraction and real-time qPCRanalysis. In May 2012, leaf samples were collectedfrom four random trees in each of the 16 plots, andtransported in dry ice to the laboratory in Gainesvillefor analysis of plant nutrients, HLB titre by real-timeqPCR, and microbial communities by PCR-DGGE(denaturing gradient gel electrophoresis).

Soil and plant nutrient analyses

Air-dried soil samples were subjected to analysis in theSoil Analysis lab at the University of Florida (UF),Gainesville, FL. Soil pH was determined with a glasselectrode (soil:water = 1:2). Soil organic C was deter-mined by the dichromate oxidation and total N by theKjeldahl digestion according to Mylavarapu and Moon(2007). Automatic colorimetric analysis was employedto determine NH3-N and NOx-N (NOx-N = NO2-N +NO3-N) using an Alpkem Flow Solution IVautoanalyzer (OI Analytical, College Station, TX,USA). Soil P, K, Ca and Mg were extracted withMehlich-1 extraction solution (soil:water = 1:4), andfiltered through Whatman 42 filterpaper as described byMylavarapu and Moon (2007). The filtrates were ana-lyzed for nutrients using an inductively coupled plasma(ICP) spectrophotometer (SPECTRO AnalyticalInstruments Inc., Mahwah, NJ, USA).

Composite leaf samples consisting of 20 leavesfrom each of 4 trees per treatment and per block wereground in a Foss Cyclotec 1093 (Eden Prairie, MN55344), and then subjected to nutrient analysis in theSoil Analysis lab at UF, Gainesville. Nitrogen wasanalyzed using the total Kjeldahl nitrogen (TKN)method according to Mylavarapu and Moon (2007).Automatic colorimetric analysis was employed to de-termine nitrogen in TKN digestates using an AlpkemFlow Solution IVautoanalyzer (OI Analytical, College

Station, TX, USA). Other nutrients were analyzedusing an ashing and acid digestion procedure as de-scribed by Mylavarapu and Moon (2007). The filtratewas analyzed for nutrients using an ICP spectropho-tometer (SPECTRO Analytical Instruments Inc.,Mahwah, NJ, USA).

Plant hormone analysis

Hormone analysis was exactly as in Birkemeyer et al.2003. Briefly, 300 mg of frozen leaves were groundunder liquid nitrogen and suspended into 3 ml Bieleskisolvent pre-cooled to −20 °C and incubated at roomtemperature for 1 h. After centrifugation (5,000 × g for5 min), the supernatant was separated and transferredto a vacuum centrifuge at 45 °C and 10 mbar untildryness. The dried residue was suspended in 30 μl ofmethanol and then, 200 μl diethyl ether were addedand sonicated in a bath for 1 min. Samples were thenapplied to an aminopropyl solid-phase extraction-cartridge (Fisher Scientific, St Louis, MN, USA).Each column was preconditioned with two times thevolume of methanol/diethyl ether followed by sampleapplication into each column. Columns were thenwashed with 250 μl of CHCl3:2-propanol = 2:1 (v/v),and the hormone fraction was eluted twice with 200 μldiethyl ether containing 2 % acetic acid. The combinedeluted fractions were mixed with 3 μl of a 0.1-mg/mlsolution of the internal standard 5α-cholestane and thentaken to dryness in a vacuum centrifuge (1 min at200 mbar, then for a further min at 10 mbar). The driedsamples were suspended in 80 μl of N-methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide (MTBSTFA) andincubated at 100 °C for 1 h. GC-MS analyses werecarried out in a HP5890 GC coupled to an HP5971series mass spectrometer (MS) from Hewlett Packard,(Santa Clara, CA). Chromatogram analysis was com-pleted using HP ChemStation software. Samples (1 μl)were injected splitlessly at 230 °C with an oven, tem-perature ramp of 6 °C/min from 70 to 350 °C, the ionsource temperature was set to 230 °C, and the transferline was at 260 °C. Hydrogen carrier gas was used at aflow-rate of 1 ml/min. Quadrupole GC-MS chromato-grams were by electron impact ionisation and total ionmonitoring, m/z 40–600, as well as selective ion mon-itoring was used with the selected fragments of m/z 133(JA), m/z 309 (SA), and m/z 217 (5α-cholestane). Allreagents were from Fisher Scientific (St Louis, MN,USA).

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Nucleic acid extractions

For HLB testing, midribs of leaves and petioles werecut with a flamed scissor into 1–2 cm long pieces,pooled per sample, and placed in polypropylene vialsprior storing at −80 °C. For microbial communityanalysis of the endophytes inside plant tissues, leafsamples were washed with sterilized-distilled waterfor 5 min using an ultrasonic cleaning system. The leafsamples were surface-sterilized for 30 s with 75 %ethanol, 5 min in a 5 % NaClO, followed by severalwashes with sterilized-distilled water. The leaf bladeswere cut into small pieces (about 2 cm diameter) andstored at −80 °C before DNA extraction.

DNA from leaf samples was extracted with aDNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA,USA) according to the manufacturer’s instructions.Briefly, 100 mg of leaf midribs and petioles were addedinto XXTuff Reinforced Microvials (Biospec,Bartlesville, OK, USA) containing four 2.3 mmChrome-Steel Beads (Biospec, Bartlesville, OK,USA). After the addition of liquid nitrogen, the sam-ples were homogenized at 3,000 rpm for 1 min using aPowerlyzer 24 Bench Top Bead-Based Homogenizer(MO BIO Laboratories, Carlsbad, CA, USA). Thesample materials were lysed, salt-precipitated, and cen-trifuged through the DNeasy Plant Mini Kit columns.Total DNAwas purified with a silica-based membraneand elution reagent (QIAGEN Inc., Valencia, CA,USA). The purified DNA was stored at −20 °C forfurther analysis.

Regular PCR and real-time PCR of Las

Regular PCR was performed using primer pair OI1 andOI2c to detect Las in leaf samples (Jagoueix et al. 1996).Real-time PCR of leaf DNA was carried out using aCFX96 Real-Time PCR Detection System (Bio-RadLaboratories, Hercules, CA, USA) according to Liet al. (2006). The primer pair HLBas and HLBr, and aTaqMan probe HLBp were used for PCR amplificationto target the 16S rRNA gene of Las (Li et al. 2006). Anadditional primer-probe set designed on the basis of theCOX gene was used as positive internal control (Li et al.2006). Two-step thermal profiles consisted of 95 °C for20 s, followed by 40 cycles of 1 s at 95 °C and 40 s at58 °C, with plate reading at 58 °C for data acquisition.Each run contained one positive and one negative con-trol sample from citrus plants in a quarantine greenhouse

at the Division of Plant Industry at Gainesville, FL(obtained from Debra Jones). Data analysis wasperformed with CFX Manager Software Version 2.0(Bio-Rad Laboratories, Hercules, CA, USA). TheHLB laboratory at the SWFREC considers positive toHLB samples with Ct values less than or equal to 32.

PCR-DGGE analysis

A nested system was used for amplification of α-proteobacterial 16S ribosomal RNA genes. The firstround of PCR was performed by applying the primerpair F203α (CCG CAT ACG CCC TAC GGG GGAAAG ATT TAT) and R1494 (CTA CGG YTA CCTTGT TAC GAC) (Weisburg et al. 1991; Gomes et al.2001). The first round of PCR products then served astemplates for a second round of PCR with primer pairF984GC (CGC CCG GGG CGC GCC CCG GGCGGG GCG GGG GCA CGG GGG G AAC GCGAAG AAC CTT AC) and R1378 (CGG TGT GTACAA GGC CCG GGA ACG) (Heuer et al. 1997),which amplified the variable V6 region of 16S rRNA.PCR was carried out with a Veriti 96 well ThermalCycler (Applied Biosystems, Foster City, CA, USA) in50 μl reaction volumes containing 0.4 μM of eachprimer, 2.5 mM MgCl2, 0.2 mM of each dNTP and1.25 U Taq DNA polymerase (Invitrogen, GrandIsland, NY, USA).

DGGE was performed at 60 °C with the DCodeTM

Universal Mutation Detection System (Bio-RadLaboratories, Hercules, CA, USA) according to themanufacturer’s instructions. DGGE was carried outusing 6.5 % (wt/vol) polyacrylamide gels (ratio ofacrylamide to bisacrylamide, 37.5:1) with a gradientof 40 % to 55 % where a 100 % denaturing solution isdefined as 7 M urea and 40 % formamide (Watanabeet al. 2001). The gels were electrophoresed at 65 V for16 h in 0.5 × TAE buffer and stained with SYBR Gold(Molecular Probes, Eugene, Oregon, USA) for 30 min(Tuma et al. 1999; Sigler et al. 2004). The stained gelswere immediately photographed on a UV transillumi-nator with a CCD camera (Bio-Rad Laboratories,Hercules, CA, USA). Digital images of the gels werefurther analysed by Quantity One® 1-D AnalysisSoftware (Bio-Rad Laboratories, Hercules, CA,USA). Removing the background intensity from eachlane, the software performs a density profile throughlanes, detects individual bands and matches bandsoccupying the same position in different lanes (Xue

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et al. 2006). The genetic diversity of endophytic mi-crobial communities was analyzed by Richness (S),Shannon diversity (H), and Evenness (EH) indices(Xue et al. 2006).

Statistical analyses

Statistical analyses were performed to test main effects,their interaction, and individual treatments using theGeneral Linear Model Procedure. Main effects wereconsidered if the interaction of the two factors was notsignificant (p>0.05). Results were subjected to analysisof variance (ANOVA) to determine the significance ofthe differences in data (plant hormones were Log10transformed) with SAS 9.2 Software (SAS InstituteInc., Cary, NC, USA). Least significant difference(LSD) post-hoc tests were conducted to determine thedifferences between the individual treatments.

Correlations of microbial communities of endophyt-ic α-proteobacteria to environmental factors wereassessed using redundancy analysis (RDA). RDA wasperformed using the ‘species data’ (individual DGGEbands) and ‘environmental data’ (leaf nutrient con-tents, leaf area, leaf fresh weight per unit of area, andleaf Las titer) with the computer software CANOCO4.5 (Microcomputer Power, Ithaca, NY, USA). AMonte Carlo permutation test was carried out basedon 499 random permutations.

Results

Soil pH, organic C and nutrient contents

Analyses of soil pH (p=0.001), NOx-N (p=0.002), andK (p=0.008) revealed significant interactions of the twofactors (insecticides and nutrients), so that individualtreatment combinations are reported separately. SoilpH was highest in control plots, lower in insecticidetreated plots and nutrition plus insecticide plots, andlowest (p=0.05) after the treatment with Boyd’s nutri-ents (Table 2). Soil organic C was significantly de-creased after the insecticide applications (p<0.001), bothwith and without nutrients compared to the control orapplication of nutrients only (p=0.05). Soil NH3-N wassignificantly increased after treatment with nutrients(p=0.042) compared to that without nutrients, but de-creased after application of insecticides (p=0.004) com-pared to the treatments without insecticides. Soil NOx-N

and NH3-N were significantly enhanced by the nutrienttreatment compared to the other treatments (p=0.05).Total Kjeldahl nitrogen (TKN) content in soil was greatestin the plots treated with nutrients only and was signifi-cantly decreased by both insecticide treatments (p=0.013).Soil K was significantly greater in the nutrition treatmentthan in the other treatments (p=0.05). Soil P, Ca and Mgcontents were not significantly different among the fourtreatments.

Leaf nutrient contents

Leaf N was significantly (p=0.031) decreased after thetreatments with insecticides compared to those withoutinsecticides, and was significantly (p=0.05) lower inthe insecticide only treatment than in the other treat-ments (Table 3). Analyses of leaf Ca (p=0.035) re-vealed significant interactions between the two factors(insecticides and nutrients), so that only individualtreatment effects are reported. The leaf Ca contentwas significantly higher after the treatment with nutri-ents only than after the combination of nutrients plusinsecticides (p=0.05). Leaf Mn and Zn were also sig-nificantly (p<0.001) increased after nutrient applica-tions, with or without insecticides, compared to thecontrol and insecticide treatments (p=0.05). In con-trast, the leaf Cu content was significantly decreasedby the nutrient treatments (p=0.009), particularly thenutrient treatment without insecticides. On the otherhand, the leaf B content was significantly increased bythe nutrient treatments (p=0.031), especially the nutri-ent treatment without insecticides. Leaf P, K, Mg andFe contents were not significantly different among thefour treatments.

Plant hormones

The salicylic acid (SA) content was significantlyhigher in old leaf samples treated with insecticides(p=0.029) or nutrients (p=0.007) than in the controltrees (Table 4). In young leaf samples, the main treat-ment effects were not significant. Yet, multiple com-parisons indicated that the SA content in those leaveswas significantly greater in the nutrition treatment thanin the control. There were significant interactions be-tween the insecticide and nutrient treatments with re-spect to the jasmonic acid (JA) contents in both old(p<0.001) and young leaf samples (p=0.029). The JAcontent in old leaf samples was greatest in the nutrition

734 Eur J Plant Pathol (2013) 137:727–742

treatment, lower in the insecticide treatments (with andwithout nutrition), and lowest in the control treatment(p=0.05). The JA content in young leaf samples wassignificantly greater after treatment with nutrients thanin any of the other treatments (p=0.05).

Regular PCR and real-time PCR analyses of Las

The 16S rRNA fragments of Las in leaf samples col-lected in May 2012 were successfully amplified in thefour treatments using primer pair OI1 and OI2c(Fig. 2). Similar results had been obtained in previousyears (data not shown). The Ct values of Las weresignificantly greater after the treatments with nutrientscompared to those without nutrients in October 2011(p=0.037) and May 2012 (p=0.001) (Fig. 3). For indi-vidual treatment combinations, the Ct values weresignificantly higher in both nutrition treatments thanin the control (p=0.05), whilst no significant differ-ences were observed between the insecticide treatmentand the other three treatments in October 2011(Fig. 3a). The Ct values were significantly greater inthe nutrition only treatment than in the control andinsecticide treatment (p=0.05), whilst no significantdifferences were found between nutrition plus insecti-cide treatment and the other three treatments in May2012 (Fig. 3b).

PCR-DGGE analysis

The Shannon index of endophytic α-proteobacteriawas significantly greater in the control than in theinsecticide treatment while it was similar for the othertreatments (Fig. S1 and Table 5). The richness indexwas significantly (p=0.05) greater for leaves that hadreceived the nutrient treatment than the insecticide

treatment; it was intermediate for the control and nu-trition plus insecticide treatments. The evenness indexwas significantly (p=0.05) greater in leaves from thecontrol trees than in those from the other treatments.

According to the main axis in the RDA ordinationplot, the communities of endophytic α-proteobacteriadiffered considerably between the nutrient only treat-ment and the other treatments, while the communitiesglobally differed between the treatments without andwith insecticide applications along the secondary axis(Fig. 4). The percentage of the variance explained bythe environmental variables was 28.6 % along axis 1and 26.1 % along axis 2. The RDA graph illustratesthat leaf Ca (p=0.076) and Mn content (p=0.004),insecticide application (p=0.040) and nutrient manage-ment (p=0.056) were important environmental vari-ables controlling the endophytic α-proteobacteriacommunity structure. The Ct value of Las was posi-tively associated with nutrition management, and leafcontents of Ca, Mn, B, Zn, Mg, and Fe. In contrast, nosignificant association was observed between Ctvalues and leaf contents of N, P and K. Nutritionmanagement was positively correlated with leaf con-tents of Ca, Mn, B, Zn, Mg, and Fe whilst insecticidemanagement was negatively correlated with leaf Ncontent.

Leaf area and fresh weight

Analysis of leaf area (p=0.010) and fresh weight perunit leaf area (p=0.027) revealed significant interac-tions of insecticide and nutrient treatments (Fig. 5).The average leaf area was significantly larger in leavestreated with foliar nutrients than in control leaves(p=0.05), while the areas were intermediate for theinsecticide and insecticide plus nutrition treatments

Table 2 Soil pH, organic C and nutrient contents (mg kg−1) under different management regimes (nutritional, insecticide, nutritionalplus insecticide and untreated control) in a field experiment in Immokalee, South Florida

Treatment pH OrganicC

NOx-N NH3-N TKN P K Ca Mg

Control 7.7±0.2a 8.7±0.4a 13.8±3.0b 1.3±0.0b 1423.6±115.2ab 231.4±18.7 54.3±7.7b 1818±168.2 167.2±24.2

Insecticide 7.2±0.0b 6.4±0.4c 29.0±2.1b 1.0±0.1b 1309.9±52.4b 184.3±46.2 89.2±9.3b 1240±297.3 118.3±17.4

Nutrition 6.6±0.2c 7.6±0.2b 75.0±13.1a 1.9±0.2a 1727.4±171.6a 205.8±23.0 185.9±41.6a 1392±199.5 134.8±21.5

Nutrition +insecticide

7.2±0.1b 5.9±0.4c 32.6±4.1b 1.0±0.0b 1110.7±133.3b 180.1±14.5 82.3±5.7b 1591±132.8 133.6±12.2

Values (Means ± SE, n=4) followed by the same letter are not significantly different within columns (p=0.05)

Eur J Plant Pathol (2013) 137:727–742 735

(Fig. 5a). The leaf fresh weight per unit leaf area wasgreatest for the insecticide plus nutrition treatment,lower in the nutrition treatment, and least in the control(p=0.05) (Fig. 5b). There were no significant differ-ences between the insecticide only treatment and thenutrition only treatment or the control.

Discussion

Huanglongbing was first detected in the experimentalarea in March 2006 by the Florida Department ofAgriculture and Consumer Services, Division of PlantIndustry. The percentage of HLB positive trees in theexperimental area averaged 29.9±1.9 in November2008, and increased to 94.7±1.3 in May 2010(Stansly et al. 2013). In May 2012, 100 % of the treestested in this study were HLB positive, despite the factthat the Asian citrus psyllid populations were consis-tently lower on insecticide-treated trees than on treesnot treated with insecticide over the entire 4-year peri-od. The Ct values of Las generally decreased especiallyin the first 2 years of the experimental period, and thepercentage of HLB positive trees sharply increasedalmost at the same time. These results suggest thatinsecticide and nutrient applications could not slowthe spread of HLB once the trees were infected byLas (Chiyaka et al. 2012). The Ct values of Las weresignificantly increased by insecticides in January 2010but not significantly thereafter (Stansly et al. 2013),suggesting that the effects of insecticides on Las titreswere significant only in the initial phase of epidemicdevelopment. Overall, the insecticide treatments didnot reduce HLB spread compared to the untreatedcontrols. Indeed, the insecticides commonly used forthe control of the psyllid were not as effective asexpected (Ichinose et al. 2010), and even inducedinsecticide resistance in field populations of D. citriin Florida (Tiwari et al. 2011).

For each of the treatments, the Ct values of Lasvaried over time but generally decreased fromNovember 2008 to October 2011 (Fig. 3; Stanslyet al. 2013). The Ct values of Las decreased in treesreceiving nutrients compared to trees not receivingnutrients in the first 2 years. This was possibly aconsequence of the high HLB incidence and Las titresin these plots at the start of the experiment.Alternatively, the higher Las titres, even compared tothe control plots, could be related to a greaterT

able3

Leafnu

trient

contentsin

leaves

of‘Valencia’orange

underdifferentm

anagem

entregim

es(nutritio

nal,insecticide,nu

trition

alplus

insecticideandun

treatedcontrol)in

afield

experimentin

Immok

alee,S

outh

Florida

Treatment

N(g

kg−1)

P(g

kg−1)

K(g

kg−1)

Ca(g

kg−1)

Mg(g

kg−1)

Fe(m

gkg

−1)

Mn(m

gkg

−1)

Zn(m

gkg

−1)

Cu(m

gkg

−1)

B(m

gkg

−1)

Con

trol

28.33±

0.99

ab1.50

±0.71

13.33±

0.53

29.90±

1.11ab

2.98

±0.18

32.75±

1.54

5.17

±1.00b

12.55±

0.37

b7.20

±0.24a

40.52±

2.81

b

Insecticide

25.75±

0.31

b1.55

±0.03

13.53±

0.32

32.10±

1.73

ab3.00

±0.11

36.40±

1.51

7.12

±1.29b

11.62±

0.75

b7.43

±0.51a

39.47±

1.81

b

Nutritio

n28

.80±

0.74

a1.53

±0.03

13.25±

0.48

34.28±

1.40

a3.08

±0.13

32.83±

1.25

26.35±

3.68

a18

.86±

1.28

a5.94

±0.37b

51.59±

4.68

a

Nutritio

n+insecticide

27.18±

1.15

ab1.45

±0.05

12.88±

0.21

28.93±

1.98

b2.65

±0.23

32.18±

1.01

21.50±

0.72

a17

.21±

0.68

a6.28

±0.22ab

44.60±

0.78

ab

Values(M

eans

±SE,n

=4)

follo

wed

bythesameletterareno

tsignificantly

differentw

ithin

columns

(p=0.05

)

736 Eur J Plant Pathol (2013) 137:727–742

attractiveness to the psyllids of trees receiving foliarnutrients but no insecticides. Nevertheless, significantdecreases in Las titres were observed in the trees re-ceiving nutrients compared to trees not receiving nu-trients in October 2011 and May 2012 (Fig. 3),suggesting that nutrients could ultimately mitigateLas titres and the severity of HLB symptoms after a3-year application. The lack of any effects of nutrientapplications in other experiments could have been dueto the relatively short experimental period (Gottwaldet al. 2012).

Most severe HLB symptom expression was ob-served in untreated control trees (3.3±0.08), followedby trees receiving insecticide only (3.0±0.7). The leastsevere symptoms occurred in trees receiving nutrientsonly (2.7±0.8) or nutrients plus insecticides (2.8±0.8)(Stansly et al. 2013). The highest fruit yields wereconsistently obtained from trees receiving insecticidesplus nutrients, but in the final year (2012), trees

receiving only nutrients also yielded significantly morethan the control (Stansly et al. 2013). These resultssuggest that insecticide and nutrient applications couldattenuate HLB symptoms and maintain infected treesproductive for a longer term. Our previous studyshowed that symptoms were absent for several yearsafter detection of Las positive D. citri in the organicgrove, suggesting that balanced nutrition by organicmanagement may not slow down HLB transmissionbut postpone symptom expression (Shen et al. 2013).

The Ct value of Las was greatest in the controlwhilst least in the nutrition only treatment inNovember 2010 (Stansly et al. 2013). Meanwhile, soilpH was highest in the control and lowest in the nutri-tion only treatment, and was significantly correlatedwith the Ct values of Las (r=0.710, n=16, p<0.01). Incontrast, soil NOx-N and K were lowest in the controland highest in nutrition only treatment, and were sig-nificantly negatively correlated with the Ct values of

Table 4 Mean concentrations of salicylic acid and jasmonic acidin old and young leaves of ‘Valencia’ orange under differentmanagement regimes (nutritional, insecticide, nutritional plus

insecticide and untreated control) in a field experiment inImmokalee, South Florida

Treatment Salicylic acid (log ng g−1 FW) Jasmonic acid (log ng g−1 FW)

Old leaf Young leaf Old leaf Young leaf

Control 4.89±0.11b 4.54±0.00b 4.79±0.06c 5.34±0.34b

Insecticide 5.94±0.29a 6.14±0.04ab 5.69±0.39ab 5.09±0.06b

Nutrition 5.78±0.30a 6.42±0.88a 6.25±0.14a 7.82±0.78a

Nutrition + insecticide 6.15±0.11a 5.81±0.03ab 5.11±0.11bc 5.46±0.07b

Means ± SE (n=4) followed by the same letter are not significantly different within columns (p=0.05)

Nutrition Insecticide Insecticide+Nutrition Control

N0I1 M M N1I0 N1I0 N1I0 N1I0 N0I1 N0I1 N0I1 N1I1 N1I1 N1I1 N1I1 N0I0 N0I0 N0I0 N0I0 PC NC

bp2000155014001000750

500400300200

100

Fig. 2 Conventional polymerase chain reaction amplification ofthe 16S rDNA gene of Ca. Liberibacter asiaticus from ‘Valencia’orange midribs/petioles under different management regimes.M=100 bp Low Scale Ladder (Fisher Scientific, Pittsburgh, PA,

USA), N1I0 = nutritionals only, N0I1 = insecticides only, N1I1 =Nutrionals plus insecticides, N0I0 = untreated control, PC = positivecontrol (DNA from a tree that tested positively previously), NC =negative control (DNA from a tree that tested negatively previously)

Eur J Plant Pathol (2013) 137:727–742 737

Las (r=−0.588 and −0.535, n=16, p<0.05). The highestCt value of Las was in the nutrition only treatment withthe greatest leaf Mn, Zn and B in May 2012, indicatingthat leaf Mn (r=0.604, n=16, p<0.05), Zn (r=0.612,n=16, p<0.05) and B (r=0.609, n=16, p<0.05) werenegatively correlated with the Las titre. Lower Fe andZn were observed in symptomatic HLB infected leafsamples than in healthy leaf samples (Masaoka et al.2011). These results are consistent with the RDA re-sults that leaf Ca, Mn, Zn and B were positively corre-lated with the Ct values of Las, suggesting that ‘Boydnutrient solution’ application could improve the citrus

nutrition status and thus lower the Las titres.Furthermore, significant increases in leaf size and cropyields were observed in the nutrition only and/or nu-trition plus insecticide treatments in comparison withthe control (Fig. 5; Stansly et al. 2013). In contrast,significantly lower leaf Cu was found in trees receivingnutrients compared to trees not receiving nutrients,possibly due to addition of copper hydroxide with theinsecticides (Qureshi and Stansly 2009).

Las belongs to the α-proteobacteria which is one ofthe largest and most extensively studied groups ofbacteria. Foliar nutrient application including salicylic

Fig. 3 Mean Ct values ofCa. Liberibacter asiaticusfrom ‘Valencia’ orange mid-ribs/petioles under differentmanagement regimes in Oc-tober 2011 (a) andMay 2012(b). Vertical lines are stan-dard errors (n≥16). Meanswith the same letters do notdiffer significantly at p=0.05

738 Eur J Plant Pathol (2013) 137:727–742

acid is assumed to change the physiology and micro-bial niches in citrus trees, which may alter the compe-t i t ion among Las and other endophyt ic α -proteobacteria. Our results showed that nutrients hadno significant effects on the Shannon diversity andevenness indices of endophytic α-proteobacteria, al-though the species richness was highest after a 4-yearapplication of ‘Boyd nutrient solution’. However, thistreatment did shift the community structure of endo-phytic α-proteobacteria according to the primary axisin the RDA analysis (p=0.056). However, insecticideapplication was not positively correlated with the Ctvalues of Las as controlling environmental variable ofendophytic α-proteobacteria. Microbial communitiesand plant pathogens are strongly affected by plant

defence responses to various stress factors like insectfeeding, phytopathogen infection, and others. Theseinduced systemic defence responses are mediated byplant hormones like SA and JA (Klessig et al. 2000;Loake and Grant 2007; Thaler et al. 2012). SA isassociated with resistance against biotrophic pathogensand some phloem feeding insects, while JAwith resis-tance against necrotrophic pathogens, some phloemfeeding insects and chewing herbivores (Thaler et al.2012). The pathways of both SA and JA are activatedin Las-infected trees (Martinelli et al. 2012). In ourstudy, SA in old leaves was significantly increasedafter nutrient applications, and was positively correlat-ed with the Ct values of Las in October 2011 (r=0.592,n=16, p<0.05). SA in young leaves and JA in both oldand young leaves were highest in trees receiving nutri-ents only. This is consistent with lower pathogen titresin both nutrition treatments than in the control inOctober 2011, and in the nutrition only treatment inMay 2012, suggesting that long-term nutrient applica-tions could modulate plant defence against HLB.

So far, effects of nutritional treatments on symptomexpression and the spread of HLB were poorly under-stood and a topic of debate in Florida (Gottwald et al.2012). The use of enhanced nutritional programs didnot sustain tree health, yield, or fruit quality of Las-infected HLB-symptomatic trees in a 2-year field ex-periment (Gottwald et al. 2012). In another study how-ever, application of Zn or Cu ions in combination withCa delayed HLB incidence and severity, and increasedfruit production and total soluble solid content (Ahmadet al. 2011). Micronutrient application together withother cultural practices had limited effects on HLBdisease progress and spread but prolonged grove pro-ductivity (Xia et al. 2011). Nutritional treatments werealso thought to give stress relief to HLB infected plants

Table 5 Shannon, richness and evenness indices of endophyticα-proteobacteria in leaves of ‘Valencia’ orange under differentmanagement regimes (nutritional, insecticide, nutritional plus

insecticide and untreated control) in a field experiment inImmokalee, South Florida

Treatment Shannon index Richness index Evenness index

Control 3.023±0.070a 26±1ab 0.926±0.009a

Insecticide 2.789±0.036b 23±1b 0.887±0.005b

Nutrition 2.919±0.052ab 28±1a 0.874±0.013b

Nutrition + insecticide 2.847±0.070ab 26±1ab 0.877±0.014b

Values (Means ± SE, n=4) followed by the same letter are not significantly different within columns (p=0.05)

Fig. 4 Redundancy analysis (RDA) of endophyticα-proteobacteriacommunities in leaves of ‘Valencia’ orange under different manage-ment regimes. N1I0 = nutritionals only, N0I1 = insecticides only,N1I1 = Nutrionals plus insecticides, N0I0 = untreated control

Eur J Plant Pathol (2013) 137:727–742 739

(Razi et al. 2011). Our study clarified that foliar nutri-ents could induce plant resistance, mitigate Las titresand expression of HLB symptoms, and increase leafsize after at least 3 years of application. Our resultscould provide a better understanding of the importanceof long-term foliar nutrient application in maintainingthe productivity of HLB-infected trees.

In conclusion, long-term insecticide applicationscan decrease the diversity of endophytic bacteria incitrus leaves in comparison with a control, but do notaffect Las titres once most of the trees are infected.Foliar nutrients plus salicylic acid and phosphite caninduce systemic resistance and/or systemic acquired

resistance, mitigate Las titres and symptom expression,and improve foliar growth after at least 3 years ofapplication. A combination of both insecticides andfoliar nutrients may be the best agricultural practicein areas where relatively few trees are infected withLas, while nutrient treatments may be more effectivelater. However, it remains to be investigated whichcomponents of the Boyd program induce ISR or SARand what their effects are on the physiology of the tree.It is also not known how long the effects of the treat-ments last and how frequently they need to be appliedin order to sustain the productivity of the trees. Finally,it is not known if young trees planted to replace

Fig. 5 Leaf area (a) and leaffresh weight per unit of leafarea (b) of ‘Valencia’ orangeunder different managementregimes (N1I0 = nutritionalsonly, N0I1 = insecticides only,N1I1 = Nutrionals plus insec-ticides, N0I0 = untreated con-trol) in a field experiment inImmokalee, South Florida.Bars represent standard er-rors. Vertical lines are stan-dard errors (n=4). Means withthe same letters do not differsignificantly at p=0.05

740 Eur J Plant Pathol (2013) 137:727–742

declined trees can be kept healthy under a regime ofnutritionals including resistance inducing agents.

Acknowledgments The authors would like to thank StephanieShea Teems for her work with real-time PCR analyses of leafsamples at Southwest Florida Research and Education Center,University of Florida. We also like to thank Debbie Jones of theDivision of Plant Industries (DPI) for providing negative and pos-itive control samples of citrus leaves and for teaching some of us thereal-time qPCR techniques used at the DPI in Gainesville. We aregrateful to Ganyu Gu, Hongling Er and Christinah Chiyaka for theirhelp with sampling. We thank Ellen Dickstein for organizing soiland plant nutrient analyses at the Soil Analysis lab of the Universityof Florida. Funding for this research was provided by the EmergingPathogens Institute and the Smallwood Foundation.

References

Ahmad,K., Sijam,K., Hashim,H., Rosli, Z., &Abdu,A. (2011). Fieldassessment of calcium, copper and zinc ions on plant recoveryand disease severity following infection of Huanglongbing(HLB) disease. African Journal of Microbiology Research, 5,4967–4979.

Birkemeyer, C., Kolasa, A., & Kopka, J. (2003). Comprehensivechemical derivatization for gas chromatography–massspectrometry-based multi-targeted profiling of the majorphytohormones. Journal of Chromatography, A, 993, 89–102.

Bonani, J. P., Fereres, A., Garzo, E., Miranda, M. P., Appezzato-Da-Gloria, B., & Lopes, J. R. S. (2010). Characterization ofelectrical penetration graphs of the Asian citrus psyllid,Diaphorina citri, in sweet orange seedlings. EntomologiaExperimentalis et Applicata, 134, 35–49.

Bové, J. M. (2006). Huanglongbing: a destructive, newly-emerging,century-old disease of citrus. Journal of Plant Pathology, 88,7–37.

Chiyaka, C., Singer, B. H., Halbert, S. E., Morris, J. G., & vanBruggen, A. H. C. (2012). Modeling huanglongbing trans-mission within a citrus tree. Proceedings of the NationalAcademy of Sciences of the United States of America, 109,12213–12218.

Coletta-Filho, H. D., Carlos, E. F., Alves, K. C. S., Pereira, M. A.R., Boscariol-Camargo, R. L., de Souza, A. A., et al.(2010). In planta multiplication and graft transmission ofCandidatus Liberibacter asiaticus revealed by real-timePCR. European Journal of Plant Pathology, 126, 53–60.

Dordas, C. (2008). Role of nutrients in controlling plant diseasesin sustainable agriculture. A review. Agronomy forSustainable Development, 28, 33–46.

Etxeberria, E., Gonzalez, P., Achor, D., & Albrigo, G. (2009).Anatomical distribution of abnormally high levels of starchin HLB-affected Valencia orange trees. Physiological andMolecular Plant Pathology, 74, 76–83.

Folimonova, S. Y., Robertson, C. J., Garnsey, S. M., Gowda, S.,& Dawson, W. O. (2009). Examination of the responses ofdifferent genotypes of citrus to Huanglongbing (CitrusGreening) under different conditions. Phytopathology, 99,1346–1354.

Francis, M. I., Redondo, A., Burns, J. K., & Graham, J. H. (2009).Soil application of imidacloprid and related SAR-inducingcompounds produces effective and persistent control of citruscanker. European Journal of Plant Pathology, 124, 283–292.

Gomes, N. C. M., Heuer, H., Schönfeld, J., Costa, R., Mendonça-Hagler, L., & Smalla, K. (2001). Bacterial diversity of therhizosphere of maize (Zea mays) grown in tropical soil stud-ied by temperature gradient gel electrophoresis. Plant andSoil, 232, 167–180.

Gottwald, T. R. (2010). Current epidemiological understanding ofcitrus Huanglongbing. Annual Review of Phytopathology, 48,119–139.

Gottwald, T. R., da Graça, J. V., Bassanezi, R. B. (2007). CitrusHuanglongbing: the pathogen and its impact. Online. PlantHealth Progress doi:10.1094/PHP-2007-0906-01-RV.

Gottwald, T. R., Graham, J. H., Irey, M. S., McCollum, T. G., &Wood, B. W. (2012). Inconsequential effect of nutritionaltreatments on huanglongbing control, fruit quality, bacterialtiter and disease progress. Crop Protection, 36, 73–82.

Gu, G., Cevallos-Cevallos, J. M., Vallad, G. E., & van Bruggen,A. H. C. (2013). Organically managed soils reduce internalcolonization of tomato plants by Salmonella entericaserovar Typhimurium. Phytopathology, 103, 381–388.

Halbert, S. E. (2005). The discovery of huanglongbing inFlorida. In: Proceedings of the 2nd International CitrusCanker and Huanglongbing Research Workshop. Orlando,FL: Florida Citrus Mutual.

Halbert, S. E., & Manjunath, K. L. (2004). Asian citrus psyllids(Sternorrhyncha: Psyllidae) and greening disease of citrus:a literature review and assessment of risk in Florida. FloridaEntomologist, 87, 330–353.

Hall, D. G., & Nguyen, R. (2010). Toxicity of pesticides toTamarixia radiata, a parasitoid of the Asian citrus psyllid.Biocontrol, 55, 601–611.

Heuer, H., Krsek, M., Baker, P., Smalla, K., & Wellington, E. M.H. (1997). Analysis of actinomycete communities by specificamplification of 16S rDNA and gel electrophoretic separa-tion in denaturing gradients. Applied and EnvironmentalMicrobiology, 63, 3233–3241.

Ichinose, K., Miyazi, K., Matsuhira, K., Yasuda, K., Sadoyama,Y., Do, H. T., et al. (2010). Unreliable pesticide control of thevector psyllidDiaphorina citri (Hemiptera: Psyllidae) for thereduction of microorganism disease transmission. Journal ofEnvironmental Science and Health. Part. B, 45, 466–472.

Inoue, H., Ohnishi, J., Ito, T., Tomimura, K., Miyata, S.,Iwanami, T., et al. (2009). Enhanced proliferation and effi-cient transmission of Candidatus Liberibacter asiaticus byadult Diaphorina citri after acquisition feeding in thenymphal stage. Annals of Applied Biology, 155, 29–36.

Jagoueix, S., Bové, J.M., &Garnier,M. (1996). PCRdetection of thetwo ‘Candidatus’ liberobacter species associated with greeningdisease of citrus.Molecular and Cellular Probes, 10, 43–50.

Kim, J. S., Sagaram, U. S., Burns, J. K., Li, J. L., & Wang, N.(2009). Response of sweet orange (Citrus sinensis) toCandidatus Liberibacter asiaticus infection: microscopyand microarray analyses. Phytopathology, 99, 50–57.

Klessig, D. F., Durner, J., Noad, R., Navarre, D. A., Wendehenne,D., Kumar, D., et al. (2000). Nitric oxide and salicylic acidsignaling in plant defense. Proceedings of the NationalAcademy of Sciences of the United States of America, 97,8849–8855.

Eur J Plant Pathol (2013) 137:727–742 741

Li, W., Hartung, J. S., & Levy, L. (2006). Quantitative real-timePCR for detection and identification of CandidatusLiberibacter species associated with citrus huanglongbing.Journal of Microbiological Methods, 66, 104–115.

Loake, G., & Grant, M. (2007). Salicylic acid in plantdefence—the players and protagonists. Current Opinion inPlant Biology, 10, 466–472.

Mangomere, T. O., Obukosia, S. D., Mutitu, E., Ngichabe, C.,Olubayo, F., Shibairo, S. (2009). Molecular characterization ofcandidatus Liberibacter species/strains causing huanglongbingdisease of citrus in Kenya. Electronic Journal of Biotechnology,12, 1–14. http://www.ejbiotechnology.info/content/vol12/issue2/full/2/.

Martinelli, F., Uratsu, S. L., Albrecht, U., Reagan, R. L., Phu,M. L.,Britton,M., et al. (2012). Transcriptome profiling of citrus fruitresponse to huanglongbing disease. PLoS ONE, 7, e38039.doi:10.1371/journal.pone.0038039.

Masaoka, Y., Holford, P., Beattie, A., Iwanami, T., Pustika, A.,Subandiyah, S., et al. (2011). Lower concentrations of microel-ements in leaves of citrus infectedwith ‘Candidatus Liberibacterasiaticus’. Japan Agricultural Research Quarterly, 45, 269–275.

Musetti, R., Polizzotto, R., Grisan, S., Martini, M., Borselli, S.,Carraro, L., et al. (2007). Effects induced by fungal endo-phytes in Catharanthus roseus tissues infected byphytoplasmas. Bulletin of Insectology, 60, 293–294.

Mylavarapu, R. S., & Moon, D. L. (2007). UF/IFAS Extension SoilTesting Laboratory (ESTL) Analytical Procedures andTraining Manual Circular 1248. Gainesville: Soil and WaterScience Department, Florida Cooperative Extension Service,Institute of Food and Agricultural Sciences, University ofFlorida.

Patt, J. M., & Sétamou, M. (2010). Responses of the Asian citruspsyllid to volatiles emitted by the flushing shoots of itsRutaceous host plants. Environmental Entomology, 39,618–624.

Pelz-Stelinski, K. S., Brlansky, R. H., Ebert, T. A., & Rogers, M. E.(2010). Transmission parameters for Candidatus Liberibacterasiaticus by Asian citrus psyllid (Hemiptera: Psyllidae).Journal of Economic Entomology, 103, 1531–1541.

Pereira, F. M. V., & Pereira Milori, D. M. B. (2009). Investigation ofthe stages of citrus greening disease using micro synchrotronradiation X-ray fluorescence in association with chemometrictools. Journal of Analytical Atomic Spectrometry, 25, 351–355.

Qureshi, J. A., & Stansly, P. A. (2009). Insecticidal control of Asiancitrus psyllid Diaphorina citri (Hemiptera: Psyllidae).Proceedings of the Florida State Horticultural Society, 172.

Qureshi, J. A., & Stansly, P. A. (2010). Dormant season foliarsprays of broad spectrum insecticides: an effective compo-nent of integrated management for Diaphorina citri(Hemiptera: Psyllidae) in citrus orchards. Crop Protection,29, 860–866.

Razi, M. F., Khan, I. A., & Jaskani, M. J. (2011). Citrus plantnutritional profile in relation to Huanglongbing prevalencein Pakistan. Pakistan Journal of Agricultural Sciences, 48,299–304.

Rogers, M. E., & Ebert, T. A. (2009). Seasonality of psyllidscarrying the HLB pathogen. Citrus Industry Update,University of Florida. October-November 2009, 4–5.

Shen,W., Halbert, S. E., Dickstein, E., Manjunath, K. L., Shimwela,M. M., & van Bruggen, A. H. C. (2013). Occurrence and in-

grove distribution of citrus huanglongbing in North CentralFlorida. Journal of Plant Pathology, 95, 361–371.

Sigler, W. V., Miniaci, C., & Zeyer, J. (2004). Electrophoresistime impacts the denaturing gradient gel electrophoresis-based assessment of bacterial community structure. Journalof Microbiological Methods, 57, 17–22.

Stansly, P. A., Arevalo, H. A., Rouse, R. E. (2011). Role of nutritionaland insecticidal treatments inmitigation ofHLB:main effects andinteractions. Page 188 in. Proceedings of the second InternationalResearch Conference on Huanglongbing (IRCHLB) at Orlando,Florida, January 2011. Plant Management Network. 292 pp.http://www.plantmanagementnetwork.org/proceedings/irchlb/2011/presentations/IRCHLB_2011_FullDocument.pdf.

Stansly, P. A., Arevalo, H. A., Qureshi, J. A., Jones, M. M.,Hendricks, K., Roberts, P. D., et al. (2013). Vector controland foliar nutrition to maintain economic sustainablility ofbearing citrus in Florida groves affected by huanglongbing.Pest Management Science. doi:10.1002/ps.3577.

Stokstad, E. (2012). Dread citrus disease turns up in California,Texas. Science, 336, 283–284.

Teixeira, D. C., Saillard, C., Couture, C., Martins, E. C., Wulff,N. A., Eveillard-Jagoueix, S., et al. (2008). Distribution andquantification of Candidatus Liberibacter americanus,agent of huanglongbing disease of citrus in Sao PauloState, Brazil, in leaves of an affected sweet orange tree asdetermined by PCR. Molecular and Cellular Probes, 22,139–150.

Thaler, J. S., Humphrey, P. T., & Whiteman, N. K. (2012).Evolution of jasmonate and salicylate signal cross-talk.Trends in Plant Science, 17, 260–270.

Tiwari, S., Mann, R. S., Rogers, M. E., & Stelinski, L. L. (2011).Insecticide resistance in field populations of Asian citruspsyllid in Florida. Pest Management Science, 67, 1258–1268.

Tuma, R. S., Beaudet, M. P., Jin, X., Jones, L. J., Cheung, C. Y.,Yue, S., et al. (1999). Characterization of SYBR Goldnucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators. Analytical Biochemistry,268, 278–288.

Wakgari, W. M., & Giliomee, J. H. (2003). Natural enemies ofthree mealybug species (Hemiptera: Pseudococcidae) foundon citrus and effects of some insecticides on the mealybugparasitoid Coccidoxenoides peregrinus (Hymenoptera:Encyrtidae) in South Africa. Bulletin of EntomologicalResearch, 93, 243–254.

Watanabe, K., Kodama, Y., & Harayama, S. (2001). Design andevaluation of PCR primers to amplify bacterial 16S ribo-somal DNA fragments used for community fingerprinting.Journal of Microbiological Methods, 44, 253–262.

Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J.(1991). 16S ribosomal DNA amplification for phylogeneticstudy. Journal of Bacteriology, 173, 697–703.

Xia, Y., Ouyang, G., Sequeira, R. A., Takeuchi, Y., Baez, I., &Chen, J. (2011). A review of huanglongbing (citrus green-ing) management in citrus using nutritional approaches inChina. Plant Health Progress. doi:10.1094/PHP-2010-1003-01-RV.

Xue, D., Yao, H. Y., & Huang, C. Y. (2006). Microbial biomass,N mineralization and nitrification, enzyme activities, andmicrobial community diversity in tea orchard soils. Plantand Soil, 288, 319–331.

742 Eur J Plant Pathol (2013) 137:727–742