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Crop Protection 84 (2016) 150e158

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Crop Protection

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Mitigating citrus huanglongbing via effective application ofantimicrobial compounds and thermotherapy

Chuanyu Yang a, b, d, Charles A. Powell b, Yongping Duan c, Robert G. Shatters c,Youjian Lin b, Muqing Zhang a, b, d, *

a State Key Lab for Conversation and Utilization Subtropical Aro-Biological Resources, Guangxi University, Nanning, Guangxi, 530005, Chinab IRREC-IFAS, University of Florida, Fort Pierce, FL, 34945, USAc USHRL, USDA-ARS, Fort Pierce, FL, 34945, USAd College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

a r t i c l e i n f o

Article history:Received 24 December 2015Received in revised form13 March 2016Accepted 15 March 2016

Keywords:HuanglongbingChemoethermotherapyDelivery method

* Corresponding author. State Key Lab for Convetropical Aro-Biological Resources, Guangxi UniversityChina.

E-mail address: mqzhang@ufl.edu (M. Zhang).

http://dx.doi.org/10.1016/j.cropro.2016.03.0130261-2194/© 2016 Elsevier Ltd. All rights reserved.

a b s t r a c t

Huanglongbing (HLB) is a serious citrus disease that threatens the citrus industry worldwide. HLB is asystemic, infectious disease and the putative causal bacterium Candidatus liberibacter asiaticus (Las)resides in citrus phloem. In this study, the effects of heat treatment, chemical formulations, and appli-cation methods on bacterial titer in the citrus host, measured by quantitative PCR, were evaluated.Thermotherapy at 45 �C (Ct ¼ 30.79 ± 6.26) was more effective against Las than thermotherapy at 40 �C(Ct ¼ 28.72 ± 6.00) or 42 �C (Ct ¼ 28.02 ± 6.25), and enhanced the delivery efficiency of effectivechemical compounds into the citrus phloem. Ampicillin sodium (Amp) and a combination of actidioneand validoxylamine A (Act þ VA) were the most effective chemical formulations against Las. When Ampor Act þ VA treatment was combined with thermotherapy, the area under disease progress curvestandardized (AUDPCs) was significantly lower than the thermotherapy treatments alone. The Las titer ofAmp and Act þ VA treatment applied by bark paint were significantly reduced, compared with those ofAmp or Act þ VA applied by root drench. Therefore, we propose a chemoethermotherapy strategycoupled with a bark paint application method for disease control in HLB-infected citrus plants.

© 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Huanglongbing (HLB), or citrus greening, is the most devas-tating disease of citrus. HLB has spread tomost citrus growing areasworldwide, including the Americas, Asia, and Africa (McClean andOberholzer, 1965), causing significant declines in both the pro-duction and profitability of the industry (Gottwald, 2010). In Floridaalone, HLB cost the state economy an estimated $3.63 billion in lostrevenue as well as 6611 lost jobs by reducing orange juice pro-duction since 2006 (Hodges, 2012).

HLB is caused by three species of uncultured, phloem-restrictedproteobacteria, Candidatus liberibacter asiaticus, C. liberibacteramericanus, and C. liberibacter africanus (Bove, 2006; Bove andAyres, 2007; Jagoueix et al., 1994), and is transmitted by either

rsation and Utilization Sub-, Nanning, Guangxi, 530005,

Diaphorina citri Kuwayama or Trioza erytreae Del Guercio(Gottwald, 2007). There are no effective strategies to control HLB inthe field in Florida due to the lack of resistant citrus cultivars, aswell as rapid disease spread due to our inability to suppress thepsyllid vectors (Lopes and Frare, 2008; Nariani et al., 1973). ’

C. liberibacter asiaticus' (Las), is a heat-tolerant bacterium and canthrive under high temperature conditions extending to 35 �C(Lopes et al., 2009). Previous studies demonstrated that HLBsymptoms on infected budwood and seedlings were eliminatedafter plant materials were exposed to moist hot air at 49 �Ce50 �Cfor 50e60 min or, alternatively, continuously treated with hotwater (at 47 �Ce50 �C, 6e12 min per exposure) three times per dayfor three days. Leaves remained asymptomatic for a minimum of 29months after treatment (Lo, 1983; Weiwen et al., 1981). Recently,continuous thermal exposure (at 40 �Ce42 �C) for a minimum of48 h was found to significantly reduce the titer of Las bacterium inHLB-affected citrus tissues (Hoffman et al., 2013). However, sinceHLB is a systemic disease, eliminating the bacterium from the entirecitrus plant, including roots and branches, is essential to achieve

C. Yang et al. / Crop Protection 84 (2016) 150e158 151

effective control. Elimination of Las in HLB-affected citrus plantsmay be achieved by thermotherapy combined with chemotherapy.

In previous studies, several antimicrobial compounds wereidentified that have potential activity against Las (Zhang et al., 2010,2011, 2012, 2014a). Both sulfadimethoxine sodium and sulfathia-zole sodium have been shown to be effective against Las (Zhanget al., 2014a). However, when these chemical compounds wereapplied to HLB-affected citrus trees by root drench without heattreatment, the Las titer was unaffected. This finding may have beendue to low absorption and utilization of the chemical compounds(unpublished). HLB-affected citrus treated with heat displayedsignificantly improved vigorous growth (Hoffman et al., 2013). Wehypothesize that chemotherapy combined with thermotherapymay increase the delivery and therapeutic efficiency of the chem-ical compounds in treatments combatting the Las bacterium ininfected citrus plants.

When ampicillin (Amp) solution was applied onto HLB-affectedcitrus by foliar spray, Las was still detected 240 days after the initialtreatment, indicating that the leaf cuticle acts as a barrier limitinguptake of the ampicillin (Yang et al., 2015). There are very few re-ports measuring uptake and delivery efficiency of chemicalsapplied to citrus plants by root drench or bark absorption. Todevelop effective strategies against citrus HLB, we combined ther-motherapy using different temperatures coupled with chemo-therapy using either bark paint or root drench application ininfected citrus seedlings.

2. Materials and methods

2.1. Materials

2.1.1. Plant materialsTwo-year-old healthy grapefruit (Citrus paradisi Macfad) seed-

lings were graft-inoculated with HLB-affected lemon (Citrus limonBurm.f.) scions. Plants were subsequently maintained in an insect-free greenhouse located at the Indian River Research and EducationCenter, the University of Florida, Fort Pierce, FL USA, and used forthe chemoethermotherapy studies. Typical HLB symptoms such asblotchy mottles appeared on the leaves of the inoculated seedlings10 months after the graft-inoculation. The seedlings with typicalHLB symptoms tested positive for the presence of Ca. L. asiaticusbacteria using quantitative real-time polymerase chain reaction(qPCR) with Ca. L. asiaticus-specific primers (HLBas, HLBr, andHLBp) (Li et al., 2006).

2.1.2. Chemical concentration and preparationThe chemical compounds and their concentrations used in the

study were based on our previous study (Zhang et al., 2014a)(Table 1). Ampicillin sodium (Amp) was purchased from FisherScientific (Waltham, MA, USA), Nicotine (Nic), Actidione (Act), andZinc sulfate (Zn) were purchased from SigmaeAldrich, Co. (St.Louis, MO, USA). Zhongshengmycin and Validoxylamine A (VA)were purchased from Fujian Kaili Bio-product Co., Ltd (Fuzhou,

Table 1Chemical formulations and their concentrations used for control of HLB by three applica

ID Chemical compounds Working conc.(

Grafting assay

Amp Ampicillin sodium 500Nic Nicotine 200Zn Zinc sulfate 200Nic þ Zn Nicotine þ Zinc sulfate 200 (Nic) þ 200Act þ VA Actidione þ Validoxylamine A 10 (Act) þ 100

Fujian, China). Solutions were freshly prepared before applicationby dissolving inwater as listed in Table 1. Water alone was includedin these experiments as a non-treatment control.

2.2. Methods

2.2.1. ChemoethermotherapyA splitesplit plot experimental designwas conductedwith three

replicates. The entire plot was distinguished by applicationmethods (bark paint and root drench). The antimicrobial chemo-therapeutic compounds (Amp, Nic, Zn, Nic þ Zn, Act þ VA or tapwater) were considered as the split plot as well as the temperaturesused in thermotherapy (40 �C, 42 �C, and 45 �C). A total of 108 2-year-old potted plants that were PCR-positive and had typicalHLB symptoms were used for these studies. For thermotherapy,HLB-infected citrus seedlings were exposed to a temperatureregime, consisting of 40 �C, 42 �C or 45 �C, for 8 h per day for oneweek, in a growth chamber. The temperature regimes of the growthchambers were set up as follows: relative humidity was a constant85%; midnight through 8 am, dark, temperature 25 �C; 8 ame10am, light, 35 �C; 10 am to 6 pm, light, at 40 �C, 42 �C, or 45 �C; 6pme8 pm, light, 35 �C; 8 pm through midnight, dark, 25 �C. Afterthermotherapy, chemotherapeutic treatments were conducted. TheHLB-affected potted plants were treated twice per week for twoweek with the solutions listed in Table 1 by root drench or barkpaint. Tap water was used as a non-treatment control (CK). Beforebark application, the trunk of HLB-affected potted plants was gentlyscrapedwith a knife andwrappedwith a 24� 12 cm sponge (Fisherscientific, Waltham, MA, USA), then a total of 500 ml of chemicalsolution (Table 1) was poured into the sponge to completely satu-rate it. Seedlings were kept in the same greenhouse at room tem-perature at IRREC, UF. Five leaf samples were collected at day0 (before treatment), day 60, and day 180 after the initial treatmentfor qPCR testing.

2.2.2. Graft-based evaluation assayGraft-based chemotherapy (Zhang et al., 2012) was used to

evaluate the effectiveness of chemicals against Las and theirphytotoxicity to citrus. Briefly, HLB-infected budsticks werecollected from symptomatic rough lemons (C. limon 'lemon#76') atthe USDA-ARS-USHRL farm in Fort Pierce, FL and confirmed posi-tive for Las by real-time qPCR (Li et al., 2006; Zhang et al., 2012). Thebudsticks were soaked in one of the chemical treatments listed inTable 1 (30 scions per treatment per concentration) overnight in afume hood under ventilation and continuous fluorescent light. Eachsoaked budstick was cut into a 2-bud scion and grafted onto indi-vidual 2-year-old HLB-free grapefruit (C. paradisi 'Duncan') root-stock seedlings and the graft covered with plastic tape for threeweeks. To improve scion growth, new flush from the rootstocks wasremoved immediately after grafting. Grafted plants were main-tained at 25 �C ± 2 �C under the shade in an insect-proofgreenhouse.

The effectiveness of the chemical treatments against Las was

tions (grafting assay, root drench, and bark absorption).

mg/L)

Root drench Bark paint

500 3000200 1000200 1000

(Zn) 200 (Nic) þ 200 (Zn) 1000 (Nic) þ 1000 (Zn)(VA) 10 (Act) þ 100 (VA) 50 (Act) þ 1000 (VA)

Table 2Variance analysis of Ct values of HLB-affected citrus treated with various applicationmethods, different chemical formulations and different temperature treatments.

Source df F value P value

Application methods(A) 1 50.668 0.0001Chemical formulation(C) 5 27.832 0.0001Temperature(T) 2 19.1316 0.0001A � C 5 19.0294 0.0001A � T 2 10.3682 0.0001C � T 10 2.828 0.0025A � C � T 10 7.2956 0.0001

C. Yang et al. / Crop Protection 84 (2016) 150e158152

determined by measuring the titer of Las in both the grafted scionand the rootstock using qPCR. Briefly, five leaves were randomlysampled from the scion (rough lemon) and rootstock (grapefruit)120 days after grafting, and again 180 days after grafting. The leaveswere washed in tap water and then rinsed three times with sterilewater. Midribs were excised, frozen in liquid nitrogen and storedat �80 �C for further processing. The midribs of five leaves fromeach sample were pooled together and used for DNA extraction andsubsequent qPCR analysis as described (Li et al., 2006; Zhang et al.,2012). After grafting, some bud scions died, others remained greenbut did not flush and others produced new flush. The percentage ofscion survival was calculated from the number of scions that sur-vived (remained green or produced flush) versus the total numberof grafted scions. The percentage of scion growth was calculatedfrom the number of scions that had newly emerging leaves orflushes versus the total number of grafted scions. The scion infec-tion percentage was calculated from the number of scions withthreshold cycle (Ct) values lower than 36.0 versus the total graftedscion number. The Las transmission percentagewas calculated fromthe number of rootstocks that tested Las positive by qPCR with Ctvalues less than 36.0.

2.2.3. Genomic DNA extraction and qPCR analysisMidribs were separated from the leaf samples and cut into

pieces 1.0e2.0 mm in size. DNA was extracted from 0.1 g of themidrib tissue using a Qiagen DNeasy Plant Mini Kit (Qiagen,Valencia, CA) following the procedure provided the manufacturer.The qPCR was performed with primers and probes (HLBas, HLBrand HLBp) that were specific for Las using an ABI PRISM 7500sequence detection system (Applied Biosystems, Foster City, CA) (Liet al., 2006). The total PCR reaction volume was 20 ml for each re-action. Each reaction included: 300 nM of each primer HLBas andHLBr, 150 nM of target probe HLBp, and 1 � TaqMan qPCR Mix(Applied Biosystems). The cycling conditions were 95 �C for 20 sfollowed by 40 cycles at 95 �C for 3 s and 60 �C for 30 s. All reactionswere performed in triplicate and each run contained one negative(DNA from a Las-infection free citrus plant) and one positive (DNAfrom a Las-infected citrus plant) sample as controls.

2.3. Data analysis

The resulting Ct values were converted to the estimated bacte-rial titers using the grand universal regression equationY ¼ 13.82�0.2866X, where Y is the estimated log concentration oftemplate and X is the Ct values from qPCR, as described by Li et al.(Li et al., 2006). The area under the disease progress curve stan-dardized (AUDPCs) per treatment was calculated using the esti-mated Las titer (Log unit per gram of plant tissue) at day 0, day 60and day 180 by software Origin 8.0. Disease severity was consideredto increase as bacterial titer increased. Disease severity wasassigned a grade from 0 to 4, where 0 ¼ Ct value � 36.0, 1 ¼ 32.0 Ctvalue< 36, 2¼ 28� Ct value< 32, 3¼ 24� Ct value< 28, and 4¼ Ctvalue < 24. The disease index (DI) for each sample day was calcu-lated as:

DI ¼ Sum of all numerical gradesTotal number of plants counted�maximum grade

� 100:

The therapeutic efficiency (TE) was calculated as:

TE ¼ 1� DI180=DI0:

The DI180 was the disease index of the tested seedlings at day180 after the initial treatment. The DI0 was the disease index of thetested plants before initial treatment.

The data were analyzed as a generalized linear mixed modelusing the SAS procedure GLIMMIX 8.1. The whole block and splitplot factors were treated as fixed effects, and the replication and itsinteraction with the whole-plot factor were treated as random ef-fects. Differences among treatments were determined with theLINES option of LSMEANS.

3. Results

3.1. Effects of application methods on the uptake and delivery ofchemicals in chemoethermotherapy for HLB control

Variance analysis showed that there were significant differencesin Las titers in fix model between application methods (P¼ 0.0001)(Table 2). Using bark paint applications (Ct¼ 30.53± 6.11) to deliverchemicals significantly lowered Las titer compared to root drenchapplication (Ct ¼ 27.82 ± 6.14) (P ¼ 0.0001).

The effect of interaction of application methods and chemicalformulations on Las titer also was significant (P¼ 0.0001) (Table 2).Using bark paint application for delivering Amp (P ¼ 0.0215), Nic(P¼ 0.0311) and Actþ VA (P¼ 0.0001), significantly suppressed Lascompared to using root drench application (Fig.1A). However, therewere no significant Las titer differences between bark paint androot drench application using Zn (P¼ 0.8765), Nicþ Zn (P¼ 0.1626)or tap water (CK) (P ¼ 0.4896) treatment (Fig. 1A). The Amp andActþ VA treatment via bark paint application had lower Area underdisease progress standard (AUDPCs) and higher therapeutic effi-ciency (TE) than root drench application (Fig. 1B and C).

3.2. Effects of chemical treatments on Las titers

Analysis of variance indicated that there were significant dif-ferences in Las titers between chemical treatments (P ¼ 0.0001)(Table 2). The HLB-infected citrus treated with Amp (P ¼ 0.0001)and Act þ VA (P ¼ 0.0384) showed a significant reduction in Lastiter at 60 and 180 days after initial treatment (Fig. 2). When HLB-infected citrus were treated with other chemical formulations suchas Nic, Zn, Nic þ Zn, as well as tap water (CK), there was non-significant influence on suppression of Las titer (Fig. 2).

The effect of the interaction of chemical formulation and tem-perature treatment was also significant (P ¼ 0.0001) (Table 2).AUDPCs analysis indicated that chemotherapy with Amp orAct þ VA when coupled with thermotherapy at 40 �C (P ¼ 0.0012),42 �C (P ¼ 0.0002) or 45 �C (P ¼ 0.0001) significantly reduced Lastiter compared to thermotherapy alone or chemotherapy with Nic,Zn, or Nic þ Zn, even when those chemicals were coupled withthermotherapy (Fig. 3).

To further confirm the efficacy of Amp, Zn, Nic, Zn þ Nic, andAct þ VA against the Las bacterium, we evaluated their phytotox-icity on citrus using a graft-based assay. The results indicated thatHLB disease indices (DI) following Amp and Act þ VA treatmentswere 0%, significantly lower than the Zn, Nic, Nic þ Zn and CK

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Fig. 1. Comparison of root drench application and bark paint application of chemicals effective against HLB. Ampicillin sodium (Amp at 500 mg/L for root drench and at3000 mg for bark paint), Nicotine (Nic at 200 mg/L for root drench and at 1000 mg/L for bark paint), Zinc sulfate (Zn at 200 mg/L for root drench and at 1000 mg/L for bark paint),combination of Nicotine and Zinc sulfate (Nic at 200 mg/L and Zn at 200 mg/L for root drench, and Nic at 1000 mg/L and Zn at 1000 mg/L for bark paint), combination ofActidione þ Validoxylamine A (Act at 10 mg/L and VA at 100 mg/L for root drench, and Act at 50 mg/L and VA at 1000 mg/L for bark paint), and tap water treatment was selected ascontrol (CK). A, Ct value of HLB-affected citrus treated by root drench application or bark paint application, using various chemical formulations (Amp, Nic, Zn, Nic þ Zn, Act þ VA,and tap water treatment (CK)), Ct < 16.0: high bacterial titer; 16.1 < Ct < 26.0: moderate bacterial titer; 26.1 < Ct < 35.9: low bacterial titer; Ct > 36.0, bacteria undetectable. B,Standardized area under disease progress curves (AUDPCs) of HLB-affected citrus treated by root drench application or bark paint application, using the same chemical formulations.C, Therapeutic efficiency (TE) of HLB-affected citrus treated by root drench application or bark paint application, using the chemical formulations. Results of experimental treat-ments denoted with the same letter are not significantly different according to Duncan's multiple range test at P < 0.05. Standard deviation of the mean is indicated by a vertical line.

C. Yang et al. / Crop Protection 84 (2016) 150e158 153

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Amp Nic Zn Nic+Zn Act+VA CK

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180

Fig. 2. Ct value at 0, 60, and 180 day after initial treatment, of HLB-affected citrus treated with Ampicillin sodium (Amp), Nicotine (Nic), Zinc sulfate (Zn), combination ofNicotine and Zinc sulfate (Nic þ Zn), combination of Actidione þ Validoxylamine A (Act þ VA) and tap water (CK). Ct < 16.0: high bacterial titer; 16.1 < Ct < 26.0: moderate bacterialtiter; 26.1 < Ct < 35.9: low bacterial titer; Ct > 36.0, bacteria undetectable. Results of experimental treatments denoted with the same letter are not significantly different accordingto Duncan's multiple range test at P < 0.05. Standard deviation of the mean is indicated by a vertical line.

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Fig. 3. Standardized area under disease progress curves (AUDPCs) of HLB-affected citrus treated by various chemical formulations, coupled with thermotherapy at varioustemperatures, 40 �C, 42 �C and 45 �C, using bark paint application. Ampicillin sodium (Amp at 3000 mg), Nicotine (Nic at 1000 mg/L), Zinc sulfate (Zn at 1000 mg/L), combinationof Nicotine and Zinc sulfate (Nic at 1000 mg/L and Zn at 1000 mg/L), combination of Actidione þ Validoxylamine A (Act at 50 mg/L and VA at 1000 mg/L), and tap water treatmentwas selected as control (CK). Results of experimental treatments denoted with the same letter are not significantly different according to Duncan's multiple range test at P < 0.01.Standard deviation of the mean is indicated by vertical line.

C. Yang et al. / Crop Protection 84 (2016) 150e158154

treatments (Table 3). Moreover, plants (scions and rootstocks)graft-inoculated using HLB-infected scions treated with Zn, Nic,Nic þ Zn or water (CK) had higher Las scion infection percentagesand transmission percentages (Table 3). Las bacterial titers inrootstock of scions treated with Zn, Nic, and Nic þ Zn were

Table 3Efficiency of chemical formulations against the Las bacterium by grafting assay. DiffDuncan's multiple range tests using the SAS software package. NS represented no scion

Chemical formulations Scion survival (%) Scion growth (%) Scion infected (%)

Amp 97.5 48.8 0.0Nic 30.0 15 100Zn 45.5 9.1 100Nic þ Zn 31.3 6.3 100Act þ Va 0.0 0.0 0.0Water(CK) 95.3 38.4 62.5

significantly lower than the water (CK) treatment (P ¼ 0.0001)(Table 3). But, graft-inoculated citrus (scions and rootstocks)treated with these chemical formulations had higher Las scioninfection percentages, transmission percentages, and the treatedscion was as infected as the water treatment (CK) (Table 3). No Las

erent letters represent significant differences at p � 0.01. All data were analyzed bysurvival.

Las transmission (%) Ct value Disease index (%)

Scion Rootstock

0.0 40.00 ± 0.00 A 40.00 ± 0.00 A 0.00% ± 0.00% B60 26.82 ± 2.40 C 32.47 ± 6.78 AB 43.53% ± 3.71% A70 25.20 ± 0.46 C 31.23 ± 6.20 B 45.29% ± 3.94% A37.5 34.73 ± 0.00 B 34.92 ± 6.53 AB 36.89% ± 20.75% A0.0 NS 40.00 ± 0.00 A 0.00% ± 0.00% B

75.0 27.26 ± 1.10 C 23.16 ± 0.67 C 43.75% ± 4.42% A

C. Yang et al. / Crop Protection 84 (2016) 150e158 155

was detected in rootstocks of scions receiving Amp or Act þ VAtreatment. The Las titers in rootstocks of scions treatedwith Nic andNicþ Znwere not significantly different from those of rootstocks ofscions treated with an effective compound (Amp or Act þ VA). TheLas-infected scions treated with Act þ VA did not survive. Only31.3%e45.5% of scions treated with Zn, Nic, or Nic þ Zn survived.Only 6.3%e9.1% of scions had new growth when treated with Zn,Nic, or Nic þ Zn (Table 3). The scions treated with Amp or water(CK) exhibited higher survival rates (95.3% and 97.5%) and sciongrowth (38.4% and 48.8%) (Table 3).

3.3. Effects of temperature on control of HLB

The effect of temperature on Las bacterial titer was significant(P ¼ 0.0001) (Table 2). The qPCR Ct value of HLB-infected citrusafter thermotherapy at 45 �C was 30.79 ± 6.26, significantly higherthan that of HLB-infected citrus treated at 40 �C (28.72 ± 6.00) or42 �C (28.02 ± 6.25). Furthermore, the bark-paint therapeutic effi-cacy of Amp, Nic, Nic þ Zn, and Act þ VA, combined with ther-motherapy at 45 �C, was much greater than these chemicaltreatment coupled with thermotherapy at 40 �C or 42 �C. The lattertreatment had no measureable effect on Las titers (Fig. 4).Chemotherapy with Nic, Amp or Act þ VA combined with ther-motherapy was also effective, up to 100% TE (Figs. 4 and 5). The Zntreatment combined with thermotherapy at 45 �C, 42 �C or 40 �Cproduced the least significant therapeutic effect (Fig. 4).

4. Discussion

HLB is a serious disease that threatens the citrus industryworldwide. HLB is a systemic disease. The presumptive causal or-ganism is a bacterium that resides in the phloem tissues of citrus,and it is difficult to completely eliminate bacteria from the infectedcitrus plants by thermotherapy or chemotherapy. A major barrier ofusing chemicals to control HLB is the low efficacy in deliveringchemicals into the citrus phloem tissues. In this paper, a combinedstrategy of chemotherapy and thermotherapy coupled with a barkapplication of chemicals is described.

Thermotherapy is a potentially effective strategy for control of

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Fig. 4. Effect of different temperatures (40 �C,42 �C and 45 �C) on therapeutic efficiencAmpicillin sodium (Amp at 3000 mg), Nicotine (Nic at 1000 mg/L), Zinc sulfate (Zn at 1000 mcombination of Actidione þ Validoxylamine A (Act at 50 mg/L and VA at 1000 mg/L), and

HLB. The efficacy of this method depends upon the duration andthe temperature of treatment (Hoffman et al., 2013). Our studyshowed that 45 �C was the most effective temperature, while 40 �Cand 42 �C were less effective. Studies have shown that thermo-therapy at 45 �C can increase the respiration rate and cell mem-brane activities of citrus and increase the concentrations of growthhormones, secondary metabolites, reactive oxygen species, andstress-related proteins (Saidi et al., 2011; Wahid et al., 2007). Thesephysiological changes can promote new flush and root growth andenhance the vigor of citrus plants, which benefit the plants byincreasing the ability to uptake chemicals through the roots or bark,resulting in increased exposure of Las to the chemotherapeuticchemicals. However, there are different heat-tolerance thresholdsamong plant species and during different development stages.Reactions to heat stress were noticeably different among differentcitrus varieties (Hoffman et al., 2013). In this study, we used only agrapefruit cultivar. The heat treatment at 45 �C did produce severalsignificant effects, but 45 �C may not be an ideal choice for othercitrus varieties. Chemoethermotherapy for other citrus varietieswill need to be investigated.

Our results indicate that the combination of chemotherapy,applied using a bark paint method, coupled with thermotherapy at45 �C is an effective strategy for combating the disease. In thisstudy, thermotherapy at 45 �C enhanced the TE of chemicals orchemical combinations such as Amp, Nic, Nic þ Zn and Act þ VA.When combined with thermotherapy at 45 �C, the TE of chemicaltreatments such as Amp and Act þ VA were up to 100.0%, signifi-cantly higher than that of thermotherapy at 45 �C alone (TE ¼ 57%)(Fig. 4).

The efficacy of chemoethermotherapy against Las also depen-ded on antibacterial activity of the chemicals or chemical combi-nations. Our chemoethermotherapy test results indicated thatAmp and Act þ VA were the most effective treatments against theLas bacterium (Fig. 2), while the other chemicals or chemicalcombinations had less effect. AUDPCs of Amp and Act þ VA treat-ments were significantly lower than those of the other chemicals orchemical combinations, even when they were combined withthermotherapy (Fig. 3).

Ampicillin (Amp) is a beta-lactam antibiotic. The major

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y (TE) of various chemical formulations against HLB, using bark paint application.g/L), combination of Nicotine and Zinc sulfate (Nic at 1000 mg/L and Zn at 1000 mg/L),

tap water treatment was selected as control (CK).

Fig. 5. HLB-affected citrus seedlings treated with various chemical formulations combined with heat treatment at 45 �C, using bark paint application. A, Ampicillin sodiumtreatment (Amp at 3000 mg); B, Nicotine treatment (Nic at 1000 mg/L); C, Zinc sulfate treatment (Zn at 1000 mg/L); D, combination of Nicotine and Zinc sulfate treatment (Nic at1000 mg/L and Zn at 1000 mg/L); E, combination of Actidione þ Validoxylamine A treatment (Act at 50 mg/L and VA at 1000 mg/L); F, tap water treatment (CK).

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mechanism of the beta-lactam antibiotics is to inhibit the growth ofsensitive bacteria by inactivating enzymes located in the bacterialcell membrane, known as penicillin binding proteins, which areinvolved in cell wall synthesis (Spratt and Cromie, 1988). Due topublic concerns regarding the emergence of antibiotic-resistantbacteria and the potential effects on human health, application ofampicillin, carbenicillin, and penicillin has not been approved foruse on crops by the Environmental Protection Agency (EPA) orother regulatory agencies. However, results from the current studyas well as our past work demonstrated that Amp was the mosteffective chemical compound against the citrus Las bacterium(Zhang et al., 2012, 2013).

Actidione (Act) has been widely studied as a protectant agent,which is produced by streptomycin-yielding cultures of Strepto-myces griseus, and hasmarked antimicrobial activities (Tukey,1948;Whiffen, 1948, 1950). The action of Act resembles that of the anti-microbial antibiotics chloramphenicol, aureomycin and terramycinin that the primary point of inhibition appears to lie in the sequenceof reactions leading to the synthesis of nucleic acid and protein inmicroorganism (Kerridge, 1958). In previous studies, Act had beenproved to be effective against bean mildew (Erysiphe polygoni D.C)and cucumber scab (Cladosporium cucumerinum Ell. et Arth)(Dezeeuw and Vaughn,1950; Felber and Hamner,1948). In recently,Zhang et al.'s study indicated that Act has an inhibitory effect onLas, but it is also phytotoxic to citrus plants (Zhang et al., 2014a).Validamycin A (VA) is a metabolite of a Streptomyces strain and isused as an inhibitor of trehalase (Kameda et al., 1987). VA is anagro-specific antibiotic, which can suppress bacterial plant diseasessuch as Chinese cabbage soft rot, and lettuce bacterial rot byinhibiting trehalase in bacteria (Ishikawa et al., 1996, 2004).Meanwhile, VA likely has other mode of action with respect tocontrol of pathogen. This mode of action may involve activation of

plant defense responses (Ishikawa et al., 2005). In our previousstudy, VA was also effective against Las bacterium and low toxicityto citrus (Zhang et al., 2014a). Therefore, combination of Act and VAcould potentially be used in citrus production in the future.

Nicotine (Nic), 3-(1-methyl-2-pyrrolidinyl) pyridine is a color-less, light pale yellow, hygroscopic oily liquid present in theleaves of tobacco. It is one of the highly toxic chemicals belonging tothe tobacco alkaloids, which have multiple biological mechanismsagainst microorganisms (Willits et al., 1950). Pavia's study showedthat Nic can be against a spectrum of bacterial and fungal patho-gens (Pavia et al., 2000). In our previous study, Nic was identified ina screening test to have a partial antibiotic effect on Las (Zhanget al., 2014b). In this study, the TE of Nic treatment coupled withthermotherapy at 45 �C was as effective as the Amp and Act þ VAtreatments also coupled with the thermotherapy. The TE of Nictreatment without thermotherapy was lower than Amp andAct þ VA treatments. These findings may result from differences inchemical absorption in the citrus phloem tissues.

A nutrient imbalance is often present in plant tissues of HLB-affected trees. Zinc (Zn) is an essential micronutrient for plant(Hafeez et al., 2013). Additional nutrients like Zn are provided toHLB-affect trees in an attempt to alleviate nutrient balance. How-ever, in a previous study, we found that zinc, especially whenprovided at a high concentration in citrus tissues, significantlystimulated the growth of Las bacterium (Zhang et al., 2016). In thisstudy, we obtained similar results. The Las bacterial titer increasedafter the treatment with Zn combined with a heat treatment at45 �C. Our explanation for this phenomenon is that Zn absorptionby the citrus plant was significantly increased by the heat treat-ment at 45 �C (Hoffman et al., 2013).

The bark paint application was a better method for deliveringchemicals in this study than was the root drench application

C. Yang et al. / Crop Protection 84 (2016) 150e158 157

method. Several studies have reported using bark paint applicationto deliver agro-antibiotics and nutrients to fruit trees (Alvarez et al.,2008; Katz et al., 1989; Timmer, 1977). However, there are few re-ports concerning the use of bark application to deliver agro-antibiotics for the control of citrus HLB. In this study, wecompared the delivery efficacy of chemicals applied by either rootdrench or bark application and found that the antimicrobial for-mulations Amp and Act þ VA were more effectively delivered intocitrus tissues by the bark, as measured by reduction in Las titers(Fig. 1A, B, and C). Bark applicationmay overcome limitations in theroot system such as few new root hairs and the death of large rootsin HLB-infected citrus plants (Etxeberria et al., 2009). Althoughthermotherapy can induce new root growth and enhance vigor ofthe plants (Hoffman et al., 2013), some antibiotics such as Act cansuppress new root growth (Norman, 1959). Therefore, root drenchapplication for some compounds such as Act þ VA may provideinefficient delivery of the chemicals into the citrus plants leading toreduced therapeutic effect against Las. Although Amp is rapidlytaken up by citrus plants (Brian, 1957), the efficacy of Amp appliedto the bark was higher than the root drench application. Therefore,bark paint may be a practical and effective application method fordelivering compounds to combat Las in infected citrus plants.

A major advantage of bark application compared to root drenchis the low level of phytotoxicity. During the 180 day test period, thechemicals and chemical combinations applied onto the citrusseedlings by bark application produced no phytotoxicity symptoms(Fig. 5). A second advantage of bark application is that it reduces theamount of chemicals used in an open environment, which helps tomitigate the potentially negative effect of chemicals on theenvironment.

In conclusion, a chemoethermotherapy strategy using barkapplication of chemicals may be a useful method for controllingHLB. In this study, the optimum temperature for effective ther-motherapy on grapefruit plants was 45 �C and the most effectivechemicals or chemical combinations were Amp and Act þ VA.Although the method of bark application used in this paper may belabor intensive for field use, it was an effective method for deliv-ering chemicals into the citrus plants. We are currently testing newchemical application techniques such as trunk spray, and devel-oping additional promising chemical formulations. We aim toidentify treatments that are more convenient, less laborious, andmore effective in delivery of chemicals for the control of HLB in thefield. Further work concerning risk assessments of the chemicals onhumans and the environment is also needed.

Acknowledgments

We greatly appreciate Bioscience Editing Solutions for criticallyreading this paper and providing helpful suggestions. Financialsupport was provided by US Citrus Research and DevelopmentFoundation (project CRDF#584), Guangxi Natural Science Founda-tion (project 2013GXNSFCB019002) and Guangxi Programs forscience and technology development (project 347004-7).

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