USDAUSDA ARSARS NationalNational ClonalClonal GermplasmGermplasm RepositoryRepository forfor CitrusCitrus andand DatesDates ReportReport onon NCGRCDNCGRCD ActivitiesActivities JuneJune 20082008 ChallengesChallenges andand OpportunitiesOpportunities MissionMission

„„ Access,Access, evaluate,evaluate, maintain,maintain, preserve,preserve, andand distributedistribute germplasmgermplasm ofof CitrusCitrus,, CitrusCitrus relatives,relatives, andand datedate palmspalms „„ MaintainMaintain informationalinformational filesfiles onon accessionsaccessions „„ ConductConduct researchresearch thatthat isis compatiblecompatible withwith primaryprimary missionmission PersonnelPersonnel

„ RichardRichard LeeLee „ Research Leader, Research Plant Pathologist (Perm) „ RobertRobert KruegerKrueger „ Curator, Horticulturalist (Perm) „ ManjunathManjunath KeremaneKeremane „ Post Doc (Temp) PersonnelPersonnel

„ Polly Balance „ Biological Technician, Lab (Perm) „ Vicki Newman „ Biological Technician, Plants (Perm) „ Jaclyn Sweet (Resigned in June) „ Biological Technician, Plants (Perm) Vicki and Jaclyn Lee Gross „ John Lehr (Started in June) „ Biological Technician, Plants (Temp) „ Leila Ruiz (.75 FTE) „ Office Assistant (Temp) „ Lee Gross (.4 FTE) „ Maintenance Mechanic Polly Leila PersonnelPersonnel

ThroughThrough UCR:UCR: ChandrikaChandrika Ramadugu,Ramadugu, VisitingVisiting AssistantAssistant ResearchResearch onon SCASCA withwith M.M. RooseRoose PartPart timetime studentsstudents Chandrika HiredHired throughthrough UCRUCR AgOpsAgOps 2.52.5 FTEFTE PersonnelPersonnel

„ NancyNancy Knap,Knap, LAOLAO „ JoAnnJoAnn Moore,Moore, LocationLocation PurchasingPurchasing OfficerOfficer „ PeggyPeggy Carroll,Carroll, LocationLocation AccountantAccountant PersonnelPersonnel changeschanges

„ PatriciaPatricia NielsenNielsen „ Retired,Retired, movedmoved toto NewNew YorkYork StateState „ BenBen RangelRangel „ 44 yearyear eligibilityeligibility endedended inin MayMay 07,07, positionposition willwill notnot bebe refilledrefilled „ JaclynJaclyn SweetSweet „ ResignedResigned toto gogo intointo businessbusiness withwith herher husbandhusband whowho justjust completedcompleted aa MBAMBA degreedegree GermplasmGermplasm collectioncollection

„ AA collectioncollection ofof collectionscollections „ Citrus variety Collection (CVC) „ Belongs to UCR GermplasmGermplasm collectioncollection

„ AA collectioncollection ofof collectionscollections „ CitrusCitrus varietyvariety CollectionCollection (CVC)(CVC) „ Belongs to UCR (Dr. T. Kahn) „ Used cooperatively by NCGRCD for characterization/evaluation, as a seed source, and occasionally material for distribution (pollen, flowers, leaves, etc; seed source trees tested for CLBV) „ SCA with UCR for maintenance and evaluation „ Over 1,010 accessions „ Endowment campaign underway by UCR to provide long term preservation of CVC GermplasmGermplasm collectioncollection

„ CitrusCitrus varietyvariety CollectionCollection (CVC)(CVC) „ 139139 totaltotal treestrees plantedplanted inin CVCCVC inin 20072007 „ Includes 27 new accessions „ Includes 10 citrus relatives „ Remainder are replants, many due to the construction relocation GermplasmGermplasm collectioncollection

„ AA collectioncollection ofof collectionscollections „ ProtectedProtected collectioncollection (PC)(PC) „ Small potted trees propagated from pathogen tested budwood „ Maintained in protected conditions in the screenhouse „ about 425 accessions „ New accessions are quarantined by USDA-APHIS and CDFA officials at the Repository, released to PC only after therapy and pathogen testing GermplasmGermplasm collectioncollection

„ AA collectioncollection ofof collectionscollections „ ProtectedProtected collectioncollection (PC)(PC) „ Pathogen testing „ CTV ELISA annually „ Tested for DMV/CLBV „ Tested for leprosis „ testing for viroids and HLB via PCR „ About 40 being re-indexed for psorosis and for viroids (5 year schedule is goal) GermplasmGermplasm collectioncollection

„„ AA collectioncollection ofof collectionscollections „ CitrusCitrus relativesrelatives collectioncollection „ MaintainedMaintained inin greenhousesgreenhouses ifif extremelyextremely coldcold sensitivesensitive „ FieldField plantingplanting inin thethe moremore moderatemoderate coastalcoastal temperaturestemperatures nearnear Irvine,Irvine, somesome alsoalso inin thethe CoachellaCoachella ValleyValley inin thethe lowlow desertdesert climateclimate „ 8080 accessionsaccessions ofof generagenera otherother thanthan CitrusCitrus representingrepresenting 2828 ofof 3333 generagenera ofof AurantioideaeAurantioideae family,family, howeverhowever somesome generagenera representedrepresented byby onlyonly oneone accessionaccession GermplasmGermplasm collectioncollection

„„ AA collectioncollection ofof collectionscollections „ CitrusCitrus relativesrelatives collectioncollection „ 1818 treestrees plantedplanted atat SouthSouth CoastCoast FieldField StationStation „ 2 new accessions „ Field 45 is being removed, accessions are being reestablished into Field 25. Accessions remaining in Field 45 are those we commonly get seed requests for. GermplasmGermplasm collectioncollection „ CitrusCitrus relativesrelatives collectioncollection „ CoachellaCoachella ValleyValley AgriculturalAgricultural ResearchResearch StationStation (CVARS)(CVARS) „ Citrus relatives „ Most lost in January 07 freeze „ Replanted 40 trees „ Total Citrus relatives accessions: 22 „ Citrons „ wild or semi wild citrons, some have no flesh similar to Yemeni type and as such are of interest to Rabbis „ Trifoliate selections: 20 GermplasmGermplasm collectioncollection „ CitrusCitrus relativesrelatives collectioncollection „ CoachellaCoachella ValleyValley AgriculturalAgricultural ResearchResearch StationStation (CVARS)(CVARS) „ Seedling populations (monoembryonic & zygotics) „ 36 total accessions, 177 total trees „ Persian lime SPB-7 „ Wood pocket-free budline? „ Determine performance in warm climate GermplasmGermplasm collectioncollection

„ AA collectioncollection ofof collectionscollections „ DateDate PalmPalm collectioncollection

„ Coachella Valley Agricultural Research Center CVARS (most recently collection) „ Brawley (Imperial Valley Agricultural Research Station) GermplasmGermplasm collectioncollection

„ AA collectioncollection ofof collectionscollections „ Date Palm collection „ Brawley—trees need clean up. Currently under contract to clean up trees and remove debris from ground. GermplasmGermplasm collectioncollection

„ AA collectioncollection ofof collectionscollections „ DateDate PalmPalm collectioncollection „ 128 accessions total, includes some related species „ About 500 trees at CVARS, about 100 trees at Brawley „ CDFA permit received which allowed accessing 10 new varieties from Arizona „ Received 6 seed populations from Baja California from exploration trip by R. Krueger in Oct 2007 AccessionsAccessions

„ NCGRCDNCGRCD recognizedrecognized asas aa quarantinequarantine facility,facility, cancan receivereceive accessionsaccessions directlydirectly fromfrom nationalnational andand internationalinternational sourcessources „ OneOne ofof threethree recognizedrecognized quarantinequarantine facilitiesfacilities forfor CitrusCitrus introductionsintroductions „ NCGRCDNCGRCD „ CitrusCitrus ClonalClonal ProtectionProtection Program,Program, CACA „ CitrusCitrus IntroductionIntroduction Program,Program, FLFL 1400 DistributionsDistributions

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0 2003 2004 2005 2006 2007 911 distributions in 2007 total of 90 different orders NewNew ProjectProject PlanPlan ApprovedApproved

„ SpecificSpecific projectproject objectivesobjectives „ StrategicallyStrategically expandexpand andand improveimprove collectionscollections ofof prioritypriority geneticgenetic resourcesresources ofof citruscitrus andand datedate palmpalm andand associatedassociated informationinformation „ ConserveConserve andand propagatepropagate citruscitrus andand datedate geneticgenetic resourcesresources efficientlyefficiently andand effectively,effectively, andand distributedistribute pathogenpathogen--testedtested samplessamples andand associatedassociated informationinformation worldwideworldwide NewNew ProjectProject PlanPlan ApprovedApproved

„ SpecificSpecific projectproject objectivesobjectives „ StrategicallyStrategically characterizecharacterize ((““genotypegenotype””)) andand evaluateevaluate ((““phenotypephenotype””)) citruscitrus andand datedate palmpalm geneticgenetic resourcesresources forfor prioritypriority geneticgenetic andand horticulturalhorticultural traitstraits „ DevelopDevelop moremore rapidrapid andand accurateaccurate diagnosticdiagnostic methodsmethods forfor prioritypriority graftgraft--transmissibletransmissible pathogenspathogens ofof citruscitrus toto promotepromote exchangeexchange ofof pathogenpathogen--testedtested stockstock andand efficientlyefficiently screenscreen forfor hosthost--plantplant resistanceresistance DatabasesDatabases

„ DatabasesDatabases maintainedmaintained onon http://www.arshttp://www.ars-- grin.gov/rivgrin.gov/riv „ LocalLocal databasesdatabases havehave beenbeen migratedmigrated toto AccessAccess „ LookingLooking forwardforward toto improvementsimprovements inin GRINGRIN-- GlobalGlobal DatabasesDatabases

„ GRINGRIN „ Most “permanent” accessions (CRC numbers) now loaded „ Many “non-permanent” accessions are loaded „ Ones not loaded are lacking critical information „ Date palms are up to date „ Improved information delivery „ When possible, provide improved passport data „ Improved readability by incorporating HTML coding „ Link to CVC, CCPP websites, other resources when available „ Older entries (1980s) need improvement SpecificSpecific CooperativeCooperative AgreementsAgreements

„ CitrusCitrus VarietyVariety Collection:Collection: Evaluation,Evaluation, Maintenance,Maintenance, andand AcquisitionAcquisition (Tracy(Tracy Kahn)Kahn) „ MolecularMolecular CharacterizationCharacterization ofof thethe CitrusCitrus VarietyVariety CollectionCollection (Mikeal(Mikeal Roose)Roose) „ BiologicalBiological ControlControl andand ManagementManagement ofof thethe GiantGiant WhiteflyWhitefly (Thomas(Thomas Bellows)Bellows) (direct(direct funded)funded) SpecificSpecific CooperativeCooperative AgreementsAgreements

„ CitrusCitrus VarietyVariety Collection:Collection: Evaluation,Evaluation, Maintenance,Maintenance, andand AcquisitionAcquisition (Tracy(Tracy Kahn)Kahn) „ CooperatingCooperating onon characterizationcharacterization ofof citruscitrus relativesrelatives „ 600600 treestrees werewere evaluatedevaluated forfor 6060 differentdifferent descriptorsdescriptors suchsuch atat growthgrowth habit,habit, spinespine length,length, laminalamina shapeshape andand width,width, flowerflower arrangement,arrangement, etcetc „ DigitalDigital imagesimages readyready toto loadload intointo GRINGRIN SpecificSpecific CooperativeCooperative AgreementsAgreements

„ MolecularMolecular CharacterizationCharacterization ofof thethe CitrusCitrus VarietyVariety CollectionCollection (Mikeal(Mikeal Roose)Roose) „ Phylogenetic analysis of Citrus based on sequences of six nuclear genes of 33 different Citrus and related accessions „ Malate dehydrogenase (MDH)—housekeeping gene „ P12 (blight-related gene) „ Aspartate transcarbamylase (ATC)—housekeeping gene „ Limonoid glucosyltransferase (LGT) (fruit debittering gene) „ CC-NBS-LRR type R (CTV resistance region) „ beta-carotene hydroxylase (HyB) SCA- Molecular Characterization of the Citrus Variety Collection (Mikeal Roose) Sequence data from 6 nuclear genes from 33 different Citrus and related accessions. High heterozygosity was observed in many samples. Phylogenetic trees derived from haplotypes showed incongruence among gene trees, inferred to be due to hybridization with some lineage sorting. The data documents origin of certain hybrid accessions, and enhances understanding variation within species.

1 Atalantia ceylanicaREL 1 Citropsis gabunensisREL 1 Naringi crenulataREL 1 Swinglea glutinosa REL 4 Atalantia ceylanica REL 1 Ichang PPD 4 Swinglea glutinosa REL 1 Hanayu A PPD 4 Rubidoux B SOR 1 Mexican A LIM 4 Winged B LIM 1 Frost eureka A LIM 4 Nagami KUM Kumquat 86 1 Arizona 861 CIT 4 Indian sour B CIT 1 Mexican B LIM Citron 100 4 Indian sour A CIT 82 1 Indian sour A CIT 1 South coast Field Station CIT 4 South coast Field Station CIT 81 90 1 Palestine sweet B LIM 87 4 Mexican A LIM 1 Nasnaran A MAN 4 Palestine sweet LIM Citron 1 Indian sour B CIT 100 4 Frost eureka A LIM 99 1 Winged A LIM 4 Arizona 861 A CIT 99 100 1 Kalpi A LIM 96 1 Kalpi B LIM 4 Arizona 861 B CIT 98 4 Mountain A CIT 1 Nasnaran B MAN 1 Palestine sweet A LIM 4 Mountain B CIT 1 Tienchieh A MAN 4 Kalpi LIM 1 Tienchieh B MAN 4 Mexican B LIM 1 Korai MAN 97 4 Nasnaran A MAN 1 Cleopatra A MAN 1 Washington navel orange A MAN 4 Winged A LIM 4 Nasnaran B LIM 100 1 Hanayu B PPD 1 Nagami KUM 4 Rubidoux A SOR 1 Mountain CIT 4 Australian finger-lime AUS Australasian 1 King B TGR Mandarin 100 4 Frost Eureka B LEM 1 Winged B LIM 4 Tienchieh MAN 99 1 Frost Owari Satsuma MAN 90 99 1 Frost Eureka B LEM 4 Scarlett Emperor MAN 1 Scarlett Emperor MAN 4 Encore MAN 1 Cleopatra B MAN 4 Korai A MAN Mandarin 1 Bouquet des Fleurs B SOR 4 Cleopatra MAN 1 Rubidoux B SOR 4 Bouquet des Fleurs SOR 1 Washington navel orange B MAN 4 Washington navel orange MAN 1 Encore A MAN 4 King TGR 1 Encore B MAN 4 Ichang PPD 1 King A TGR 1 Bouquet des Fleurs A SOR 4 Hanayu A PPD Papeda 4 Korai B MAN 1 Rubidoux A SOR 99 1 Kaopan PUM Pummelo 97 4 Hanayu B PPD 100 1 Kaopanne PUM 4 Frost owari satsuma A MAN 1 Matobuntan PUM 4 Kaopan A PUM 1 Australian finger-lime A AUS Australasian 4 Matobuntan A PUM 1 Australian finger-lime B AUS group 4 Kaopanne PUM Pummelo 90 1 Flying dragon B TRF 4 Kaopan B PUM 1 Pomeroy B TRF 99 1 Flying dragon A TRF Trifoliate 4 Frost owari satsuma B MAN 100 1 Pomeroy A TRF 100 4 Matobuntan B PUM 99 4 Flying dragon TRF 4 Pomeroy TRF Trifoliate 100 Taxonomy of Murraya is confusing.

In California, plants with different morphologies (leaf shape and size) are sold as “Murraya paniculata” or Orange jessamine (jasmine).

The Citrus variety collection at Riverside has several accessions with different phenotypes (external appearances) that are labeled as Murraya paniculata.

There is a need to resolve the taxonomic identification of Murraya species for local use and also for understanding of the potential role of Murraya in spreading citrus greening.

If the Murraya species that is responsible for greening spread is Identified, only this can be regulated. It will be much easier to control certain varieties of Murraya than regulating all citrus relatives.

Photos: Chandrika Ramadugu Identification of molecular markers to differentiate between different Murraya plants

Gene fragments (both nuclear and chloroplast DNA) were amplified by PCR using primers for conserved sequences. The PCR products are now being sequenced to compare and identify SNPs (single nucleotide polymorphisms) that can be used in proper identification of the species and differentiation of the accessions. G 58-63: Plants identified by CDFA as Murraya and brought to USDA lab for HLB testing. RC 98-104 Plants collected from CVC collection. Murraya and close relatives. PCR products of chloroplast gene fragment RPS16 PCR products of nuclear gene fragment MDH

3 Kb

1 Kb 1 Kb G61 G61 G58 G59 G60 G62 G63 G62 G63 G58 G59 G60 RC98 RC99 RC98 RC99 RC100 RC101 RC102 RC103 RC104 RC101 RC102 RC103 RC104 RC100 1Kb ladder 100bp ladder 1Kb ladder PCR products of chloroplast gene fragment RBC L PCR products of nuclear gene fragment LGT 3 Kb 3 Kb

1 Kb 1 Kb 1Kb G61 G61 G62 G63 G62 G63 G58 G59 G60 G58 G59 G60 RC98 RC99 RC98 RC99 RC100 RC101 RC102 RC103 RC104 RC100 RC101 RC102 RC103 RC104 1Kb ladder G58 to G63, RC 100-101 are Murraya paniculata samples. RC 99 is M.koenigii. RC 98, 102,103 are Clausena sp. RC 104 is Mex. Lime control. Developing diagnostic methods for Citrus Leprosis Virus: an Exotic Disease that is spreading in Central America In cooperation with R. Brlansky and A. Guerra, Univ. of FL, Lake Alfred, FL

Leprosis will kill trees in 3 yrs RT-PCR assay developed Spread by Brevipalus mite species M1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M2 Disease management adds 25% to 4361 the cost of production in Brazil 2072 2027 Rapid diagnostic method needed for control 1500

600 564 300 Leprosis gives 400bp product INF INF INF Healthy

2-fold dilutions of samples

Leprosis lesions on fruit, leaves, and twigs Serological methods being developed GenomeGenome organizationorganization ofof CCLVCCLV

ORF 2 ORF 1

5’ M7 3’ G OH *AG1-H10 *AG1-C09 CCLV RNA 1 8730 nt

ORF 2 ORF 4 ORF 1 ORF 3 CCLV RNA 2 5’ M 3’ 4969 nt 5’ M7 3’ G OH

*AG2-D08 *AG1-A01

Conserved domains of ORF 1, RNA 1 Methyltransferase OTU-like cysteine transferase Open Reading Frame (ORF) FtsJ-like methyltransferase Subgenomic RNAs Helicase Dig-labeled probes used in Northern blot analysis RdRP ConfirmedConfirmed graftgraft--transmissibletransmissible naturenature ofof CitrusCitrus measlesmeasles andand reportedreported inin CaliforniaCalifornia forfor thethe firstfirst timetime DevelopedDeveloped aa robustrobust diagnosticdiagnostic realreal timetime PCRPCR assayassay forfor detectiondetection ofof greeninggreening ((HLBHLB)) fromfrom pysllidspysllids;; applyingapplying thisthis approachapproach forfor earlierearlier detectiondetection ofof HLBHLB

Assay developed in cooperation with Susan Halbert, FL DPI, Susan Webb, Univ. of FL, and NCGRCD, Riverside

Assay will be used to screen germplasm for relative tolerance/resistance to HLB Early detection Sampling for PCR: Plant vs psyllid

•Detection from plants is mainly based on visualization of symptoms followed by PCR confirmation

•HLB bacteria can be detected in psyllid nymphs and adults •Bacteria reach detectable levels in nymphs within a few days •Are psyllids better targets than About 20 days on the same plant plants for early detection of HLB Sucking continuously….. activity? A PCR machine on plant… Early detection of HLB bacteria in psyllids Development of a real-time PCR detection method

™Modification of method developed by Wenbin Li et al., 2006

™A primer‐probe combination for the psyllid DNA as internal control

™Preliminary standardization was done using psyllid samples from infected areas in Miami

™Samples shown to be positive by conventional PCR were used for standardization Standard curve

Plasmid Ct (dR) dilution 10-1 9.08 10-2 13.1 10-3 16.63 10-4 19.88 10-5 23.04 10-6 26.83 10-7 30.44 10-8 33.9 40 35 y = -3.502x + 37.373 30 2 ™A plasmid DNA with HLB target sequence R = 0.9994 25 (16s rDNA) was used as template 20 ™Serial dilutions were used as templates 15 10 ™Standard curve was prepared

cycle threshhold (Ct) 5 0 -8 -7 -6 -5 -4 -3 -2 -1 Starting plasmid DNA dilution Real time PCR from a psyllid DNA sample

Plasmid sample Ct dilution (dR) 10-1 62 22.93 10-2 62 26.34 10-3 62 29.38 10-4 62 32.54 10-5 62 34.81

40

HEX 35 ™Most psyllid DNA samples can be y = -3.1982x + 41.487 R2 = 0.9931 used for detection when diluted up to 30 FAM 1000 times 25 y = -2.9965x + 38.19 R2 = 0.9956 cycle threshhold (Ct) threshhold cycle ™FAM is the fluor used for HLB 20 probe, and HEX is the fluor used for -4 -3 -2 -1 0 the psyllid DNA Log10 extract starting quantity Real time PCR in a 96-well format in Stratagene Mx3005p machine

AB

Detection of HLB- LAS Detection of psyllid with FAM probe DNA with HEX probe (internal control) ResearchResearch ActivitiesActivities

„ DevelopedDeveloped antibodiesantibodies specificspecific forfor CitrusCitrus leafleaf blotchblotch virusvirus (CLBV)(CLBV) (Dweet(Dweet mottlemottle virus)virus) „ ExpressedExpressed coatcoat proteinprotein usedused asas thethe injectinject antigenantigen „ CLBVCLBV isis seedseed--borneborne „ antibodies should be useful to verify freedom from CLBV before seed collection „ CooperativeCooperative withwith Dept.Dept. ofof PlantPlant Pathology,Pathology, UCRUCR PublicationsPublications

„ RefereedRefereed journals:journals: 1313 „ NonNon--refereedrefereed journals:journals: 1414 „ AbstractsAbstracts published:published: 22 FacilitiesFacilities

„ GreenhouseGreenhouse (5,948(5,948 ftft2)) „ Additional 5,000 ft2 in UCR greenhouses „ ScreenhouseScreenhouse (16,200(16,200 ftft2)) „ HeadhouseHeadhouse (1,375(1,375 ftft2)) „ LaboratoryLaboratory (538(538 ftft2)) „ Office/laboratoryOffice/laboratory spacespace inin trailertrailer (480(480 ftft2)) FacilitiesFacilities

Greenhouse 16-3, July when the cooling system/power failed CCMP located in this greenhouse, also lost somatic hybrids from Jude Grosser being evaluated for CTV tolerance, also CTV inoculated plants from a cross from M. Roose. FacilitiesFacilities

„ 2020 XX 2020 quarantinequarantine screenhousescreenhouse constructedconstructed inin 20072007 „ StillStill needsneeds water,water, gas,gas, benches,benches, etcetc „ TwoTwo functionalfunctional growthgrowth chamberschambers forfor thermotherapythermotherapy useuse

ChallengesChallenges

„ BudgetBudget——increasedincreased costscosts „ AsianAsian citruscitrus psyllid/HLBpsyllid/HLB „ RecoveryRecovery ofof germplasmgermplasm exposedexposed toto HLBHLB „ FloridaFlorida „ California?California? PreservingPreserving CitrusCitrus GermplasmGermplasm forfor FutureFuture GenerationsGenerations USDA ARS National Clonal Germplasm Repository for Citrus and Dates, Riverside

Thank you for your attention! [email protected]