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Orchard and nursery dynamics of the effect of interplanting citrus with guava for huanglongbing, vector, and disease management T.R. Gottwald a, * , D.G. Hall a , A.B. Kriss a , E.J. Salinas b , P.E. Parker b , G.A.C. Beattie c , M.C. Nguyen d a U. S. Horticultural Research Laboratory, USDA-ARS, 2001 South Rock Road, Fort Pierce, FL 34945, USA b Mission Laboratory, USDA-APHIS-PPQ-CPHST, 22675 N. Mooreeld Rd., Bldg. 6414 Edinburg, TX 78541, USA c Centre for Food and Plant Science, University of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, Australia d Southern Horticultural Research Institute, P.O. Box 203, My Tho, Tien Giang, Viet Nam article info Article history: Received 27 March 2014 Received in revised form 10 June 2014 Accepted 12 June 2014 Available online Keywords: Citrus greening Diaphorina citri Disease progress Survival analysis abstract The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important pest of citrus in the United States of America primarily because it vectors Candidatus Liberibacter asiaticus, the bacterium putatively responsible for Asiatic huanglongbing (HLB). Asiatic HLB is considered one of the most serious diseases of citrus. In the United States where Asiatic HLB was rst found in the state of Florida, vector control is considered an essential component to mitigate pathogen infection and spread of the disease. Therefore commercial citrus growers in Florida have adopted intensive insecticide programs to manage psyllid populations. However, the repetitive use of insecticides for ACP control is expensive and interferes with biological control of ACP and other citrus pests. As an alternative to insecticides, reports from Vietnam indicated that infestations of ACP in citrus (and consequently incidence of HLB) were reduced when citrus was interplanted with white guava, Psidium guajava L. Speculations were that guava volatiles reduced ACP infestations in citrus by either repelling ACP or interfering with ACP ability to locate and infest citrus grown next to guava. We present the results of two studies conducted in Florida (where both ACP and HLB occur) to assess ACP infestations and HLB incidence in citrus inter- planted with either white or pink guava compared to infestations and disease incidence in citrus grown as a monoculture, both in orchards and nurseries. In the eld study, the effect of guava on ACP in- festations was assessed alone and in combination with insecticide or oil applications. Signicant re- ductions in ACP infestations in citrus interplanted with pink guava were identied, but there was no reduction in citrus interplanted with white guava. The effect of pink guava on ACP infestations could be investigated further. However, intercropping citrus with either white or pink guava did not prevent the introduction and spread of HLB. Conclusions from eld studies regarding guava as a management tactic against ACP were difcult to make due to persistent nematode problems and freeze damage to guava, which could have interfered with the production of guava volatiles responsible for deterring ACP in- festations. Conversely, citrus nursery trees interspersed with guava did show reduced HLB incidence and disease progression over time. However, vector and disease reduction resulting from guava intercropping in citrus nurseries was not adequate to recommend it as a management strategy. Published by Elsevier Ltd. 1. Introduction Huanglongbing (HLB) is a devastating, insect-vectored disease of citrus putatively caused by phloem-limited bacteria within the genus Candidatus Liberibacter.Citrus trees infected by this disease decline in productivity; produce misshapen, inedible or off- avored fruit; decline over multiple years and eventually die, with tree mortality rates generally more rapid in young trees (Gottwald et al., 2007a,b; Hall and Gottwald, 2011). In Asia (China) * Corresponding author. Tel.: þ1 772 462 5883; fax: þ1 772 462 5986. E-mail addresses: [email protected] (T.R. Gottwald), david.hall@ars. usda.gov (D.G. Hall), [email protected] (A.B. Kriss), elma.j.salinas@aphis. usda.gov (E.J. Salinas), [email protected] (G.A.C. Beattie), [email protected] (M.C. Nguyen). Contents lists available at ScienceDirect Crop Protection journal homepage: www.elsevier.com/locate/cropro http://dx.doi.org/10.1016/j.cropro.2014.06.009 0261-2194/Published by Elsevier Ltd. Crop Protection 64 (2014) 93e103
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Page 1: Orchard and nursery dynamics of the effect of ... · Vietnamese guava effect on ACP or that the small cage studies may be inadequate for investigating effects of guava on ACP because

lable at ScienceDirect

Crop Protection 64 (2014) 93e103

Contents lists avai

Crop Protection

journal homepage: www.elsevier .com/locate/cropro

Orchard and nursery dynamics of the effect of interplanting citruswith guava for huanglongbing, vector, and disease management

T.R. Gottwald a, *, D.G. Hall a, A.B. Kriss a, E.J. Salinas b, P.E. Parker b, G.A.C. Beattie c,M.C. Nguyen d

a U. S. Horticultural Research Laboratory, USDA-ARS, 2001 South Rock Road, Fort Pierce, FL 34945, USAb Mission Laboratory, USDA-APHIS-PPQ-CPHST, 22675 N. Moorefield Rd., Bldg. 6414 Edinburg, TX 78541, USAc Centre for Food and Plant Science, University of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, Australiad Southern Horticultural Research Institute, P.O. Box 203, My Tho, Tien Giang, Viet Nam

a r t i c l e i n f o

Article history:Received 27 March 2014Received in revised form10 June 2014Accepted 12 June 2014Available online

Keywords:Citrus greeningDiaphorina citriDisease progressSurvival analysis

* Corresponding author. Tel.: þ1 772 462 5883; faxE-mail addresses: [email protected] (T.R

usda.gov (D.G. Hall), [email protected] (A.B.usda.gov (E.J. Salinas), [email protected] (G.A.C(M.C. Nguyen).

http://dx.doi.org/10.1016/j.cropro.2014.06.0090261-2194/Published by Elsevier Ltd.

a b s t r a c t

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important pest ofcitrus in the United States of America primarily because it vectors ‘Candidatus Liberibacter asiaticus’, thebacterium putatively responsible for Asiatic huanglongbing (HLB). Asiatic HLB is considered one of themost serious diseases of citrus. In the United States where Asiatic HLB was first found in the state ofFlorida, vector control is considered an essential component to mitigate pathogen infection and spread ofthe disease. Therefore commercial citrus growers in Florida have adopted intensive insecticide programsto manage psyllid populations. However, the repetitive use of insecticides for ACP control is expensiveand interferes with biological control of ACP and other citrus pests. As an alternative to insecticides,reports from Vietnam indicated that infestations of ACP in citrus (and consequently incidence of HLB)were reduced when citrus was interplanted with white guava, Psidium guajava L. Speculations were thatguava volatiles reduced ACP infestations in citrus by either repelling ACP or interfering with ACP abilityto locate and infest citrus grown next to guava. We present the results of two studies conducted inFlorida (where both ACP and HLB occur) to assess ACP infestations and HLB incidence in citrus inter-planted with either white or pink guava compared to infestations and disease incidence in citrus grownas a monoculture, both in orchards and nurseries. In the field study, the effect of guava on ACP in-festations was assessed alone and in combination with insecticide or oil applications. Significant re-ductions in ACP infestations in citrus interplanted with pink guava were identified, but there was noreduction in citrus interplanted with white guava. The effect of pink guava on ACP infestations could beinvestigated further. However, intercropping citrus with either white or pink guava did not prevent theintroduction and spread of HLB. Conclusions from field studies regarding guava as a management tacticagainst ACP were difficult to make due to persistent nematode problems and freeze damage to guava,which could have interfered with the production of guava volatiles responsible for deterring ACP in-festations. Conversely, citrus nursery trees interspersed with guava did show reduced HLB incidence anddisease progression over time. However, vector and disease reduction resulting from guava intercroppingin citrus nurseries was not adequate to recommend it as a management strategy.

Published by Elsevier Ltd.

: þ1 772 462 5986.. Gottwald), [email protected]), elma.j.salinas@aphis.. Beattie), [email protected]

1. Introduction

Huanglongbing (HLB) is a devastating, insect-vectored diseaseof citrus putatively caused by phloem-limited bacteria within thegenus ‘Candidatus Liberibacter.’ Citrus trees infected by this diseasedecline in productivity; produce misshapen, inedible or off-flavored fruit; decline over multiple years and eventually die,with tree mortality rates generally more rapid in young trees(Gottwald et al., 2007a,b; Hall and Gottwald, 2011). In Asia (China)

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where HLB was first described, the disease is attributed to ‘C. Lib-eribacter asiaticus’ (Las) and is vectored by the Asian citrus psyllid(ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) (Bov�e,2006; Gottwald, 2010; Halbert and Manjunath, 2004). ACP andAsiatic HLB have slowly spread from Asia to other citrus producingregions around the world including South, Central and NorthAmerica, and the Caribbean. Other species of ‘C. Liberibacter’ (C. L.americanus (Lam), C. L. africanus (Laf), and C. L. africanus cv.Capensis (Lafc) causing HLB in citrus occur in some countries andanother psyllid (Trioza erytreae Del Guercio) can transmit HLBpathogens, however, the most severe cases of HLB worldwide areusually related to ‘C. Liberibacter asiaticus’ and ACP (Bov�e, 2006).

Following the discovery of HLB in Brazil and the United States in2004 and 2005, respectively, most scientists recommended thefollowing three-tiered management approach, as recommended bythe UNDP-FAO, Asian Citrus Rehabilitation Project (Aubert, 1990):(1) intensively manage ACP, (2) regularly identify and remove treesinfected byHLB, and (3) only plant nursery stock known to be free ofthe disease (Gottwald, 2007). Currently, biological control of ACP byparasitoids and predators is considered insufficient for mitigatingthe disease (Yang et al., 2006). Many growers therefore adoptedintensive insecticide programs. The three-tiered HLB managementscheme is expensive, and the repetitive use of insecticides for ACPcontrol interferes with biological control of citrus insect pestsincluding ACP. Additionally, many growers are reluctant to removeinfected trees that are still marginally productive. This retainsinfected trees in situ that act as inoculumsources and exacerbate theepidemic. Citrus in urban residential areas also act as recalcitrantinoculum sources and reservoirs for ACP. Therefore, biological con-trol is believed to be of some benefit in urban plantings whereregular insecticide applications are impractical and rarely used.

As an alternative to the three-tiered HLB management program,reports from Vietnam indicated that infestations of ACP in citrus(and consequently incidence of HLB) were reducedwhen citrus wasinterplanted with white guava, Psidium guajava L. (plant familyMyrtaceae). Guava is quick to bear fruit, and was interplanted inorder to improve farm income prior to the citrus trees bearing fruit,(Beattie et al., 2006; Gottwald et al., 2007a,b). In addition, inter-planting citrus with white guava resulted in reduced infestations ofcitrus aphids (species not reported) and Asiatic citrus leafminer(Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae). Theseobservations were made in high density citrus plantings that wereintercropped with guava at a one-to-one tree ratio [2.5 m rowspacing, 2.5 m tree spacing along rows]. During a 2007 trip to theMekong Delta, ACP was frequently observed on citrus in mono-culture plantings but none were observed on citrus interplantedwith guava (D. G. Hall, personal observations), and HLB was prev-alent in citrus in these monoculture plantings but little HLB wasobserved in citrus planted next to guava (T. R. Gottwald, personalobservations), consistent with reports from the region (Gottwaldet al., 2010). It was speculated that guava volatiles were respon-sible for reducing infestations of the psyllid in citrus by eitherrepelling psyllids or interfering with the ability of ACP to locate andinfest citrus grown next to guava.

Idstein and Schreier (1985) characterized the chemical compo-sition and concentrations of individual chemical components of thevolatile signature given off by guava. Rouseff et al. (2008) examinedguava leaf volatiles to determine potential components responsiblefor guava's protective effect against ACP and found seven candidatesulfur volatiles including dimethyl disulfide (DMDS), an insecttoxic, defensive volatile not associated with citrus leaves. Onagbolaet al. (2011) reported that responses of ACP to citrus volatiles wereinhibited by guava volatiles and that DMDSmay have been partiallyresponsible for this effect. However, DMDS is not volatilized indetectible concentrations from non-wounded trees, but rather only

from trees that have sustained foliar damage. Thus, volatized DMDSmay not exist in sufficient concentration in citrus orchards to repelACP. In greenhouse cage studies conducted in Florida, ACP survivalwas reduced when they were confined to guava in no-choice sit-uations as compared to survival on citrus, but survival was alsoreduced when ACP were confined to either cotton or tomato, twonon-citrus neutral hosts (Hall et al., 2008). While significant re-ductions in infestations of adults on young citrus sometimesoccurred in cages containing both citrus and guava, the reductionswere not enough to validate the Vietnamese guava effect. In theirstudies, Hall et al. (2008) speculated that the guava germplasmstudied was possibly deficient in the traits responsible for theVietnamese guava effect on ACP or that the small cage studies maybe inadequate for investigating effects of guava on ACP because ofthe inability to concentrate sufficient guava volatiles in the prox-imity to caged citrus plants. Zaka et al. (2009) demonstrated thatboth young and old guava leaves produced volatiles that had equalrepellent activity on adult ACP in Y-tube olfactometer experiments.

The objective of research presented here was to investigate theinfluence of guava on infestation levels of ACP in citrus in Floridaunder field conditions in young plantings and in a field nurserysetting. In addition, the influence of guava on the temporal inci-dence of HLB was assessed in Florida.

2. Materials and methods

2.1. Field study

Citrus [‘Valencia’ sweet orange, Citrus sinensis (L.) Osbeck] wasgrown as a monoculture or interplanted with one of two guavacultivars. The two guava cultivars were Vietnamese ‘Thai Xaly nghi’white guava (Stover et al., 2008) and ‘Beaumont’ pink guava, bothseeded selections. Guava trees for the study were grown from seedsof fruit obtained from commercial plant sources in South Florida.The following treatments were investigated:

1. Citrus planted as a monoculture and subjected to a minimal(conventional) insecticide program.

2. Citrus planted as a monoculture and subjected to an insecticidaloil program.

3. Citrus interplanted with pink guava with no insecticides or oils.4. Citrus interplanted with pink guava and subjected to an insec-

ticidal oil program.5. Citrus interplantedwith white guavawith no insecticides or oils.6. Citrus interplantedwith white guava and subjected to aminimal

(conventional) insecticide program.7. Citrus interplanted with white guava and subjected to an

insecticidal oil program.

Because citrus growers in Vietnam reported that they plantedguava up to a year in advance of planting citrus in interplantedsituations, guava was planted during August 2008 and citrus wasplanted a year later during August 2009. The planting was estab-lished at the USDA, ARS U.S. Horticultural Research Service, PicosFarm in St. Lucie County, Florida, latitude 27.432947longitude �80.4290003, elevation 5.8 m. The treatments indicatedabove were initiated within 30 days of planting the citrus. Indi-vidual plots were three rows wide (2.4 m row spacing) and 136 mlong, with 3 m between citrus trees (a total of 132 citrus trees per0.1 ha plot). For plots interplanted with guava, there was one guavaplant between each pair of citrus trees along a row. There were twoplots (replications) for each treatment, and treatments wererandomly assigned to the plots with 10.5 m between plots.

Prior to establishing the citrus trees, fertilizer was applied fourtimes to the guava trees on 20March 2008 (0.227 kg/tree 6-6-6); 16

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July 2008 [125 cc/tree 8-10-10withmicronutrients (ProSource One,Plant City, FL)]; 6 November 2008 (136 kg/ha 8-10-8); and 6 March2009 [18.35 kg/ha 4-2-2 (Diamond R Special 4-2-2, chickenmanurewith chitosan, Diamond R Co., Fort Pierce, FL)]. One conventionalfarm spray was applied on 10 July 2008 consisting of 22.7 L horti-cultural oil (citrus soluble oil 99.3%, Loveland Products Inc., Greeley,CO); 1.33 L abamectin 2.0%, (Reaper 0.15 EC, Loveland Products Inc.,Greeley, CO); and 1.8 kg copper hydroxide 53.8%, (Kocide® 2000,Dupont Chemical, Wilmington, DE) in a 1893 L tank mix. Oneinsecticide spray was applied 10 July 2008 consisting of 0.907 kgBacillus thuringiensis 10.3% (Xen Tari BT 2, Valent USA Corporation,Walnut Creek, CA) and 2.84 L carbaryl 43.0% (Sevin SL, BayerEnvironmental Science, Montevale, NJ) per 946 L tank mix. Nem-atodes established on weed species began to move to the guavaplants, therefore nematicides were applied five times on 15 October2008, 30 October 2008, 22 May 2009, 18 December 2009 and 17November 2010 consisting of 112 kg/ha fenamiphos (Nemacur® 10%granular, Bayer Environmental Science, Research Triangle Park,NC); 18.35 kg/ha fenamiphos; 4.67 L/ha spray oxamyl 24% (Vydate®

L drench, Dupont Chemical, Wilmington, DE); 4.67 L/ha oxamyl bydrip irrigation; and 10 ml fenamiphos/guava tree by soil injection,respectively.

Following the establishment of the citrus interplanting, theseven treatments above were instituted and, for those plotsreceiving insecticides and/or oils, these were applied to citrus treesonly coincident with citrus new growth flushes on 8 September2009 and 7 May, 12 July, and 30 September 2010. The minimalinsecticide program consisted of ultra-fine hort oil 1% v/v (UltraPure Oil 98%, Prescription Treatment Brand, Whitmore Micro-GenResearch Laboratories, Inc., St. Luis, MO) and 0.15 L abamectin2.0% (Reaper 0.15 EC, Loveland Products Inc., Greeley, CO) in com-bination with nutritionals [0.95 L 30-0-0 liquid fertilizer (SureOn30, Plant Food Systems, Zellwood, FL); 0.9 Kg copper hydroxide53.8%; 0.47 L 28-0-0 (N-SURE® Urea-Triazone fertilizer, Fresno, CA);and 4.72 L bulk liquid fertilizer 7-0-7] in a 227 L tank mix applied toeach tree until run off. The oil program consisted of ultra-fine hortoil 1% v/v in combinationwith 4.72 L bulk liquid fertilizer 7-0-7 in a227 L tank mix, applied to each tree until run off. In addition tothese insecticide/oil/nutrient treatments to citrus, the citrus treesin each plot also received nutritionals on 10 November 2009(125 cc/tree 6-6-6); 25 May 2010 (18.35 kg/ha 4-2-2); 23 June 2010[1.89 L chelated microelements and proprietary biostimulants(RTRx Plus, Diamond R Co., Fort Pierce, FL), 3.79 L 28-0-0, 2.27 kgcopper hydroxide 53.8%, and bulk fertilizer 7-0-7 1.89 L in a 227 Ltank mix]; and 12 October 2010 [250 cc/tree 14-4-9 (Harrell's 13-4-9 slow release, Harrell's Inc, Lakeland, FL]. Occasionally individualguava trees declined due to nematode infestation and werereplaced. Prior to guava tree replacement, the soil immediatelyunder each nematode-infested tree location was treated with 16ml/tree space soil fumigant (Telone II, Dow AgroSciences LLC,Indianapolis, IN) by soil injection. New guava trees were replantedtwo weeks after soil fumigation.

Windbreaks were established around the perimeter and inter-nally within the experiment to enhance the retention of guavavolatiles within plots where guava and citrus were interplanted.This was accomplished by erecting 2.4-m screen walls (60% knitshade fabric) running parallel to the rows in each plot (a screenwallbetween every two plots) as well as perpendicular to the ends ofeach row. The screen was supported by wire cables attached towooden poles (4.3 m tall) at top and bottom.

2.2. Nursery study

Two citrus nurseries were established on 18 June 2009with foursweet orange, C. sinensis (L.) Osbeck, cultivar/rootstock

combinations (‘Hamlin’ on ‘Carizzo’ x Citroncirus sp., ‘Hamlin’ on‘Swingle’ x Citroncirus sp., ‘Valencia’ on ‘Carizzo’, and ‘Valencia’ on‘Swingle’) and one grapefruit C paradisi Macfad. ‘Ruby Red’ oncitrumelo rootstock. The planting was established at the USDA, ARSU.S. Horticultural Research Service, Picos Farm in St. Lucie County,Florida, latitude 27.43367 longitude �80.436621, elevation 5.8 m.All citrus were grown in 20.3 cm diameter by 30.5 cm tall blackplastic pots approximately 6 months of age when the experimentbegan. Each nursery consisted of a randomized complete blockwith two replicates, each replicate consisting of 18 rows with threetrees per row composed of six rows of grapefruit and 12 rows ofsweet orange. Rows were 10.6 cm apart with trees 10.6 cm apartwithin row. White guava plants of Vietnamese cv ‘Thai Xaly nghi’were grown from seed in 10.6 cm square by 30.5 cm tall blackplastic pots. One nursery was grown as a monoculture consisting of216 citrus trees only. The second nursery was composed of 216citrus interplanted with 228 guava, such that guava plants were atthe beginning and end of each replicate and three guava plantsbetween each pair of citrus rows. Both nurseries were establishedwith ca. 18-mo-old citrus and ca 6-mo-old guava. Canopies of citrusand guava were allowed to intermingle through time. Due to aDecember 2010 freeze that damaged the original guava plants, allplants were replaced on 6 January 2011 with guava from an in-ventory of the same age as originally placed in the nurseries. Nofreezes occurred during the period of fall 2011 to spring 2012.

Windbreaks were established around the perimeter of eachnursery to enhance the retention of guava volatiles within thenursery where guava and citrus were interplanted. This wasaccomplished by erecting 2.6-m screen walls of 60% knit shadefabric supported by wooden poles 12.7 cm diameter, 4.3 m tall and0.6 m seated in the ground in concrete. Poles supporting an indi-vidual wall were spaced 6.1 m apart. To secure the screen, wirecables were used to secure the top and bottom of the screen to thepoles. One end of a cable was attached to a pole with an eye bolt,and the other was secured to a grommet affixed in the screen.

2.3. Vector and pathogen assays

2.3.1. ACP surveys of field and nursery studiesThirty citrus trees in each field plot and within each nursery

were monitored on a three-week schedule from September 2009through November 2010 for the field study and July 2009 throughApril 2012 for the nursery study, for infestations of ACP, citrusaphids, and citrus leafminer. Adult ACP counts were made on a pairof mature leaves (including the stem between the leaves), 5 pairs ofleaves per tree. One flush shoot was examined on each of the 30trees to record the number of adults present and to determine if theshoot was infested by ACP eggs, ACP nymphs, aphids (species notnoted) or citrus leafminer (P. citrella). Due to variation in flushabundance, fewer than 30 trees had to be sampled for insects onflush on some sample dates, and sometimes no flush was presenton any trees. The following variables were investigated to gaugetreatment effects for insect suppression: 1) mean number of adultACP per pair of mature leaves; 2) mean number of adult ACP perflush shoot; and 3) mean percentages of flush shoots infested byACP eggs, ACP nymphs, aphids or leafminers.

2.3.2. HLB assays of field and nursery studiesOnly citrus trees grown in a disease-free enclosed nursery and

confirmed to be free of HLB via q-PCR assay were planted in thisexperiment. After planting in the field or established in nurseries,the trees were sampled for HLB approximately once every threemonths. Trees infected by theHLBpathogenwere identified using q-PCR primers and protocols developed by Li et al. (2006). Briefly, 2-4leaves with petioles were detached from a tree (leaves with HLB

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symptoms were chosen if available). DNA was extracted from themid-rib/petiole of the sampled leaves. For each tree, a combinedsample of 100e180 mg of mid-rib was excised prior to DNAextraction. After the DNA was extracted, samples were quantifiedwith a nano-drop spectrophotometer (Nanodrop 100, Thermo Sci-entific, USA) and standardized to 25 ng/ul with nuclease-free sterilewater. The PCR primer sequences used for all analysis were asdescribed by Li et al. (2006). The quantitative TaqMan PCR methoduses 16S rDNA-based primer-probe sets specific to Las. The primer/probe system employed was labeled with NED/MGB, i.e., it uses aTaqManMGB (minor groove binding) probe, which incorporates a 5'reporter dye and a 3' non-fluorescent quencher. The reporter dyeusedwasNED. This systemwas optimal for use on the Real-time PCRinstrument with a FAST platform (Applied Biosystems, Life Tech-nologies Corporation, Carlsbad, CA). InvitrogenExpress qPCRMasterMixwas usedwith the addition of the primers, probe, nuclease-freewater and PVP (polyvinylpyrrolidone). The primers were used at aconcentration of 0.3uM, the probe at 0.15 mMand the PVP (added tobind PCR reaction inhibitors) at 25 mg/mL mix. Total volume pereach of the 96 wells/plate was 20 ml.

The cycle parameters of the real-time program were: 95 �C for20 s followed by 40 cycles of 95 �C for 3 s and 60 �C for 30 s. Theplacing of the baseline was done automatically (ABI software, In-ternational Trade Systems, Portland, OR). The baseline was placedabove the fluorescent background signals created by the PCR re-action in the early cycles, before the target amplification was suf-ficiently above the background signal. The threshold must also beassigned by the automatic analysis software. This threshold is thenumerical value assigned by the program to represent a statisticallysignificant point above the calculated baseline. The point at whichthe target DNA in each sample well reaches this threshold is thethreshold cycle (Ct) value. Lower Ct values indicate higher initialtemplate in the well. If a sample contains pathogen DNA, ampliconwill be created in the PCR reaction and the amount will be reflectedin the Ct value. For our studies, a Ct value of �36 was consideredpositive for the pathogen.

2.4. Statistical analyses of field and nursery studies

For the field planting and nursery studies, percentages of flushshoots infested by insects were arcsine-transformed and subjectedto time-series analyses of variance with PROC GLM (SAS Institute,2008), and significant differences among treatments were investi-gated using Tukey's test. Analyses of variance (PROC GLM) wereconducted on final cumulative numbers of ACP per plant sample,and significant differences among treatments were investigatedusing Tukey's test.

Las-infected tree incidence (number of Las-infected treesdivided by the total number of trees) of each treatment in the fieldand nursery studies was calculated for each assessment. The in-crease in Las incidence for all treatments was assessed by linearregression analysis of transformed disease incidence data. Theappropriateness of each model tested, i.e. exponential, logistic, andGompertz, was determined by examining the coefficient ofregression, the correlation coefficient of observed vs. predictedvalues, and the plots of standardized residual values vs. predictedvalues (Madden et al., 2007). Las-infected tree increase among plotswas compared via t-test of rates (¼ slopes b of the regressionmodel) of Las-infected tree increase determined by linear regres-sion of the most appropriate model to determine if there weresignificant differences in Las-infected tree increase relative totreatment (Madden et al., 2007). To provide a straightforwardcomparison for Las-infected tree increase over time among treat-ments, the D50ti (time to reach 50% Las-infection of the population)was calculated.

For the field study, the increase in Las-infected trees within theinterior of each plot was also compared to the increase of trees onthe periphery of the plot. This was done to determine if citrus-guava intercropping provides differential protection to the treesbased on within orchard location compared to those on the pe-riphery via examining for an edge effect, i.e., those trees withgreater exposure to ACP immigration.

Survival analysis methods were used to examine the effect ofpredictor variables such as ACP population and treatment on theresponse variable time to infection (Tinf). As previously indicated,trees were assessed for HLB approximately once every threemonths at 217, 302, 395, 485, and 528 days after planting for thefield study and 18, 120, 233, 305, 391, 481, 573, 754, 850, 938, 1027,and 1118 days after planting for the nursery study. Initial survivalcurves for each treatment estimated from the KaplaneMeiermethod (Proc lifetest, SAS Inc., 2008) were generated to express theprobability that an individual tree present at time 0 will survive(remain Las-free or at least Las PCR negative) until time t (Allison,1995). Differences in survival among the treatments wereexplored using the Wilcoxon test. Tinf data was censored for treesthat died due to some other cause and never became infected priorto the end of the study, with exception of the last interval where thedata were censored for trees that never became Las-infected. A Coxproportional hazards model (Cox and Oakes, 1984) was thendeveloped to evaluate the relationship between ACP population(LP ¼ the mean number of adult psyllids per five leaf pairs) andtreatment on Tinf. The midpoints of the assessment intervals (e.g.,the midpoints for the field study were 108.5, 259, 347.5, 439, and506.5 days, respectively) were used as the observed date of infec-tion in lieu of assessment day post planting, to account for treesbecoming Las-infected at an unknown point during the intervalbetween assessments. Additionally, the effect of ACP population ontime to infectionwas examined over various lengths of time (e.g., 3,6, and 9 weeks) and retrospectively, that is the effect that ACPpopulations sometime in the past have on Las-infection at a givenpoint in time. It was determined for these studies that the mostappropriate value for LP was derived from the average number ofadult psyllids per five leaf pairs collected over the previous 9weeks.Wald tests were used to determine which predictor variables hadan effect on the hazard (i.e., risk) of HLB infection. Differencesamong treatments were also evaluated with Wald tests and theassociated hazard ratio (ratio of the estimated hazard for treessubjected to a certain treatment versus a different treatment) wasdetermined. A hazard ratio equal to one indicates there is no effecton Tinf due to the treatment (Allison, 1995).

3. Results

3.1. Vector population dynamics

3.1.1. Field studyThe mean ± SEM daily air temperature during the study aver-

aged 22.0 ± 0.3 �C with minimum and maximum daily tempera-tures averaging 17.1 ± 0.3 and 27.3 ± 0.2 �C, respectively. Freezesoccurred during three nights in January 2010 (as low as �2.2 �C)and during eight nights in December 2010 (as low as�2.3 �C duringtwo nights). Relative humidity averaged 79.4 ± 0.3% during thestudy. The average daily wind speed was 10.7 ± 0.2 kph, withmaximum daily wind speed exceeding 50 kph on 27 days duringthe study (highest wind speed was 74.8 kph). Daily rainfall aver-aged 0.26± 0.03 cm, with a cumulative total of 146.5 cm rain duringthe study.

Infestation levels of ACP in citrus were low for up to four monthsafter planting regardless of whether citrus was interplanted withwhite or pink guava (Fig. 1 A,B). Thereafter, ACP infestations

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T.R. Gottwald et al. / Crop Protection 64 (2014) 93e103 97

generally increased under each treatment. There were no signifi-cant differences among treatments over all sample dates withrespect to percentages of flush shoots infested by immature ACP,aphids or citrus leafminer (Table 1), and there were no significantdifferences among the treatments with respect to mean infestationlevels of adult ACP on flush shoots (F ¼ 1.1, Pr > F ¼ 0.4, 6 df). Overall treatments and sample dates, a mean ± SEM of 0.06 ± 0.01 ACPadults were observed per pair of mature leaves and a mean of0.32 ± 0.04 adults were observed per flush shoot. Significantlygreater numbers of ACP on mature leaves were observed in plots of

Fig. 1. Dynamics of HLB and survival analysis results for the field experiment (AeF) and nursmature citrus leaves, and B) young citrus flush shoots. Monoculture plots of citrus were treaunder different insecticide and oil programs (Florida field study). The insecticide treatmhuanglongbing in citrus monoculture and citrus/guava interplantings under different insectiJ) Rate of change in disease incidence for each treatment. E and K) Estimated survival functionare: 1) Citrus planted as a monoculture and subjected to a minimal (conventional) insectiprogram, 3) Citrus interplanted with pink guavawith no insecticides or oils, 4)Citrus interplawith white guava with no insecticides or oils, 6) Citrus interplanted with white guava and subwhite guava and subjected to an insecticidal oil program.

citrus interplanted with white guava than in plots of citrus eitherinterplanted with pink guava or interplanted with white guava andtreated with insecticides (F ¼ 2.4, Pr > F ¼ 0.03, 6 df). These dif-ferences were reflected in cumulative numbers of ACP observed onmature citrus leaves, which were greater in plots either inter-planted with white guava (no insecticides or oils) or plantedwithout guava and treated with insecticidal oils (Table 2). Cumu-lative numbers of ACP observed on citrus flush shoots were greatestin plots of citrus monocultures treated with oils, or in plots of citrusinterplanted with white guava either with or without oil

ery experiment (GeL). Infestation densities over time of adult Asian citrus psyllid on A)ted with insecticides compared to plots of citrus interplanted with white or pink guavaent was a minimal psyllid control program. C and I) Incidence of trees infected bycide and oil programs (refer to section 2.2) or a minimal psyllid control program. D andfor each treatment. F and L) Estimated hazard function for each treatment. Treatments

cide program, 2) Citrus planted as a monoculture and subjected to an insecticidal oilnted with pink guava and subjected to an insecticidal oil program, 5) Citrus interplantedjected to a minimal (conventional) insecticide program, and 7) Citrus interplanted with

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Table 1Percentages of flush shoots infested by the Asian citrus psyllid (ACP), aphids and Asiatic citrus leafminer in citrus alone and in citrus interplanted with guava, September 2009through November 2010 (Florida study).

Treatment Mean (SEM) percentage of citrus flush shoots infested

ACP Adultsa ACP eggsb ACP nymphsc Aphidsd Leafminere

Trt 1: Citrus monoculture þ conventional spray 7.7 (2.7)a 23.1 (5.7)a 24.2 (5.8)a 7.9 (3.0)a 32.8 (5.8)aTrt 2: Citrus monoculture þ oil spray 10.0 (3.0) 20.4 (4.7)a 15.9 (4.0)a 6.1 (2.7)a 34.4 (6.2)aTrt 3: Citrus þ pink guava only 3.7 (1.1)a 12.4 (3.4)a 10.2 (2.7)a 5.2 (2.1)a 33.7 (7.3)aTrt 4: Citrus þ pink guava þ oil spray 7.3 (2.1)a 14.8 (5.7)a 14.3 (4.5)a 9.4 (4.3)a 24.0 (5.5)aTrt 5: Citrus þ white guava only 7.4 (2.4)a 25.6 (6.1)a 25.3 (6.2)a 4.9 (1.4)a 34.2 (7.4)aTrt 6: Citrus þ white guava þ conventional spray 9.4 (2.7)a 13.6 (4.8)a 13.3 (5.0)a 3.5 (1.9)a 31.5 (6.1)aTrt 7: Citrus þ white guava þ oil spray 12.0 (3.6)a 15.6 (5.0)a 17.8 (5.6)a 9.3 (3.4)a 35.0 (6.8)a

Means in the same column followed by the same letter are not significantly different (P ¼ 0.05), Tukey's test. Time-series analyses on arcsine-transformed percentages, rawpercentages shown.

a F6,252 ¼ 1.4, Pr > F ¼ 0.23.b F6,252 ¼ 1.7, Pr > F ¼ 0.12.c F6,252 ¼ 2.2, Pr > F ¼ 0.04.d F6,252 ¼ 1.0, Pr > F ¼ 0.46.e F6,252 ¼ 0.5, Pr > F ¼ 0.84.

T.R. Gottwald et al. / Crop Protection 64 (2014) 93e10398

treatments. The presence of white or pink guava did not eliminateACP infestations. Lack of ACP control/population reduction is re-flected in data on percentage of trees infected by HLB at the end ofthe experiment (Table 2,Fig. 1 C,I).

3.1.2. Nursery studyThe nursery and field studies were physically located within

0.5 km of each other; therefore the nursery study experienced thesame temperatures, rainfall, and freeze events. ACP populationswere slow to build up in both citrus monoculture and citrusinterplanted with guava nurseries. However, once ACP invaded thenurseries, cumulative ACP populations increased much faster andresulted in much higher visitation by and infestation of ACP in thecitrus monoculture nursery compared to the citrus nursery inter-planted with guava (Fig. 1 G,H).

3.2. Temporal analysis of Las infections

3.2.1. Field and nursery studiesFor both the field and nursery trials, the logistic model was the

most appropriate to describe the rate of increase in Las incidenceover time for the majority of treatments tested and t-test com-parisons of the rates (b¼ slopes of the regressionmodel) were usedto evaluate differences among the treatments (Table 3). Results forthe field trial were inconclusive and did not show a significantdifference between citrus monoculture treatments versus those

Table 2Cumulative numbers of Asian citrus psyllid (ACP) adults per plant sample, and HLB diseasthrough November 2010 (Florida study).

Treatment Mean (SEM)

Cumulative numberACP adults persample of matureleavesa

Trt 1: Citrus monoculture þ conventional spray 7.9 (4.2) bcTrt 2: Citrus monoculture þ oil spray 15.9 (0.2) abTrt 3: Citrus þ pink guava only 2.5 (2.4) cTrt 4: Citrus þ pink guava þ oil spray 5.5 (3.5) bcTrt 5: Citrus þ white guava only 19.6 (6.0) aTrt 6: Citrus þ white guava þ conventional spray 3.9 (2.3) cTrt 7: Citrus þ white guava þ oil spray 7.8 (4.9) bc

Means in the same column followed by the same letter are not significantly different (P¼analysis, raw percentages presented.

a F6,6 ¼ 11.0, Pr > F ¼ 0.005.b F6,6 ¼ 7.5, Pr > F ¼ 0.01.c F6,6 ¼ 1.1, Pr > F ¼ 0.4.

treatments with citrus interplanted with either white or pink guava(Fig. 1AeD). Those treatments with citrus interplanted with pinkguava had slightly lower increase in Las incidence, but this mayhave been a location affect since these were the farthest away froma strong source of ACP and inoculum. Within the citrus-guavainterplanting treatments, there was a slight decrease in rates ofincrease for plots treated with either conventional sprays or oilsprays compared to those that were left unsprayed (Fig. 1 D).However, results from the nursery trial did show the rate of in-crease in Las incidence was significantly greater for the mono-culture compared to the citrus-guava intercropping over the threeyear duration of the study (Table 3). Utilizing the logistic rates of Lasincrease, the citrus monoculture nursery reached a Las incidence of0.5 after 780 days, whereas the citrus guava intercropping required859 days to reach the same incidence of Las.

Las-incidence increased during the study period for all treat-ments in both the field and nursery trials with the greatest rate ofincrease occurring during late summer to fall in each year (Fig. 1D,J). Although psyllids in the nursery trial were detected on thefirst vector assessment on July 21, 2009, it was not until thefollowing April that significant populations began to appear indi-cating a prolonged 10-mo period between the beginning of thestudy and the first detection of Las infections via PCR. If the firstpsyllids to arrive in the plots gave rise to the first PCR detection, thiswould be interpreted as an approximately 10 month incubationperiod between putative infection and Las detection via PCR. The

e incidence (%) in citrus alone and in citrus interplanted with guava, September 2009

Cumulative numberACP adults per flushshoot sampleb

Final percentage of plants infected by HLBc

108.0 (76.0) b 41.7 (25.0)a305.5 (0.5) a 53.3 (24.1)a42.0 (34.0) b 22.7 (1.5)a94.5 (66.5) b 3.8 (0.8)a

181.0 (45.0) ab 37.1 (25.0)a108.0 (50.0) b 13.9 (2.8)a184.0 (98.0) ab 12.2 (6.1)a

0.05), Tukey's test. Percentage data for trees infected by HLB arcsine-transformed for

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Table 3Paired t-test of logistic models for increase of Las infection in different treatments in guava citrus intercropping.

Field trial t-values X D50Ti

Treatment Parameterestimate

Std error N Trt 2 Trt 3 Trt 4 Trt 5 Trt 6 Trt 7

Trt 1: Citrus monoculture þ conventional spray 0.0146 0.00302 12 0.93 �5.30** 67.60** �0.04 �2.84** 5.24** 565.8Trt 2: Citrus monoculture þ oil spray 0.0137 0.00213 12 �1.31 �3.04** 2.58** �0.44 �4.59** 501.4Trt 3: Citrus þ pink guava only 0.0119 0.00352 12 1.33 �2.52* 120.00** �0.03 617.0Trt 4: Citrus þ pink guava þ oil spray 0.0112 0.00297 12 �6.67** 3.45** �1.69 631.1Trt 5: Citrus þ white guava only 0.0146 0.00246 12 �1.38 �44.00** 504.8Trt 6: Citrus þ white guava þ conventional spray 0.0131 0.00353 12 1.16 625.1Trt 7: Citrus þ white guava þ oil spray 0.0119 0.00252 12 505.6

Field Trial e Exterior Trees t-valuesT2 T3 T4 T5 T6 T7

Trt 1: Citrus monoculture þ conventional spray 0.0190 0.00394 12 �7.21** 27.69** 6.16** 1.64 3.54** 73.53** 543.4Trt 2: Citrus monoculture þ oil spray 0.0177 0.00413 12 12.96** 2.53* �3.45** 1.16 30.92** 563.8Trt 3: Citrus þ pink guava only 0.0118 0.00368 12 �16.24** 58.08** �11.78** �58.89** 719.2Trt 4: Citrus þ pink guava þ oil spray 0.0159 0.00343 12 7.86** �4.82** �27.53** 554.7Trt 5: Citrus þ white guava only 0.0188 0.00380 12 4.04** 409.00** 551.3Trt 6: Citrus þ white guava þ conventional spray 0.0167 0.00327 12 �20.26** 529.8Trt 7: Citrus þ white guava þ oil spray 0.0065 0.00377 12 951.8

Field trial e interior trees t-valuesT2 T3 T4 T5 T6 T7

Trt 1: Citrus monoculture þ conventional spray 0.0179 0.00373 12 1.06 �6.07 52.13 �1.55 �3.63 5.27 558.5Trt 2: Citrus monoculture þ oil spray 0.0168 0.00266 12 �1.36 �3.07 3.81 �0.54 �4.76 494.8Trt 3: Citrus þ pink guava only 0.0146 0.00428 12 1.32 �3.81 na �0.06 612.3Trt 4: Citrus þ pink guava þ oil spray 0.0137 0.00365 12 �10.68 3.44 �1.67 631.3Trt 5: Citrus þ white guava only 0.0187 0.00318 12 �2.60 58.86 487.0Trt 6: Citrus þ white guava þ conventional spray 0.0159 0.00428 12 1.09 624.2Trt 7: Citrus þ white guava þ oil spray 0.0146 0.00311 12 511.5

Nursery trial t-valueWith guava

Citrus monoculture 0.0073 0.00027 7 �678.71** 780.2Citrus þ guava 0.0039 0.00028 7 858.7

*And ** indicate differences detected by t-test for P ¼ 0.05 and 0.01, respectively.na not applicable; test statistic is not defined.X-D50n ¼ time in days to 50% disease.

T.R. Gottwald et al. / Crop Protection 64 (2014) 93e103 99

more pronounced increase in Las-incidence did not occur untilapproximately 15 months after initiation of the experiment.

Similar to comparisons of the logistic models, the Wilcoxon testindicates there are some significant differences (P < 0.0001) be-tween the seven treatments in the field study and the two treat-ments in the nursery study. Estimates for the KaplaneMeiersurvival and hazard functions attributed to each treatment arepresented in Fig. 1 E,F,K,L. The survivor function indicates theprobability that an individual tree subjected to a certain treatmentwill not be found to be Las-positive until a given point in time, andthe hazard function is a conditional probability function describingthe instantaneous risk that an individual tree will be Las-positive,

Table 4Parameter estimates, test statistics, and estimated hazard ratios for a Cox proportional haFlorida from 2009 to 2011.

Parameter df Estimate Standard error Chi-square P

Average psyllid count 1 0.099 0.028 12.665 <Trt 1 1 2.340 0.611 14.653 <Trt 2 1 2.307 0.614 14.112 <Trt 3 1 0.619 0.720 0.739Trt 4 1 1.253 0.658 3.621Trt 5 1 2.651 0.605 19.192 <Trt 6 1 1.931 0.624 9.585Trt 7 e e e e e

a Example of the estimated hazard ratio for trees subjected to Treatment 1 versus Treb Bold font indicates the 95% Wald confidence limits do not include the value 1.

given that it has not previously been found diseased. For example,at the end of the nursery study, the survival functions indicate thereis approximately a 70 percent probability that a tree in the citrus-guava intercropping would be free of Las, as opposed to approxi-mately a 10 percent probability an individual tree in the citrusmonoculture is disease free (Fig. 1 L).

3.2.2. Survival analysis with ACP population and treatment methodThe Cox proportional hazards model of Las infection over time

described the field and nursery data well and indicated ACP pop-ulations and some treatments had a significant effect (Tables 4 and5). Most of the same differences among treatments identified from

zards model describing time to infection of individual citrus trees in a field study in

> Chi-square Estimated hazard ratio

0.001 Trt 2 Trt 3 Trt 4 Trt 5 Trt 6 Trt 70.001 1.033a 5.589b 2.966 0.733 1.505 10.3790.001 5.410 2.871 0.709 1.456 10.0460.390 0.531 0.131 0.269 1.8570.057 0.247 0.507 3.5000.001 2.053 14.1650.002 6.898

atment 2.

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Table 5Parameter estimates, test statistics, and estimated hazard ratios for a Cox propor-tional hazards model describing time to infection of individual citrus trees in anursery study in Florida from 2009 to 2012.

Parameter df Estimate Standarderror

Chi-square P > Chi-square Estimatedhazardratio

Averagepsyllidcount

1 0.219 0.051 18.508 <0.001 1.244

Treatment 1 �1.403 0.148 89.284 <0.001 0.246

T.R. Gottwald et al. / Crop Protection 64 (2014) 93e103100

the t-test results in section 3.2.1 were also found with this model.However, the hazard ratios aim to better quantify the magnitude ofthese differences. For the field study, the hazard of infection for atree in treatments 3 or 4, where citrus was interplanted with pinkguava, was only 19 to 35 percent of the hazard for trees in treat-ments 1 and 2, where citrus is planted as a monoculture (Fig. 1F).Treatment 7 (citrus interplanted with white guava and treated withinsecticidal oil) maintained fairly low levels of disease throughoutthe study and reduced the hazard, as compared to treatments 1 and2, by nearly 100 percent. The three treatments that were subjectedto an insecticidal oil program (treatments 2, 4, and 7) all reducedthe hazard as compared with the minimal or no insecticide pro-grams with the same intercropping method. For the nursery study,the estimated hazard of infection for plants in the citrus-guavainterplant plot was only about 25 percent of the hazard for plantsin the citrus only (control) plot (Fig. 1L).

The parameter estimate for ACP population (i.e., average psyllidcount) for the field study was b ¼ 0.099 and as the response vari-able in the proportional hazards model is log(Tinf), the percentchange in time to infection for each additional psyllid (on average)can be found by taking 100*(eb-1) (Allison, 1995; Scherm andOjiambo, 2004). Therefore, for each one psyllid increase (on

Fig. 2. Estimates for the survivor functions describing infection of citrus due to ACP popuintervals for incremental days post planting of citrus.

average), the hazard of infection increased by 10.4 percent (Fig. 2).Similarly, for each one-psyllid increase in the nursery study, thehazard of infection increased by 24.4 percent (Fig. 3). The hazarddue to ACP populations within each treatment can be identified byevaluating linear combinations of the parameter estimates. Assurvivor functions are often easier to interpret visually than hazardfunctions, Figs. 2 and 3 show the survivor functions for each of theassessment dates (refer to section 2.4). As expected, all survivorfunctions decrease as the average ACP population increases.

4. Discussion

4.1. Field study

4.1.1. Temporal analyses of ACP infestation and Las infection anddisease expression

Analysis of variance results indicated that the field study didnot show any reduction in ACP infestation levels in citrus inter-planted with white guava. In fact, in the absence of insecticide oroil treatments to citrus, ACP infestations were elevated in citrusintercropped with white guava although this elevation not sig-nificant (Table 1). ACP infestations were significantly reduced incitrus under some treatments, notably in citrus intercropped withpink guava. ACP infestations on both mature leaves and flushshoots were largest in citrus intercropped with white guava andgenerally lowest in citrus intercropped with pink guava especiallyearlier in the study. The effect of pink guava against ACP in-festations could therefore be investigated further. No reductionsin infestation levels of aphids or citrus leafminer were observed incitrus interplanted with either white or pink guava during thestudy.

With respect to HLB epidemiology in the field study, by 18months after planting HLB incidence was generally lowest (4e23%trees infected) in plots of citrus interplanted with white guava

lation in the field study. Individual plots shown are at the midpoints of the sampling

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Fig. 3. Estimates for the survivor functions describing infection of citrus due to ACP population in the nursery study. Individual plots shown are at the midpoints of the samplingintervals post first exposure to psyllid vectors.

T.R. Gottwald et al. / Crop Protection 64 (2014) 93e103 101

treated with oils or insecticides or in citrus interplanted with pinkguava; ACP infestations during the experiment were generallylower in these same plots. HLB incidence was generally greatest(37e53% trees infected) in plots of citrus either grown as a mono-culture or interplanted with white guava and not treated with in-secticides, and ACP infestations were highest in two of the non-insecticide treated plots Treatments 2 and 5) during the experi-ment. However, plot-to-plot variability in the final incidence of HLBwas pronounced and precluded declaring any significant differ-ences among treatments. This variability might have been negatedhad greater than two replications of each treatment and possiblylarger plot sizes been studied. Variability among plots in HLBincidence may have also been related to changes over time in thehealth and size of the guava plants, which was continually chal-lenged by freezes and nematodes.

4.2. Nursery study

Both temporal analysis and survival analyses results for thenursery study were able to identify differences among treatmentswhere such differences were less or not apparent in the field study.The citrus-guava interplanting nursery significantly outperformedthe citrus monoculture nursery by depressing Las-infected citrusincidence over time. This was directly related to a pronouncedsuppression of ACP adults, nymphs, and eggs populations in thenursery plot where potted citrus plants were interspersed withpotted guava. Even so, the effect was a delay in disease onset and areduction in disease increase. Although the effect concurred withthe field studies, the effect of interspersing guava among citrusnursery plants was not sufficient to completely protect citrusnurseries. Therefore, the authors do not recommend the use ofguava as a deterrent to enclosed insect-resistant greenhousenursery production currently required in Florida and in transitionin Texas, Arizona and California.

4.3. Survival analyses

The Cox proportional hazards model of Las infection over timeindicated ACP populations and some treatments had a significanteffect on Las-infection for both the field and nursery data. In thiscontext, hazardwas defined as the estimated risk of a tree in each ofthese treatments becoming infected with Las. For the field study,treatments with citrus interplantedwith pink guava only had 19-35percent of the hazard ratio (i.e. risk of infection) that citrusmonocultures have and when citrus was interplanted with whiteguava, the risk was only 1e2%, i.e., 50e100% reduction compared tothe citrus monocultures. If we examine those treatments whereinsecticidal oils were applied, the risk of infection was greatlyreduced compared to treatments with minimal to no insecticidaloil. For the nursery study, there was a 4-fold reduction of theestimated risk of infection for plants in the citrus-guava interplantplot compared to the risk for plants in the citrus monoculture.Survival analysis was more sensitive compared to analysis of vari-ance and capable of identifying the benefit of pink or white guavainterplanting and the use of insecticidal oils is measureable. Thiscould have been due to a simple barrier effect of the guava plantsbetween citrus trees reducing ACP spread. However, the measure-able benefit still remains too small to be of benefit for diseasecontrol in the field.

4.4. Prognosis of the use of guava repellency in subtropical climatesto control HLB

Detailed information on the cultural and nutritional practicesused to cultivate guava during these experiments is presented forthe benefit of future efforts to investigate guava as a deterrent toACP infestations. The nutritional programs generally appearedeffective for stimulating growth of guava. In spite of a healthyappearance and good growth, both white and pink guava were

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damaged by freezes that were too mild to cause much damage tocitrus. Nematode problems (Meloidogyne spp.) persisted in spiteof applications of nematicides. Freezes and nematodes during theexperiment resulted in difficulties establishing and maintaininghealthy, large guava plants. Even though trees severely damagedby freezes or nematodes were replaced as quickly as possible,during cool but non-freezing weather, guava plants did not flushor grow and essentially remained in quiescence. Only whentemperatures began to climb significantly and remained warm forprolonged periods of time did the guava resume its growth. Thisgrowth often lagged considerably behind the typical resurgenceof ACP populations stimulated by increasing daily temperatures,as described by Liu and Tsai (2000). Guava is a tropical plant butFlorida is subtropical in climate. Plant volatile production isdirectly related to temperature and growth. When guava plantsare quiescent during lower temperatures (non-tropical condi-tions) or killed back by freezing temperatures, volatile productionis much reduced or terminated, respectively. Therefore, prolongedintermittent periods occurred when non-tropical conditionspersisted in the plots during which repellence of psyllid vectorswould have been minimal or nonexistent. During these periods,the citrus would have been unprotected by volatiles. This is quitedifferent compared to the continual tropical conditions thatpersist in the Mekong Delta of Vietnam where the guava effectwas first reported and where guava is maintained in a continualstate of rapid growth. In contrast to Florida, the Mekong Deltaregion has perpetually high RH, low wind movement, and densetropical vegetation surrounding citrus plantings that is variable intype, height and density. All of these factors potentially serve toincrease the concentration and retention of guava volatiles in theDelta region.

Finally, it was possible that the white guava cultivars used inthe Florida experiments differed genetically from the cultivargrown in, Vietnam, either with respect to specific volatiles orvolatile titers which could have affected psyllid infestations. Anadditional experiment was conducted in the Rio Grande Valley ofTexas using citrus ‘Rio Red’ grapefruit, Citrus paradisi Macf.,grown in two treatments, as a monoculture or interplanted withwhite guava of a similar cultivar as grown in Vietnam. At the timeof the study, HLB did not exist in the region, so only ACP pop-ulations were monitored (Salinas, unpublished data). Two addi-tional large experiments with guava were undertaken incollaboration with private industry to investigate the negativeeffect reported from Vietnam of guava on ACP infestations incitrus. These were composed of large, replicated plots of citrusgrown either in monoculture or interplanted with white guava, inHendry and Martin Counties. For both experiments, guava wasplanted one year in advance of when citrus was to be planted.However, in both of these Florida experiments, problems withnematodes and freeze damage to guava were so extensive thatthe experiments were abandoned. The combined results of thetwo failed Florida experiments in combination with difficultiesexperienced establishing guava for the experiments conducted inFlorida reflect the challenges of growing guava interplanted withcitrus in subtropical areas where citrus is grown in the UnitedStates.

Concurrent with this study, the efficacy of interplanting of citruswith guava was examined in Cai Be, Vietnam (Ichinose et al., 2012).In the Vietnam study, two orchards demonstrated that citrusinterplanted with guava effectively protected citrus trees from Lasinfection, but only for the first 12e16 months. The authors attrib-uted the lower number of Las infections to protection of new shootsto ACP infestation. Although the guava interplanting effect wasephemeral, farmers in southern Vietnam have been performingguava interplanting with an expectation that yields from both the

Citrus nobilis and guava in interplanted orchards promise to begreater than those from only C. nobilis in non-interplanting or-chards. In particular, guava provides yields in about a half year afterplanting, while C. nobilis cannot yield fruits until one and a halfyears later.

The advantages of mixed species cropping to control arthropodsand diseases as well as improved soil fertility have been known formillennia. The reliance on cropmonocultures is a recent occurrencein human agricultural history brought into vogue during the in-dustrial revolution. Trenbath (1993) concluded, “The presence ofassociated plants in the intercrop can lead to attack escape in threeways, all involving lower population growth rate of the attacking or-ganism. In one, the associates cause plants of the attacked componentto be less good hosts; in the second, they interfere directly with ac-tivities of the attacker; and in the third, they change the environmentin the intercrop so that natural enemies of the attacker are favored”.Andow (1991) reviewed 209 studies of crop mixtures versusmonocultures encompassing 287 pest species. He showed that 149insect pest species populations were reduced and the population ofnatural enemies of the pests was higher in 53 percent of theintercrop studies and only lower in 9 percent. For disease man-agement, Vilich-Meller (1992), demonstrated that wintererye/winter-wheat and spring-barley/oats mixtures reduced fungal leafdiseases. Lennartsson (1988), showed a wheat cultivar mixturereduced the incidence of the soilborne pathogen of take-all diseaseof wheat. Garrett and Mundt (1999), used epidemiologicalmodeling to demonstrate that varietal mixtures reduced the chanceof fungal spores encountering a susceptible plant in a mixture.Thus, there are additional benefits to crop mixtures such as citrusinterplanted with guava. Although guava potentially may be re-pellent to ACP, the crop mixture could cause confusion and areduction in ACP finding citrus hosts.

As a group, plants in the Myrtaceae in addition to guava havebeen recognized for their insect repellent and insecticidal qualities(Lee et al., 2004; Isman, 2000). For example, the essential oils ofEucalyptus sp. have reported insect repellency and even toxicityagainst many insect species (Batish et al., 2009). In Florida there area number of native, ornamental, and invasive Myrtaceous species(Wunderlin, 1982). Many of these are better suited to Florida'ssubtropical to temperate climate and can withstand lower tem-peratures. Some of these Florida Myrtaceous species may have re-pellent capabilities as well and could be explored for their efficacyagainst the Asiatic citrus psyllid and other psyllid species thatvector Liberibacter species.

Acknowledgments

We acknowledge and thank Matt Hentz, Kathy Moulton, EarlTaylor, Len Therrien, Joanne Hodge, Kim Poole, Ashley Ford, TadDallas, Kyle Berk, Steve Mayo, Sean Reif, and Montserrat Watson(USDA-ARS, U. S. Horticultural Research Laboratory, Fort Pierce, FL)for their assistance during the Florida experiment. In Texas, wewish to acknowledge and thank Daniel Martinez, Albino Chavarria,Jose Renteria, Andrew Parker, Steven Rodriguez, and JohnnyRodriguez for their critical technical support. Thanks are alsoextended to Mike Irey, Tim Gast and Jim Snively (Southern GardensCitrus) and to Mike Stewart, Charlie Lucas, John Merritt and RobertUnderbrink (Consolidated Citrus) for their support.

This article reports the results of research only. Mention of atrademark or proprietary product is solely for the purpose ofproviding specific information and does not constitute a guaranteeor warranty of the product by the U.S. Department of Agricultureand does not imply its approval to the exclusion of other productsthat may also be suitable. This research was in-part sponsored bythe Florida Citrus Research and Development Foundation.

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