Early Detection of Huanglongbing-Associated Bacteria in Psyllids

Early Detection of Huanglongbing-associated Bacteria in Psyllids

Keremane Manjunath, Chandrika Ramadugu, Susan Halbert* and Richard Lee

USDA-ARS, National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA and *Division of Plant Industry, FDACS, Gainesville, FL Liberibacters may have evolved with psyllids, probably in the family Triozidae Association of HLB and true citrus may be very recent Movement of citrus into new areas has resulted in new diseases- CVC, leprosis, sudden death. HLB?

Origins of citrus, psyllids and Liberibacters Liberibacters introduced into cultivated citrus recently?

Origin of citrus and its relatives

Diaphorina

Trioza

Worldwide distribution of ruataceous plants

Liberibacters in psyllids? Evolution of plants, psyllids and Liberibacters

Citrus ichangensis, C. latipes C. Jambhiri, C. limonia, C. assamensis, C. karna, C. limettoides, C. aurantium and Zantoxylum progenitors of citron. Lemon and Calodendrum some mandarins Clausena

Microcitrus, Eremocitrus, Clymenia, Oxanthera in Australia, New Calodenia and Papua New Guinea

Zantoxylum Clausena 35 35 Ravenia spectabilis (6915) 4 99 Erythrochiton brasiliensis (6922) Ruta 4 14 99 CasimiriaCasimiroa tetrameria tetrameria(6212) (6212) 14 Calodendrum capense (6919) chalepensis 87 87 ZanthoxylumZanthoxylum flavum flavumflavum (6222) (6222(6222)) 104 (7203) 78 78 104 26 Micromelum minutum minutum (7211) (7211) 100 Ruta 26 11 100 11 13 Glycosmis trichanthera (7197) graveolens 99 22 13 Glycosmis pentaphylla (7195)(7195) (7204) 99 22 94 94 Glycosmis perakensis (7198) 5 100 100 GlycosmisGlycosmismauritiana mauritiana (7196)(7196) 99 5 6 Clausena anisata (7201)(7201) 99 7 6 Clausena excavata (7202)(7202) 8 8 7 88 88 Clausena hardmandiana (6714)(6714) 10010014 Clausena lansium(6211lansium(6211)) 98 98 14 Clausena lansium(6725lansium(6725)) 100 100 Clausena lansium(6814lansium(6814)) 9 Merrillia caloxylon (6724)(6724) 9 Murraya sp. Ex-wongarra (6777 (6777) ) 8 Murraya paniculata G59 (6818) 100 100 8 Murraya paniculata OrangeOrng. Jasmine Jasmine (6705 (6705) ) 100 100 Murraya paniculata (6813) 3 Murraya paniculata (6190) Non-Aurantioideae 94 94 Murraya paniculata (6909) 11 11 Murraya paniculata (6817) Murraya paniculata (6812) Murraya paniculata 10 Hawaiian mock orng.(6703) 10 ParamignaParamignya lobata lobata (6726) (6726) 4 ParamignyaParamigna scandens scandens (6708) (6708) Micromelinae 70 704 Microcitrus papana(6741)papuana(6741) 5 100 Wenzelia dolichophylla (6710) 3 3 82 100 Triphasia trifolia (6709) 8218 18 Pamburus missionis (6216) 3 3 Pamburus missionis (6702) 100 16 100 Aegle mamelosmarmelos Bael Bael fruit fruit(6904)(6904) Clauseninae 22 16 12 Aegle mamelosmarmelos (6229) (6229) 12 Afraegle paniculata Nig.powder flask (6903) 100 2 99 99 2 Afraegle paniculata (6213) Phylogenetics for properAegelopsis chevalieri (7199) 9090 Afraegle gabonesis (7200) Merrillinae 2 Pleiospermium latialatum(6718) 2 4 Swinglea glutinosa (4043) 4 5 Hard shelled citrus (7209) 6161 5 21 Limonia acidissima (7210) 8585 21 Feroniella sp. (6926) 100 100 Feroniella oblata (6773) Triphasiinae Naringi crenulata (2879) identification 4 of6 hostCitropsis gillettiana (6746) 6 8 3 8 Citropsis gabunensis(3286) 3 99 99100 100 Microcitrus warburgiana(6750) 6 5 Severinia disticha (6775) 6 5 Severinia buxifolia(6208) Citrinae 6 99 99 Severinia buxifolia(6700) 88 99 99 Atalantia roxburghiana(6727) 88 4 7 8 Atalantia monophylla(6784) 4 100 8 Pleiospermium alatum(6707) 100 Atalantia ceylanica(3287) Citrus ichangensis(2327)97 6 Poncirus trifoliata Flying dragon (3330) Balsamocitrinae 6 Poncirus trifoliata Pomeroy(1717) 100 100 Severinia sp. (6736) 7 Microcitrus inodora (6740) 7 Citrus alata (6780) 98 98 Oxanthera neo-caledonica(7194) Citrus maxima Kao Panne pummelo (2248) Microcitrus australasica (3672) Eremocitus glauca (7193) Phylogeny of citrus relatives based on Microcitrus autralisaustralis (6771) (6771) 57575 Citrus macrophylla (6779) 5 Citrus medica Arizona citron (3878) 94 94 Limocitruslittoralis(6699)Limnocitrus littoralis(6699) Maximum Parsimony analysis Citrus medica SCFS citron (3546) 9 Hard shelled citrus (6749) Gene used: nuclear malate dehydrogenase. 9 Balsamocitrus daweii (6745) Clymenia polyandra(6715) Region used has introns, exons, about 1 Kb. 100 100Fortunella hindsii (6774) 44 Citrus reticuata Tien ChiehChiehmandarin mandarin(2590)(2590) Citrus myrtifolia (6787) Numbers in red indicate bootstrap support. Citropsis daweana (6747) Fortunela polyadra (7208) Fortunela margarita(3877) Citrus sinensis (1241) Evolution of psyllids, endosymbionts and Liberibacters

Psyllids and their primary endosymbints have coevolved (Thao et al., 2000) Evolution of Liberibacters may not be synchronous with psyllids? Primary evolution of Liberibacters in Triozidae? Higher titers in Bactericera Transovarial transmission Occasional movement of Liberibacters to other psyllid groups like Diaphorina? Introduced plants and pests Through host passage via dodder or parasitic plants (Loranthus)? HLB

Longer incubation period in plants

A few months to over 2 years Prolonged latency of bacteria

When trees were severely pruned, a large number of non-symptomatic trees developed symptoms Longer latency with HLB?

In collaboration with Jose Luiz and Fernando Tersi, Cambuhy Farms, Brazil

No Plants Greening / Date % Erradicated Eradication 145 27 5/19/200519%=52% 19% 118 3 11/22/2005 3%

115 8 3/14/2006 7%

107 10 5/20/2006 22 9% 35 MTHS. 97 12 9/22/2006 12% 85 1 12/20/2006 1% HLB bacteria are systemic 84 1 3/24/2007 1% andAsymptomatic the disease Symptomatic has a long trees retained 83 0 11/29/2007 0% incubation treesperiod removed Total 52% This field trial suggested a latency of up to two years under field conditions An early symptom may not mean a recent infection

The tree may act as a source of inoculum for psyllid transmission well before the symptoms become visible

Manjunath et al., 2008 Lopes et al., 2009 Testing psyllids

David Zeisk Garnier

In a low titer situation, HLB bacteria may some times not be able to survive

Photo by Michael Rogers Garnier

In a low titer situation, HLB bacteria may move around without being encountered by the psyllids

Photo by Michael Rogers Garnier

In a low titer situation, HLB bacteria may be acquired by psyllids leading to rapid multiplication

Photo by Michael Rogers Different scenarios of sampling: Plant vs psyllid Sampling for PCR: Plant vs psyllid

About 20 days on the same plant Sucking continuously….. A PCR machine on plant… Sampling methods

1. Hand collection 2. Suction traps 3. Yellow sticky traps (not preferred) Psyllid DNA Extractions

Las can be detected from single adult psyllid, or single nymph Psyllid samples analyzed in 2007-08

9.95% Positive for Las in FL samples Seasonality of psyllid transmission of Las

35 140 30 120 25 100 Detection of Las in psyllid 20 80 adult (A) and nymph (B) 15 60 samples collected from 10 40

5 20 Samples tested Percent infected Florida in different months. 0 0 Total number of samples 123456789101112 Month tested each month and 30 100 percent samples (line graph) 25 80 20 with HLB incidence (bar 60 15 40 graph) are shown in each 10 5 20

graph. Samples tested Percent infected 0 0 123456789101112

Monthly data on the number 900 Month of psyllids collected from a 800 700 suction trap from March 600 2006 to February 2007, 500 400 located in a citrus grove in 300 psyllids 200 Palm Beach is shown in 100 graph C. 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Manjunath et al., 2008 Both psyllids and Las moved around the state and probably to other states on retail nursery plants and on Murraya plants

Big box stores provide nearly ideal place for multiplication of Las-infected psyllids

¾Friendly temperature conditions ¾Ideal young flush for psyllid growth ¾Psyllid infestation doesn’t attract attention ¾Infected plants are not likely to show symptoms while at store D. citri first find 1998

D. citri moved to Murraya nursery within a year

HLB first find Aug 2005 Early detection of Las in psyllids Early detection of HLB movement by testing psyllids

County Psyllid Total Plant Recorda Recordb Brevard 5/31/06 10/62 12/7/06 Collier 1/30/06 13/133 11/29/05 Dade 10/08/05 26/139 8/23/05 De Soto 2/13/06 7/75 12/7/05 Hendry 1/28/06 7/104 10/11/05 Highlands 8/2/05 2/12 11/15/05 Marion 6/2/06 13/56 5/17/07 Monroe 10/19/05 2/8 10/15/05 Nassau 9/15/05 1/14 No commercial citrus Palm Beach 10/5/05 6/31 9/28/05 Polk 9/2/05 12/135 9/20/07 St. Lucie 5/26/06 3/31 10/13/05

In several Counties, orchards, HLB activity was first detected in psyllids well before plant symptoms could be found Some first records were in big box stores Sample No. Dt Coll. Host Source County psyllids/extrn Ct/Las Ct/DC Las Psy DNA F0136 1.30.2006 C. sinensis Grove Collier 50 23.20 29.50 P P •An isolated grove with 3-yr old trees •No HLB in Jan 06 •No HLB found in follow up visits in April, June and August 06 •Nov 06- Found initial symptoms of HLB •LaterF0216 confirmed2.13.2006 C. sinensisby the DPIGrove De Soto 50 24.30 26.17 P P •Feb 06- Psyllid samples positive from a large grove •July 06- Follow up- Two trees with HLB, one PCR positive

F0018n 9.22.2005 C. sinenis Grove Polk 5 18.75 29.94 P P •Positive samples since Sept. 2005 •From four different sites collected several times •Positive results confirmed by repeat multi and single psyllid extractions •Confirmed by conventional PCR and sequencing •Some unique sequences! •Plant positive finds after two years! Psyllids as a tool For management of HLB Procedure Blocks were selected in the three large farms with different management practices in Brazil

Psyllid adults and nymphs were collected every week

Ricardo Harakava, Pedro Yamamoto and Fernando Tersi organized the psyllid collections and send them to Sao Paulo

One extraction per sample, each with 10 adults, or 10 nymphs in Sao Paulo and further extractions in Riverside

USDA Riverside: Real time PCR for HLB-Las and Lam Summary of psyllid tests for HLB-Las

Marchasan Cambuhy Agrindus 35 Samples Positive Samples Positive Samples Positive tested tested tested 30 Marchasan 2006/07 4 2 4 0 0 0 25 2006/08 15 1 12 0 0 0 2006/09 15 3 12 0 0 0 20 2006/10 32 7 22 0 5 0 Samples 15 tested 2006/11 23 1 15 0 4 0 Positive 2006/12 24 6 16 0 4 0 10 2007/01 26 8 18 0 0 0 2007/02 29 4 19 0 0 0 5 2007/03 24 5 9 0 3 1 0 2007/04 6 1 4 0 7 2 2007/05 6 1 7 0 2 0 Total 204 39 138 0 25 3 25 7 Agrindus Cambuhy 6 20 5

15 4 Samples Samples 10 tested 3 tested Positive 2 Positive 5 1

0 0

Monthly data of number of samples tested and number of Las positive samples found Monthly Average and Minimum Cycle threshold values

Marchasan Cambuhy 80 40 40 35 35 30 30 25 25

20 20

15 15 2006/07 2006/08 2006/09 2006/10 2006/11 2006/12 2007/01 2007/02 2007/03 2007/04 2007/05 2007/06 2007/07 2007/08 2008/01 2008/02

Min Avg Min Avg

Agrindus Cambuhy 93

40 40

Ct values 35 35

30 30

25 25

20 20

15 15 2006/07 2006/08 2006/09 2006/10 2006/11 2006/12 2007/01 2007/02 2007/03 2007/04 2007/05 2007/06 2007/07 2007/08 2008/01 2008/02

Min Avg Min Avg Cycle threshold values from qPCR of psyllid samples

Marchasan Cambuhy 80

35 35 30 30 25 25 20 20 15 15 10 10 5 5 0 0 1516171819202122232425262728293031323334353637383940 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 35 36 37 38 39 40

Agrindus Cambuhy 93

35 35 30 30 25 25 20 15 20 10 15 5 10 0 5 15 16 17 18 19 20 21 22 23 25 26 27 28 30 31 32 35 36 37 38 39 40 0

Agrindus 1516171819202122232425262728293031323334353637383940

Distribution of Ct values tested during one year period starting July 2006 (number of extractions on Y-axis, and Ct values on x-axis; not all samples with Ct value of 40 are shown) Two neighbors 2006 2007 2008

Cambuhy Farm, Brazil Jose Luiz and Fernando Tersi a neighboring farm

Fundecitrus: Silvio Lopes and Pedro Yamamoto Instituto Biologico: Ricardo Harakava Las-free psyllids

15 4 Samples Samples 10 tested 3 tested Positive 2 Positive 5 1

0 0 Important considerations

Las free plant may not mean it is free from HLB Las free psyllids on a tree may not mean the bacterium is absent Time of sampling Number of insectstestedfromasite Sampling method There is always a possibility that other known and unknown Liberibacters may be pesent Conclusions

Infected psyllids were found in orchards well before the appearance of symptoms-MONITOR BOTH PSYLLIDS AND PLANTS

Psyllid transmission of HLB bacteria exhibit seasonality with maximum infection in fall followed by spring, summer and winter- MONITOR CONTINUOUSLY

Regular monitoring psyllids in addition to scouting and eradication would strengthen HLB management

FUTUTE: qPCR may not be the best option for efficient monitoring of psyllids. Need better trapping, faster and cheaper assay Acknowledgements

Wayne Dixon, Matt Brodie and several members of the Division of Plant Industry, Gainesville, FL Pedro Yamamamoto and Sylvio Lopes, Fundcitrus, Brazil Ricardo Harakava, Biologico Instituto, Sao Paulo, Brazil Jose Luiz, Fernando Tersi, Cambuhy, Brazil Marchasan and Agrindus farms, Brazil

Ed Stover, USDA-ARS, Fort Pierce Phil Stansly, J. Qureshi, UF Immokalee R. Brlansky, CREC, UF Lake Alfred

FUNDING USDA TTAR Citrus Research Board, California FCPRAC, Florida