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CPHST LABORATORYCPHST LABORATORYCPHST LABORATORY Beltsville Beltsville Beltsville

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2009 annual report2009 annual report2009 annual report

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CPHST Beltsville Laboratory

2009 Annual Report

U. S. Department of Agriculture Animal and Plant Health Inspection Service

Plant Protection & Quarantine Center for Plant Health Science & Technology

National Plant Germplasm and Biotechnology laboratory

BARC-East, Bldg-580

Powder Mill Road Beltsville, MD 20705-2350

PHONE: (301) 504-7100 FAX: (301) 504-8539

CPHST WEBSITE: http://www.aphis.usda.gov/plant_health/cphst/index.shtml

BELTSVILLE WEBSITE: http://www.aphis.usda.gov/plant_health/cphst/npgqbl.shtml

USDA Nondiscrimination Statement The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an indi-vidual’s income is derived from any public assistance program. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA’s TARGET Center at (202) 720–2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, S.W. , Washington , D.C. 20250–9410 , or call (800) 795–3272 (voice) or (202) 720–6382 (TDD). USDA is an equal opportunity provider and employer. Mention of companies or commercial products does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned. USDA neither guarantees nor warrants the standard of any product mentioned. Product names are mentioned solely to report factually on available data and to provide specific

Cover: Photos: HLB in citrus (top left provided by CHRP); Ralstonia solanacearum primers on ice (top right); PPV in the field in NY (second from top); PPV conventional PCR (third from top, left); PT SOD analyst performance comparison for one PT sample (third from top, right); Serial dilution of a target in Real-time PCR (bottom).

TABLE OF CONTENTSTABLE OF CONTENTSTABLE OF CONTENTS 2009 ANNUAL REPORT

REPORT SUMMARY ............................................................................................................................................................. 1 INTRODUCTION .................................................................................................................................................................... 3 STAFF UPDATES AND DIRECTORY……………………………………………………………………………………………… ...4

Methods development/adaptationMethods development/adaptationMethods development/adaptation

FOREIGN PLANT GERMPLASM DIAGNOSTICS…………………………………………………………………………… ……..5 Potato yellow vein crinivirus .................................................................................................................................................... 6 Columbian datura potyvirus .................................................................................................................................................... 7 Sweet potato mild speckling potyvirus ................................................................................................................................... 8 Sweet potato chlorotic fleck carlavirus .................................................................................................................................... 9 Sweet potato chlorotic stunt crinivirus. ................................................................................................................................... 10 Blackcurrant reversion nepovirus. .......................................................................................................................................... 11

CITRUS PLANT PATHOGENS Citrus Greening ...................................................................................................................................................................... 12 Citrus Varigated Cholorosis .................................................................................................................................................... 15 Citrus Leprosis Virus .............................................................................................................................................................. 16 POTATO CYST NEMATODE ................................................................................................................................................. 17 PLUM POX VIRUS ................................................................................................................................................................. 18

Biotechnology platforms evaluation/adaptation

CANARY ................................................................................................................................................................................ 20

Methods validationMethods validationMethods validation

Phytophthora kernoviae .......................................................................................................................................................... 23 Potato Wart ............................................................................................................................................................................. 24

Proficiency test panel developmentProficiency test panel developmentProficiency test panel development

Proficiency Testing Program For High Consequence Plant Pathogens ................................................................................. 25

Quality management/quality assuranceQuality management/quality assuranceQuality management/quality assurance

Released Work Instructions .................................................................................................................................................... 28

Plant pathogens detection activitiesPlant pathogens detection activitiesPlant pathogens detection activities

Pathogen Detections Diagnosed by CPHST- NPGBL in 2009 ............................................................................................... 30

THECHNOLOGY TRANSFERTHECHNOLOGY TRANSFERTHECHNOLOGY TRANSFER

Hands-on Laboratory Training ................................................................................................................................................ 32 Publications, Presentations and Meetings .............................................................................................................................. 33 STAFF DIRECTORY .............................................................................................................................................................. 35

This Annual Report is an overview document that highlights the many diverse activities at this Plant Protection & Quaran-tine (PPQ) Laboratory; please view it as an attempt to provide a “snapshot” of our high visibility work. Our mission is to develop and transfer scientifically-based methods, innovative tools, and state-of-the-art technologies to PPQ and other state and federal agencies to reduce risk levels associated with potential, new, and established problem species

METHODS DEVELOPMENT/ADAPTATION - Foreign Germplasm diagnostics. Development and adaptation of real-time PCR assays for targeted viruses in foreign Germplasm: Developed six conventional RT-PCR assays and six RT-qPCR assays for the PHP Germplasm Program [Sweet potato mild speckling potyvirus (SPMSV), Columbian datura potyvirus (CDV), Blackcurrant reversion nepovirus (BRV), Potato yellow vein crinivirus (PYVV), Sweet potato chlorotic fleck carlavirus (SPCFV), and Sweet potato chlorotic stunt crinivirus (SPCSV-WA)]. Released work instructions to PPQ.

Development of group-specific nucleic acid-based diagnostic assays for the improved detection of Geminiviruses and Potyviruses infecting imported Germplasm: Developed internal control for real-time PCR assay for the detection of RNA viruses. The assay is based on Nad5 gene RNA and is multiplexed with virus specific primers and probes for the de-tection of RNA viruses in foreign Germplasm. Released two work instructions to PPQ for the total RNA extraction from sweet potato for the detection of Potyviruses and for the preparation of cDNA from total RNA for the detection of Potyvi-ruses.

METHODS DEVELOPMENT/ADAPTATION - Citrus plant pathogens. HLB PCR diagnostics development and improvement: Compared validated PCR protocols to new PCR methods for detection of HLB bacteria. Developed a genomescan system to identify candidate genes for HLB additional real-time PCR confirmation assays and diagnosis at the genus, species and isolate levels.

CVC validation and implementation: Completion of six work instructions for the non-validated DNA extraction, bacterial isolation, ELISA, conventional PCR and RFLP, real-time PCR, and biological indexing. Designed new primers and probes specific to Xylella fastidiosa CVC strains based on bio-informatic analyses on the gene islands. Preliminarily validation of a qPCR and one conventional PCR protocols with DNA from various X. fastidiosa isolates

Validation and improvement of Citrus Leprosis Virus diagnostics: Reviewed two conventional PCR methods pub-lished for CiLV diagnostics and completed the bio-informatics analyses on the genome of citrus leprosis virus cytoplasmic type. Began validation of existing conventional RT-PCR method and designed new primers and probes for development of a qPCR and improvement of the existing conventional PCR to detect the virus. Established new international collabora-tions to support the method development and validation activities.

METHODS DEVELOPMENT/ADAPTATION - Potato cyst nematodes. Validation of the ARS-developed PCR detection method for PCN and development of real-time PCR assay: Opti-mized the RFLP analysis for PCN diagnosis from overnight down to 2 hrs and expanded to differentiation between G. rostochiensis and G. tabacum. Developed a multiplex real-time PCR for the detection and identification of potato cyst nematode (PCN) and tobacco cyst nematodes (G. tabacum). Manuscript in press.

METHODS DEVELOPMENT/ADAPTATION - VIRAL PLANT PATHOGENS. Advanced development of PPV diagnostics: A PPV W-like isolate from illegally introduced Ukrainian prunus germplasm have been identified. Working on the molecular and serological characterization of this isolate is underway. Improved PPV W sub-group specific RT-PCR method (designed new forward primers). Select PPV isolates from USA (NY State) Bul-garia, Ukraine and Egypt have been preserved by grafting on woody indicators or lyophilization of PPV infected tobacco or prunus tissue. Three international cooperative agreements with countries from Eastern Europe aimed at finding and char-acterization of PPV-W like, Cherry and other unusual PPV isolates.

BIOTECHNOLOGY PLATFORMS EVALUATION/ADAPTATION– CANARY. Evaluation and adaptation of CANARY technology to plant pathogen targets: Established basic lab methods and procedures for detection of plant pathogens by CANARY technology. These methods include B cell and pathogen prepara-tion, sample testing, data analysis and adaptation of hybridoma cells for monoclonal antibody production. Determined that the CANARY assay detection limit of as few as 3 CFUs Ralstonia in a single test and that only

REPORT SUMMARYREPORT SUMMARYREPORT SUMMARY 2009 ANNUAL REPORT

2009 ANNUAL REPORT CPHST BELTSVILLE, MD PAGE 1

10 minutes or less were required for sample preparation and sample testing. Demonstrated the detection of Phy-tophthora ramorum by CANARY from infected Rhododendron leaves. Tested 9 other species of Phytophthora using mycelial dilutions prepared from pure cultures.

METHODS VALIDATION - Validation of diagnostic qPCR assays for detection and diagnosis of Phytophthora kernoviae, an exotic pathogen of Beech, Oak and other Hosts: Modified two established Real-time (q) PCR assays developed in by Fera (Great Britain), to include internal amplification quality control assays targeting both Phytophthora and Plant DNA targets. Work instructions in progress for final release.

Validation of a conventional PCR test and a Real-time (q) PCR test for rapid on-site detection of potato wart (Synchytrium endobioticum): Completion of the validation of conventional and 2 qPCR assays for diagnosing S. endobioticum. Initiated development of a reliable inoculation protocol for potatoes under containment conditions that, once refined will give us the ability to generate pathogen-infected materials to simplify sample extraction from potato wart infested soils and subsequent testing.

PROFICIENCY TEST PANEL DEVELOPMENT Development of proficiency testing reagents and administrating Proficiency Testing Program for high con-sequence plant pathogens

Provided the NPPLAP with Phytophthora ramorum PT panels for sixth year (started in 2005) and with HLB panels for third year (started 2007).

Tissue samples were introduced to the PT panels in addition to the DNA samples to evaluate analysts’ profi-ciency in DNA extraction procedure.

Twenty two (22) analysts from 14 laboratories were certified for P. ramorum diagnostics as a result of the PT09 program.

Sixteen analysts from 10 laboratories have been certified for HLB screening or confirmatory diagnostics for 2009.

QUALITY MANAGEMENT/QUALITY ASSURANCE– ISO. The NPGBL ISO 17025 Quality manual was near completion by the end of FY 09. The Quality Manager authorized 29 new or edited work instructions. In addition the Quality Manager conducted internal auditing and provided annual training to the staff. The QM continues in the next FY to complete all documentations and prepare for the register audit before the close for calendar 2010.

PLANT PATHOGENS DETECTION ACTIVITIES. The Lab performed confirmation diagnostics for 401 samples including potato cyst nematode (in parallel with NIS-MDL), Ralstonia solanacearum Race 3 biovar 2, CVC, HLB in psyllids, and Plum Pox Virus. The Lab work to transi-tion the operational (or routine) Plum Pox Virus testing to MDL. That should be completed in FY 10. Forensic analy-sis of PPV isolates (stain and sub-strain determination/ examination) will continue to be done by the NPGBL.

TECHNOLGY TRANSFER. The laboratory held 13 training sessions, one of which was delivered in Mexico. The lab and NPGBL Quality Man-ager delivered over 29 new or updated work instructions. Four of the work instructions for HLB were translated into Spanish.

REPORT SUMMARY REPORT SUMMARY REPORT SUMMARY (CONTINUED)(CONTINUED)(CONTINUED) 2008 ANNUAL REPORT

PAGE 2 2009 ANNUAL REPORT CPHST BELTSVILLE, MD

Scientists at the Laboratory have a demonstrated experience and expertise and promote interdisciplinary collaboration within and outside of the USDA APHIS with scientists that possess diverse professional backgrounds within the disciplines and associated disciplines of plant pathology, molecular biology, genomics, and advanced diagnostics.

Programs at the Laboratory utilize cutting-edge technologies from the fields of plant pathology, molecular biology, human and animal biochemical and molecular clinical diagnostics, and bio-detection to develop, adapt, and improve methods for accurate and rapid diagnosis of plant pathogens. Our scientists validate plant pathogen diagnostic methods prior to stake-holder release to assure their performance and fit for purpose in regulatory programs. We strive to achieve timely transfer of bio-technology and cutting-edge diagnostic tools that are field deployable and ap-propriately uncomplicated in operation primarily for PPQ emergency response and eradication programs. Tools are de-ployed to stakeholders following validation and inter-laboratory validations through clearly written standard operating pro-cedures; hands-on laboratory training for our end users within and outside of PPQ; and contribution from the CPHST NPGBL quality assurance program. The Laboratory not only deploys cutting-edge diagnostic methods but utilizes these and other methods to accurately and rapidly diagnose and differentiate high consequence and select agent plant pathogens with non-routine status, or for which operational methods have not been deployed but require federal confirmation. In these situations the NPGBL conducts sample testing in parallel with the PHP Molecular Diagnostic Laboratory (MDL). We are committed to quality in biochemical and molecular diagnostics and are nearing completion of ISO 17025:2005 cer-tification and soon pursuing ISO certification as a Proficiency Test provider. The Laboratory performs its work with a high proficiency level, and is proficiency tested in the operation of performed diagnostic methods. The Laboratory conducts out-reach to the plant pathology diagnostic community by providing technical support to scientists within the National Plant Pathogen Diagnostic Network (NPDN), PPQ port and regional identifiers, the PPQ Molecular Diagnostics Laboratory (MDL), and the state departments of agriculture in the detection of regulatory plant pathogens by providing SOPs, hands-on laboratory training, and providing any troubleshooting required for PPQ validated diagnostics. The Laboratory is a key component of the PPQ National Plant Pathogen Laboratory Accreditation Program (NPPLAP). Our laboratory is responsible for proficiency test panel development, delivery, and first-level evaluation of proficiency tests con-ducted by scientists who perform diagnostics on behalf of APHIS PPQ using CPHST-validated methods. Our scientists contribute their expertise as members of scientific committees of the North American Plant Protection Or-ganization; the Integrated Consortium of Laboratory Networks (ICLN) representing the National Plant Diagnostics Network (NPDN) on the Methods, Proficiency Testing/Quality Assurance, and Training Sub-groups; the NPDN Diagnostics Commit-tee; the Quadrilateral Scientific Collaboration (Quads) Diagnostic Tools Collaboration Project; several committees of the American Phytopathological Society and other governmental and discipline related scientific groups and committees.

IIINTRODUCTIONNTRODUCTIONNTRODUCTION


Beltsville Lab The CPHST Laboratory was established in 1994 under the previous APHIS PPQ Methods Development Laboratory concept. In 1999, the Laboratory became part of the newly developed Center for Plant Health Science and Technology. The Laboratory mission is to develop, adapt, validate, implement, deploy, and use advanced biochemical and mo-lecular methods for the detection of high consequence plant pathogens, including the APHIS Select Agents and plant pathogens in foreign Germplasm.

National Plant Germplasm and Biotechnology Laboratory (NPGBL) Director: Laurene Levy Location: Beltsville, MD Phone: (301) 504-7100 Fax: (301) 504-8539

SSSTAFFTAFFTAFF UPDATESUPDATESUPDATES ANDANDAND DIRECTORYDIRECTORYDIRECTORY


Laboratory Administration • Dr. Laurene Levy, Plant Pathologist & Laboratory Director

• Ms. Renee DeVries, Plant Pathologist & Quality Manager

Laboratory Administrative Support • Ms. Hazel Goodwin, Laboratory Support Assistant

Senior Scientific Staff • Dr. Wenbin Li, Plant Pathologist, Citrus Pathogen Detection Program Leader

• Dr. Zhaowei Liu, Plant Pathologist, Biotechnology Evaluation Program Leader

• Dr. Vessela Mavrodieva, Plant Pathologist, Proficiency Test Program Manager & Plum Pox Virus Detection Program Leader

• Dr. Mark Nakhla, Plant Pathologist, Foreign Germplasm Pathogen Detection Program Leader

• Dr. Kurt Zeller, Plant Pathologist, Fungal Pathogen Detection Program Leader

Junior Scientific Staff • Ms. Sarika Negi, Agriculturist, Proficiency Test Panel Development Manager

• Ms. Kristina Owens, Plant Biologist, Foreign Germplasm Pathogen Detection Program

• Mr. Deric Picton, Agriculturist, Foreign Germplasm Pathogen Detection Program

• Ms. Kate Rappaport, Plant Biologist, Biotechnology Evaluation Program

• Ms. Elizabeth Twieg, Plant Pathologist, Citrus Pathogen Detection Program

• Dr. Gang Wei, Agriculturist, Foreign Germplasm Pathogen Detection Program

• Ms. Karen Williams, Plant Biologist, Proficiency Test & Plum Pox Virus Detection Programs

• Dr. Ping Yang, Plant Biologist, Fungal Pathogen Detection Program

PPPLANTLANTLANT PATHOGENSPATHOGENSPATHOGENS METHODSMETHODSMETHODS DEVELOPMENTDEVELOPMENTDEVELOPMENT

Foreign Plant Germplasm Diagnostics


Development and adaptation of real-time PCR assays for targeted viruses in foreign germplasm CPHST STAFF: Mark Nakhla (lead); Deric Picton, Kristina Owens, Gang Wei CHAMPIONS: Joseph Foster , Jorge Abad, Margarita Licha, Clarissa Maroon-Lango CONTACT: Mark Nakhla ([email protected], 301-504-7100)

carlavirus (SPCFV), Sweet potato mild speckling potyvirus (SPMSV) and Sweet potato chlorotic stunt crinivirus (SPCSV) to be delivered on 2009. Since all these viruses have RNA genome, NPGBL developed reverse-transcription real-time PCR (RT-qPCR) work instruction for the detection of each virus. PGQP requested that in addition to real-time PCR we provide a conventional PCR assays for each real-time target so PGQP can sequence PCR prod-ucts from a conventional assay conducted as a result of any real-time PCR positive test. Therefore in addition to the pro-posed real-time PCR assays, we also adapted or developed reverse-transcription conventional PCR assays using avail-able sequences. During FY 09, NPGBL delivered to PQQP fourteen work instruction (Table 1) describing assays we developed for the detection of targeted viruses in foreign germplasm.

Pome fruits, stone fruits, ribes, rubus, potato, sweet potato, corn, sugarcane and grasses represent impor-tant part of American agriculture. Foreign germplasm is needed in order to incorporate genes of value from outside the U.S. gene pool. Germplasm may contain many diseases and insects not found in the U.S. there-fore the importation of these plant species is generally prohibited. However, small quantities of germplasm may be introduced into the United States under quar-antine while undergoing a set of pathogen detection tests and if needed therapeutic treatments. When completed, pest-free plant germplasm be released and distributed to the U.S. user community. Molecular di-agnostic methods are much faster than traditional bio-logical indexing methods and very sensitive comparing to serodiagnostic techniques. There is a need to adapt, develop and validated molecular diagnostic tests for several virus groups as well as few individual viruses which are important for PGQP. The availability of such molecular diagnostic methods will speed up the detection of infected material and will shorten the time needed to insure the release of disease-free germplasm to the user community.

Many viruses can be transmitted to and found in im-ported germplasm causing serious problems to Ameri-can agricultural, rural, or forest ecosystems. State of the art molecular diagnostic tools improve their detec-tion in the imported germplasm screened by APHIS. Real-time PCR (qPCR) offers a sensitive and accurate diagnostic technology which can be developed, adapted, validated, and transferred to the PPQ germ-plasm screening program (Plant Germplasm Quaran-tine Program). The rapid nature of the real-time PCR format, in addition to streamlined extraction methods, will increase PGQP capacity to rapidly screen foreign germplasm for these viruses. PGQP identified a list of several virus groups and independent viruses that needed development of molecular tests.

The list identified an immediate need for individual virus tests for Columbian datura potyvirus (CDV), Black currant reversion nepovirus (BRV), potato yellow vein crinivirus (PYVV), Sweet potato chlorotic fleck

Date Work Instructions

October 08 Total RNA Extraction

January 09 Preparation of cDNA from Total RNA

January 09 SPMSV-RT-qPCR

February 09 CDV-RT-qPCR

March 09 SPMSV-RT-conventional PCR

March 09 CDV-RT-conventional PCR

April 09 BRV-RT-qPCR

April 09 PYVV-RT-qPCR

May 09 BRV-RT-conventional PCR

May 09 PYVV-RT-conventional PCR

June 09 SPCFV-RT-qPCR

June 09 SPCSV-RT-qPCR

July 09 SPCFV-RT-conventional PCR

July 09 SPCSV-RT-conventional PCR

Table 1. List of work instructions NPGBL delivered to PGQP during FY 2009.


Foreign Plant Germplasm Diagnostics


Potato yellow vein crinivirus (PYVV) molecular detection CPHST STAFF: Mark Nakhla (lead); Deric Picton CHAMPIONS: Joseph Foster , Jorge Abad CONTACT: Mark Nakhla ([email protected], 301-504-7100)

2. Martelli, G.P., A.A. Agranovsky, M. Bar-Joseph, D. Salazar, L.F., G. Muller, M. Querci, J.L. Zapata and R.A. Owens. 2000. Potato yellow vein virus: its host range, distribution in South America and identification as a crinivirus transmitted by Trialeurodes vaporariorum. Ann. Appl. Biol., 137: 007-019.

Introduction PYVV is not recorded in the U.S. and can be transmitted to and found in imported germplasm causing serious prob-lems to American agricultural. Potato yellow vein virus (PYVV) is considered a quarantine pathogen in the Euro-pean and Mediterranean Plant Protection Organization (EPPO) and the USA. This virus is widespread and damag-ing at its centre of origin in South America. PYVV is recog-nized as an important constraint to potato production in Ecuador and Colombia and is spreading to neighboring countries (Salazar et al., 2000). PYVV is irregularly distrib-uted in the tuber and in low concentrations. The host range appears to be limited to the genus Solanum, but other non-Solanum ornamentals have been identified as potential inoculum sources. At present, PYVV remains as a tentative species in the genus Crinivirus (Martelli et al., 2002). It is transmitted semi-persistently by the greenhouse whitefly. PYVV particles are highly flexuous filamentous and contain a positive-stranded RNA genome. Reverse Transcription (conversion of RNA to cDNA) is required for detection and identification of PYVV using PCR.

Methods & Results Reverse-transcriptions (RT) is needed to reverse the RNA of PYVV to cDNA prior to PCR amplification. We adapted primers and a probe developed by Lopez et al., 2006 for the detection of PYVV using RT-PCR (Fig. 1) and real-time PCR (Fig. 2). We multiplexed the reaction to include internal amplification quality control assays targeting plant RNA. Both of our assays are able to detect femtogram quantities of the virus cDNA. NPGBL work instructions are delivered to PGQP to be used in testing potato germplasm.

Future Once PGQP secure a source of PYVV-infected potato we will need to reevaluate the detection methods using in-fected plants.

References 1. Lopez, R., C. Asensio, M.M. Guzman and N. Boonham. 2006. Development of real-time and conventional RT-PCR assays for the detection of Potato yellow vein virus (PYVV). J. of Vir. Meth., 136: 24-29.

Figure 1. PYVV detection using conventional multiplex RT-PCR. The virus-specific band size is 257bp and the 180bp band is specific for plant Nad5 gene and used as internal control.

Figure 2. Standard curve for PYVV detection using TaqMan real-time multiplex RT-PCR.


Germplasm program


Columbian datura potyvirus (CDV) molecular detection CPHST STAFF: Mark Nakhla (lead); Kristina Owens; Deric Picton CHAMPIONS: Joseph Foster , Jorge Abad CONTACT: Mark Nakhla ([email protected], 301-504-7100)

3. Fry, C.R., M.T. Zimmerman and S.W. Scott. 2004. Occurrence of Columbian dature virus in the Terrestrial Orchid, Spiranthes cernua. Journal of Phytopathology 152:200-203 4. Kahn RP, Bartels R. 1968. The Colombian datura virus - A new virus in the Potato virus Y group. Phytopathology 58: 587-592.

Introduction Colombian datura virus (CDV) [synonym Petunia flower mottle virus (PetFMV)] is an emerging potyvirus which may present a risk to Solanaceous crops. Inoculation studies have shown that potato could be a potential host of CDV. CDV was first isolated and described in 1968 in Datura species imported from Colombia to the USA. While data is still lacking on host range, geographical distribu-tion, and economic impact of the disease in Solanaceous crops, CDV is emerging in different parts of the world. In 1996, CDV was reported to occur in Germany and the Netherlands on ornamentals and tomato. In 2004, CDV was detected in tobacco in Hungary, Germany and Po-land. Recent studies in Hungary have showed that CDV causes severe disease in field-grown Cape gooseberry and can naturally infect pepino plants. In the USA, CDV was detected in several states in 2003 and 2004 in symp-tomatic ornamentals. In Australia, CDV was first detected in 2007 on Brugmansia in New South Wales, and subse-quently in Victoria. CDV is an RNA virus thus conversion of RNA to cDNA is required for detection and identification of CDV using PCR.

Methods & Results We developed RT-PCR and RT-real-time PCR assays for the detection of CDV. We multiplexed our developed virus-specific primers with primers based on a plant gene as an internal control in a two-step, RT-PCR. Both of our assays are able to detect femtogram quantities of the virus cDNA. NPGBL work instructions are delivered to PGQP to be used in testing potato germplasm.

Future Once PGQP secure a source of CDV-infected potato we will need to reevaluate the detection methods using in-fected plants.

References 1. Adkins S, Chellemi DO, Annamalai M, Baker CA. 2005. Colombian datura virus diagnosed in Brugmansia spp. in Florida. Phytopathology 95:6, S2. 2. Feldhoff, A., T. Wetzel, D. Peters, R. Kellner, and G. Krczal. 1998. Characterization of petunia flower mottle virus (PetFMV), a new potyvirus infecting Petunia x hybrida. Archive of Virology 143:475-488.

Figure 1. CDV detection using conventional multiplex RT-PCR. The virus-specific band size is 431bp and the 180bp band is specific for plant Nad5 gene and used as internal control.

Figure 1. Standard curve for CDV detection using TaqMan real-time multiplex RT-PCR.


Germplasm program


Sweet potato mild speckling potyvirus (SPMSV) molecular detection CPHST STAFF: Mark Nakhla (lead); Kristina Owens CHAMPIONS: Joseph Foster , Jorge Abad CONTACT: Mark Nakhla ([email protected], 301-504-7100)

Introduction SPMSV is not recorded in the U.S. SPMSV is a potyvirus which is known for having synergistic effect on sweet po-tato when co infected with other viruses. Both SPMSV and SPCSV (Crinivirus), not so important in their isolated form, however, they produce severe symptoms when co-infecting sweet potatoes. SPMSV, SPFMV and SPCSV infecting together produce the Chlorotic dwarf disease causing 70% reduction of sweet potato production. SPMSV has not been completely described yet. There is one sequence published in GenBank for an isolate of SPMSV from Argentina, accession # U61228 (August 2001), that consists of 1103 nucleotides encompassing the complete capsid protein gene and 3’ non-coding region. The CP sequence has a 63% homology with Sweet potato latent virus and 73% homology with Potato virus Y. Methods & Results An isolate of SPMSV is not available in the U.S. We de-cided to use a synthetic partial virus sequence (minigenes) as a viral target for our assays. We developed real-time and conventional PCR assays for the detection of SPMSV. Reverse-transcriptions (RT) is needed to reverse the RNA of SPMSV to cDNA prior to PCR amplification. We multi-plexed our virus-specific reagents with an internal control to confirm the quality of the sample preparation. Both of our assays are able to detect femtogram quantities of the virus cDNA. NPGBL work instructions are delivered to PGQP to be used in testing sweet potato germplasm. Future Once PGQP secure a source of SPMSV-infected sweet potato we will need to reevaluate the detection methods using infected plants. References Alvarez V, Ducasse DA, Biderbost E, Nome SF. 1997. Sequencing and characterization of the coat protein and 3' non-coding region of a new sweet potato potyvirus. Arch Virol. 142:1635-1644.

Figure 1. SPMSV detection using conventional multi-plex RT-PCR. The virus-specific band size is 425bp and the 180bp band is specific for plant Nad5 gene and used as internal control.

Figure 2. Standard curve for SPMSV detection using TaqMan real-time multiplex RT-PCR.


Germplasm program

Sweet potato chlorotic fleck carlavirus (SPCFV) molecular detection CPHST STAFF: Mark Nakhla (lead); Kristina Owens; Deric Picton CHAMPIONS: Joseph Foster and Jorge Abad CONTACT: Mark Nakhla ([email protected], 301-504-7100)

Introduction Sweet potato chlorotic fleck virus (SPCFV), also referred to as C-2 and sweet potato symptomless virus, is one of sev-eral viruses naturally infecting sweet potato (Aritua et al., 2009). SPCFV has a wide geographic distribution, occur-ring in Brazil, Bolivia, China, Columbia, Cuba, Indonesia, Japan, Panama, Peru, Philippines, Kenya, Tanzania, Uganda, Rwanda, and recently Australia (Aritua et al., 2009, Jones and Dwyer, 2007, Njeru et al., 2008,). In East Africa, SPCFV is the fourth most common plant virus, in-fecting about 3-8% of sweet potato plants. The host range of SPCFV includes members of the Convolvulaceae (Ipomoea spp.), Chenopodiaceae and Solanaceae (Nicotiana spp. and Solanum spp.). SPCFV is not recorded in the U.S. and it identified as targeted virus for PGQP which molecular diagnostic methods are needed. SPCFV is considered a putative new member of the Car-lavirus genus in the Flexiviridae family. To date, there are 18 published sequences in the National Center for Biotech-nology Information (NCBI); however, all but one are partial sequences. SPCFV isolates are genetically diverse and, depending on the part of the genome, can be clearly sepa-rated to 2-4 different groups. Reverse transcriptase-conventional PCR has been used to amplify parts of SPCFV genome (Aritua et al., 2009, Jones and Dwyer, 2007).

Methods & Results An isolate of SPCFV is not available in the U.S. We de-cided to use a synthetic partial virus sequence (minigenes) as a viral target for our assays. We developed real-time and conventional PCR assays for the detection of SPCFV. Reverse-transcriptions (RT) is needed to reverse the RNA of SPCFV to cDNA prior to PCR amplification. We multi-plexed our virus-specific reagents with an internal control to confirm the quality of the sample preparation. Both of our assays are able to detect femtogram quantities of the virus cDNA. NPGBL work instructions are delivered to PGQP to be used in testing sweet potato germplasm. Future Once PGQP secure a source of SPCFV-infected sweet potato we will need to reevaluate the detection methods using infected plants.

Figure 1. SPCFV detection using conventional multiplex RT-PCR. The virus-specific band size is 331bp and the 180bp band is specific for plant Nad5 gene and used as internal control.

Figure 2. Standard curve for SPCFV detection using TaqMan real-time multiplex RT-PCR.

References 1. Aritua, V, E. Barg, E. Adipala, R. W. Gibson, D.E. Lesemann and H.J. Vetten. 2009. Host Range, Purification, and Genetic Variability in Sweet potato chlorotic fleck virus. Plant Dis. 93:87-93. 2. Jones, R.A.C., and G.I. Dwyer. 2007. Detection of Sweet potato chlorotic fleck virus and Sweet potato feathery mottle virus-strain O in Australia. Australasian Plant Pathology, 36: 591-594. 3. Njeru, R.W., M.C. Bagabe, D. Nkezabahizi, D. Kayiranga, J. Kajuga, L. Butare & J. Ndirigue. 2008. Viruses infecting sweet potato in Rwanda: occurrence and distribution. Ann. Appl Biol 153: 215-221.



Germplasm program

Sweet potato chlorotic stunt crinivirus (SPCSV) molecular detection CPHST STAFF: Mark Nakhla (lead); Gang Wei CHAMPIONS: Joseph Foster and Jorge Abad CONTACT: Mark Nakhla ([email protected], 301-504-7100)

Introduction Sweet potato chlorotic stunt virus (SPCSV) typically stunts sweet potato plants and causes vein yellowing or sunken veins on leaves. Symptoms may be very mild or even ab-sent. This virus is commonly found in combination with other viruses and in synergy with many of these viruses (Untiveros, et al., 2007). For instance, in the case of co-infection of SPCSV with Sweet potato feathery mottle virus (SPFMV) there is an interaction that causes the severe synergistic disease sweet potato virus disease (SPVD). SPCSV is not recorded in the U.S. and it identified as tar-geted virus for PGQP which molecular diagnostic methods are needed. SPCSV is a phloem-associated virus transmitted by the whitefly Bemisia tabaci. It is a positive single-stranded RNA virus with a bipartite genome, being classified in the genus Crinivirus of the family Closteroviridae (Kreuze et al. 2002). Serological studies and Phylogenetic analysis of the SPCSV isolates indicate that there are 2 distinct genetic groups – East African (EA) and West African (WA) groups (Tairo et al. 2005).

Methods & Results We developed real-time and conventional PCR assays for the detection of SPCSV. Reverse-transcriptions (RT) is needed to reverse the RNA of SPCSV to cDNA prior to PCR amplification. We multiplexed our virus-specific re-agents with an internal control to confirm the quality of the sample preparation. We evaluated our protocols designed for West African isolates and successfully detected SPCSV in sweet potato foreign germplasm. Both of our assays are able to detect femtogram quantities of the virus cDNA. NPGBL work instructions are delivered to PGQP to be used in testing sweet potato germplasm.

Future Once PGQP secure a source of SPCSV-EA infected sweet potato we will start evaluating our developed protocols for these isolates using infected plants.

References 1. Kreuze, J.F., E.I. Savenkov, and J.P.T. Valkonen. 2002. Complete genome sequence and analysis of the subgenomic RNAs of Sweet potato chlorotic stunt virus reveal several new features for the Genus Crinivirus. Journal of Virology 76:9260-9270.

Figure 1. SPCSV detection using conventional multi-plex RT-PCR. The virus-specific band size is 423bp and the 180bp band is specific for plant Nad5 gene and used as internal control.

Figure 1. Standard curve for SPCSV detection using TaqMan real-time multiplex RT-PCR.

2. Tairo, F., S.B. Mukasa, R.A.C. Jones, A. Kullaya, P.R. Rubaihayo, and J.P.T. Valkonen. 2005. Unraveling the genetic diversity of the tree main viruses involved in Sweet Potato Virus Disease (SPVD), and its practical implications. Molecular Plant Pathology 6:199-211. 3. Untiveros, M., S. Fuentes, and L.F. Salazar. 2007. Syner-gistic interaction of Sweet potato chlorotic stunt virus (Crinivirus) with Carla-, Cucumo-, Ipomo-, and potyviruses infecting sweet potato. Plant Disease 91:669-676.


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Germplasm program

Blackcurrant reversion nepovirus (BRV) molecular detection CPHST STAFF: Mark Nakhla (lead); Kristina Owens CHAMPIONS: Joseph Foster and Margarita Licha CONTACT: Mark Nakhla ([email protected], 301-504-7100)

Introduction Blackcurrant reversion virus causes Blackcurrant reversion disease (BRD). BRV has an economically devastating effect on Blackcurrants and other Ribes spp. BRV is con-sidered the most important disease of blackcurrant crops worldwide (Jones & Mc Gavin 2002). While BRV is cur-rently not present in the Western Hemisphere, it is en-demic in Western and Eastern Europe, especially in areas where Ribes spp. are commercially produced. BRV is a Nepovirus of the Comovirideae virus family and is transmitted by an eriophyid gall mite. Due to the devastating effects of the disease, it has be-come important to develop rapid and effective methods to detect BRV, especially in foreign plant germplasm.

Methods & Results Currently there are more than 50 partial sequences listed in the Genbank, along with a few fully-sequenced genome fragments [RNA1 (NC_003509), RNA2 (NC_003502) & satellite RNA-viral segment (NC_003872)]. We developed real-time and conventional PCR assays for the detection of BRV. Reverse-transcriptions (RT) is needed to reverse the RNA of BRV to cDNA prior to PCR amplification. We multiplexed our virus-specific reagents with an internal control to confirm the quality of the sample preparation. We evaluated our protocols using Blackcurrants plants provided by PQQP and successfully detected BRV in in-fected germplasm. Both of our assays are able to detect femtogram quantities of the virus cDNA. NPGBL work in-structions are delivered to PGQP to be used in testing Blackcurrants and Ribes germplasm. Future We are now in the process of testing Blackcurrants and other Ribes germplasm from PGQP. We are demonstrat-ing the testing to PGQP prior to completely transfer the protocol for PGQP operation. References 1. Jones, A. Teifon. (2000) Black currant reversion disease-the probable causal agent, eriophyid mite vectors, epide-miology and prospects for control. Virus research 71:71-84.

Figure 1. BRV detection using conventional multiplex RT-PCR. The virus-specific band size is 390bp and the 180bp band is specific for plant Nad5 gene and used as internal control.

Figure 1. Standard curve for BRV detection using TaqMan real-time multiplex RT-PCR.

2. Jones, A. Teifon and Wendy J. Mc Gavin. (2002) Im-proved PCR Detection of Blackcurrant reversion virus in Ribes and further Evidence that it is the Casual Agent of Reversion Disease Plant Dis. 86:1333-1338. 3. Susi, Petri. (2004). Pathogen Profile: Black currant re-version virus; a mite transmitted nepovirus. Molecular Plant Pathology 5:167-173.



CITRUS HEALTH RESPONSE PROGRAM


PCR and sampling methods for improved detection of ‘Candidatus Liberibacter spp.’ associated with citrus huanglongbing (HLB) CPHST STAFF: Wenbin Li (lead); Elizabeth Twieg CHAMPIONS: Patrick Gomes, PPQ, EDP, CHRP CONTACT: Wenbin Li ([email protected], 301-504-7100)

PCR inhibitors from citrus plants and insects decreased the detection sensitivity and PCR amplification efficiency. This effect varied among plant tissue types, citrus species, and geographic locations. Based on these sample effects, uni-versal standard curves have been established for quantifi-cation of the bacteria in infected plants (Plant Disease 92:854-861, 2008).

Quantification of HLB bacteria in plants. We systemically quantified the distribution of the HLB bacterium in tissues of six citrus species in the field in Florida. The bacterial popu-lations in leaf midribs, leaf blades, and bark samples varied by a factor of 1,000 among samples prepared from the six citrus species and by a factor of 100 between two sweet orange trees. In naturally infected trees, above-ground por-tions of the trees averaged 1010 HLB bacteria per gram of tissue. The bacteria were also quantified in infected roots, fruits and seeds. The results demonstrate both the ubiqui-tous presence of the bacterium in symptomatic citrus trees as well as great variation between individual trees and among samples of different tissues from the same tree (Phytopathology 99:139-144, 2009).

Development of real-time PCR. It has been difficult to detect and identify the HLB bacteria. We developed quanti-tative TaqMan real-time PCR for detection and identifica-tion of the bacteria. The assays do not cross-react with other pathogens or endophytes commonly resident in citrus plants. The methods are at least 100 fold more sensitive than conventional PCR and detect the bacteria in 20 ng of midrib tissue of infected plants. The methods were suc-cessfully used in the first confirmation of the disease in Florida (2005), and has been widely used in disease sur-veys in the USA and many other countries. The real-time PCR paper was among the top 10 cited articles published in Journal of Microbiological Methods in the last five years.

Optimization of conventional PCR. We optimized and standardized three conventional PCR methods for diagno-sis of HLB under the U.S conditions, and compared these methods to the newly-developed real-time PCR and the loop-mediated isothermal amplification. The detection sen-sitivity of the validated conventional PCR assays were sig-nificantly improved compared with the original protocols. All of the validated conventional and the newly developed real-time PCR methods were reliable for confirmatory diagnosis of the disease in symptomatic samples. The disease bacte-ria could be also detected one and three months before the disease symptoms by the conventional and real-time PCR assays, respectively. We have published the evaluation of HLB detection methods in Plant Disease 91:51-58, 2007. These validated methods have also been used for the dis-ease surveys around the world.

Optimization of HLB bacterial quantification. No quanti-tative information has been available on the HLB bacterial populations either in host plants or vector psyllids. We evaluated the effects of sample composition on quantifica-tion of the HLB bacteria in citrus plants and Asian citrus psyllids by TaqMan real-time PCR. We have established various standard curves and regressions to absolutely and relatively estimate the HLB bacterial populations in plants and insects. We found that nontarget DNA and putative

Figure 1. Greenhouse HLB symptoms on sweet orange seedling and on pommelo grafted as the inoculum from the first HLB infected tree found in Miami Beach, Florida. Source: Wenbin Li




Genomewide search and evaluation of candidate genes for detection and identification of ‘Candidatus Liberibacter spp.’ CPHST STAFF: Wenbin Li (lead); Elizabeth Twieg CHAMPIONS: Patrick Gomes, PPQ, EDP, CHRP CONTACT: Wenbin Li ([email protected], 301-504-7100)

Previously only a few gene sequences such as 16S rRNA, 16S/23S ITS, β-operon protein, DNA polymerase, and the outer membrane protein genes were available for use in detection and identification of ‘Candidatus Liberibacter spp’ associated with citrus huanglongbing (HLB) (ex. Greening). Only small parts of these gene sequences are conserva-tive, specific enough for use in diagnosis of the HLB asso-ciated bacterium. To look for additional candidate genes for detection and identification of Liberibacters, we selected 156 genes in various functional categories from the whole genome of ‘Can. L. asiaticus’ (Las) sequenced in 2009. The selected candidate genes represent 13.8% of the whole genome, distributing evenly on the circular chromosome of the bac-terium (Fig.1). TaqMan real-time PCR primers and probes and conventional PCR primers for each of the selected genes were developed and tested against 28 Liberibacter-free DNA extracts from citrus plants in China, Japan, India, Brazil, Mexico, Costa Rica, Saudi Arabia, South Africa, and

Information storage and processing (33 ORFs, 26.4%)Cellular processes and signaling ( 44 ORFs, 35.2%)

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125 ORFs screened

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Figure 1. Even distribution of the selected candidate genes for Libeibacter detection and identification in the whole chromosome of ‘Candidatus Liberibacter asiaticus’.

10 US States. None of the primer/probe sets produced any false positive results. When screened against known- Las positive DNA extracts, all the designed TaqMan primer/probe sets could be confidently multiplexed with the COXfpr, the positive internal control targeting the host plant DNA. To scan the selected candidate genes against a variety of Liberibacters, we have collaborated with scientists world-wide in Brazil, China, Japan, India, Mexico, Taiwan, Ja-maica, Belize, and South Africa. We obtained more than 400 DNA extracts of four liberibacter species, ‘Can. L. asi-aticus’, ‘Can. L. africanus’, ‘Can. L. americanus’ and ‘Can. L. solanacearum’, from 18 countries. We have finished over 18,000 real-time PCR reactions screening the candidate genes against the DNA extracts. Based on the preliminary scanning results, the candidate genes were classified into three Groups for diagnostic purposes (Fig. 2)

Figure. Gene groups for Liberibacter diagnosis

Liberibacter isolates

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The Group I genes consistently produced quantitative real-time PCR (qPCR) Ct values for all the liberibacter DNA extracts of certain species or all four species tested. These genes will be suitable candidates for additional diagnostic tools besides the 16S rRNA gene. The Group 2 genes could also detect liberibacters in all positive DNA extracts, but their qPCR Ct values were not consistent among the extracts. So, they are not good candidates for diagnostic purposes but are very useful for genetic diversity study of the bacterium. The Group 3 genes yielded false negative results for some Liberibacter-positive DNA extracts be-cause they produced zero or very high (>37.00) qPCR Ct values. The genes in this group should not be used for HLB diagnosis. However, they may be useful in tracing Liberi-bacter origins and HLB molecular epidemiology. The 16S rRNA, β-operon, outer membrane protein and DNA polymerase genes are commonly targeted by scien-tists for Liberibacter diagnostic assay development. As shown in Fig. 2, to date, no candidate gene produced qPCR Ct values reliably lower than those of 16S rRNA, a Group 1 gene, demonstrating that diagnostic assays based on the 16S rRNA are very sensitive.

The β-operon and outer membrane protein genes be-long to Group 2, while the DNA polymerase belongs to Group 3. All these three genes behave too inconsistently between isolates to be used for a general diagnostic assay. New HLB detections rely on using conventional PCR to generate PCR bands that can be sequenced from sam-ples that produce 16S rRNA qPCR positive Ct values. Although the 16S rRNA gene target is used in both qPCR and conventional PCR, the level of sensitivity in the qPCR assay is greater than the conventional PCR assay. During the confirmation process, a few samples with qPCR Ct values greater than 30 inconsistently pro-duce conventional PCR bands for sequencing, resulting in the need to retest or resample trees. The work de-scribed in this project should help to identify two addi-tional HLB-specific genes that can be validated and used in tandem with the 16S assay as a trio of qPCR assays for HLB confirmation. In addition, this project may identify useful gene targets for species or isolate specific for Liberibacter detection and identification.




Development and validation of methods for detection of Xylella fastidiosa strains causing citrus variegated chlorosis CPHST STAFF: Wenbin Li (lead); Elizabeth Twieg CHAMPIONS: Patrick Gomes, PPQ, EDP, CHRP CONTACT: Wenbin Li ([email protected], 301-504-7100)

technology followed by developing of a new CVC strain-specific molecular diagnostic test that will be sensitive, easy to use and inexpensive. Reference Davis et la. 1978. Science 199:75-77 Hopkins et al. 1978. Plant Dis 62:442-445 Li et al. 1996. ISC Proc I:272-279

Xylella fastidiosa is the causal agent of several important plant diseases in the Americas. The bacterium first was isolated from grapevines with Pierce’s Disease (PD) in North America. It also causes diseases like honey peach, leaf scorch of several fruit tree, ornamental, and forest plants the U.S. In Brazil, it has become extremely important due to citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). In 2005, about 45% of the orange trees in the State of Sao Paulo showed CVC symptoms and its control costs in that State were above US $110 million. The disease is a threat to the U.S citrus industry. One X. fastidiosa PD strain was detected but did not cause CVC disease in orange tress in Florida Hopkins et al., 1978). X. fastidiosa CVC strains caused CLS in coffee plants (Li et al., 2001) and both the CVC and CLS strains could induce PD in grapevines (Li et al., 2002). In addition, X. fastidiosa CVC strains can be transmitted through citrus seeds (Li et al., 2003). No reliable methods are available to distinguish the CVC strains from other ones. Based on the previous publications, we have selected, for the project, the putative methods for Xylella isolation (Davis et al., 1978), biological indexing (Li et al., 1996), patho-genicity tests (Li et al., 1999), ELISA (Li et al., 1999), DNA extraction ( Li at al., 2002), conventional (Pooler & Hartung, 1995) and real-time PCR (Olivera et al., 2002). We have written work instructions for each of the methods, and pre-liminarily validated some of the work instructions using CVC suspect samples collected in U.S. citrus areas. To further validate all these work instructions, we are es-tablishing additional collaboration with the scientists in Bra-zil and other nations in Central America where the CVC disease is present. We are also working closely with sev-eral USDA-ARS research laboratories on this bacterial select agent. The project priority is to validate existing

Figure 1. Xylella fastidiosa CVC strain in citrus xylem (source: Fundecitrus)

Figure 2. CVC foliar symptoms (source: Wenbin Li)

Figure 3. CVC symptoms on sweet orange fruits: left– diseased, right– healthy (source: Wenbin Li)




Conventional and real-time PCR for diagnosis of citrus leprosis CPHST STAFF: Wenbin Li (lead); Elizabeth Twieg CHAMPIONS: Patrick Gomes, PPQ, EDP, CHRP CONTACT: Wenbin Li ([email protected], 301-504-7100)

2003) and to develop a new, specific, sensitive and reli-able TaqMan real-time PCR for CiLV detection in host plants and vector mites. We have already acquired all the conventional PCR primers and designed more genome-based primers and probes for conventional and real-time PCR. We have also established collaborations with sci-entists in Brazil, Mexico, Costa Rica, Panama, and else-where in the U.S. Reference Bastianel et al. 2010. Plant Dis 94:284-292 Locali et al. 2003. Plant Dis 87:1317-1321 Locali et al. 2006 J Gen Virology 87:2721-2729

Citrus leprosis is one of the most economically serious diseases of Central America citrus industries. In Brazil, the disease affects more than 50% of orange trees in Sao Paulo state and has the highest cost of citrus disease control (over US $120 millions per year) (Bastianel et al. 2010). The disease is caused by the Citrus leprosis virus cytoplasma type (CiLV-C: the prevalent form) and nuclear type (CiLV-N) (Fig. 1); both transmitted by tenuipalpid mites of the genus Brevipalpus (Fig. 2). Although citrus leprosis has never been endemic in the U.S., the disease was first described from the state of Florida at the beginning of the twentieth century, where it was originally called scaly bark, because of the char-acteristic stem lesions and bark scaling symptoms. Be-cause the virus does not spread systemically in the host plants, it causes typical localized chlorotic or necrotic light yellow to dark brown lesions where the virus parti-cles accumulate on leaves and fruits (Fig. 3). Citrus leprosis is an unusual disease caused by two completely distinct viruses with similar morphology and vector. CiLV-C is a ss+RNA bipartite virus of the newly denominated virus genus Cilevirus, while CiLV-N belongs to the Rhabdoviridae family. All active stages of the Brevipalpus mite can acquire and inoculate both viruses, but the transmission is not transovarial. Recent studies confirmed that CiLV-C circulates, but does not replicate in the mite vector. The vector has spread to almost all citrus producing areas in the U.S. and the disease has recently become endemic in Mexico, making it a great threat to the U.S. citrus industry, especially in California. Traditional cit-rus leprosis diagnosis was based on symptom analyses and virus observation by electron microscopy. Conven-tional PCR with virus-specific primers was developed for diagnosis of the disease (Locali et al., 2003). The complete CiLV-C genome was recently available for development of new molecular tools for CiLV detection in host plants and vector mites (Locali et al., 2006). The main objectives of this project are to validate the published conventional PCR method (Locali et al.,

Figure 3. CiLV-C symptoms on sweet orange– A: on stem; B: on leaves; C: on fruits (source: Wenbin Li)

Figure 2. Brevipalpus obovatus

Figure 1. CiLV in citrus: left CiLV-C; right CiLV-N (source: Kitajima)


POTATO CYST NEMATODE PCR METHODS


Multiplex real-time PCR assays for the identification of the potato cyst and tobacco cyst nematodes CPHST STAFF: Mark Nakhla (lead); Kristina Owens, Gang Wei CHAMPIONS: Osama El-Lissy CONTACT: Mark Nakhla ([email protected], 301-504-7100)

References

Nakhla, M.K., Owens, K.J., Carta, L., Skantar, A., Levy, L. 2007. Validation of potato cyst nematode (PCN) molecular identifica-tion methods and development of laboratory work instructions for a national survey. Phytopathology 97: S82. (Abstract) Skantar, A. M., Handoo, Z. A., Carta, L. K., and Chitwood, D. J. 2007. Morphological and molecular identification of Globodera pallida associated with potato in Idaho. J. Nematol. 39:133-144. Subbotin, S. A., Halford, P. D., Warry, A., and Perry, R. N. 2000. Variations in ribosomal DNA sequences and phylogeny of Glo-bodera parasitizing solanaceous plants. Nematology 2:591-604.

Introduction

The pale potato cyst nematode Globodera pallida and the golden potato cyst nematode G. rostochiensis are regulated pathogens of potato in the United States. Previously, the distribu-tion of G. pallida in North America was limited to Newfoundland, Canada, but in 2006, G. pallida was discovered in northern Bing-ham County, Idaho, U.S. The presence of this nematode repre-sents a significant threat to the U.S. potato industry; therefore, accurate identification of Globodera spp. found in North America is essential for rational regulatory decisions to eradicate and prevent the spread of potato cyst nematodes (PCN).

Methods & Results We developed TaqMan primer-probe sets for the detection and identification of PCN (G. pallida and G. rostochiensis) using two-tube, multiplex real-time PCR. One tube con-tained a primer-probe set specific for G. pallida multiplexed with another primer-probe set specific for G. rostochiensis . A second tube consisted of the G. pallida-specific primer-probe set multiplexed with a primer-probe set specific for G. tabacum (the morphologically similar tobacco cyst nematode). This two-tube multiplex qPCR system we de-veloped could detect, identify, and quantify even a single juvenile of the three Globodera species most likely to be encountered during North American surveys for PCN. This ITS rDNA-based system was specific for the Globodera species of interest and successfully identified several popu-lations of PCN. This rapid, sensitive and specific quantita-tive PCR assay presents a useful tool for PCN regulatory response and management programs. Future It is realistic to expect that the future may bring new diag-nostic challenges as additional PCN populations are de-scribed both morphologically and molecularly. G. pallida originating from the cordillera of South America is of par-ticular interest, considering the genetic diversity of those populations relative to the majority of European and North American populations. It is expected that development of PCN diagnostic protocols will continue to evolve as infor-mation about new populations is revealed.

Figure 1. Standard curves of the qPCR for detection of: A, G. pallida, B, G. rostochiensis, and C, G. tabacum.



Advanced development of Plum pox virus (PPV) diagnostics CPHST STAFF: Vessela Mavrodieva (lead); Sarika Negi CHAMPIONS: Don Albright (PPV Eradication Program Manager) , Steven Poe (PPQ EDP) and Joel Floyd (Domestic Diagnostic Coordinator, PPQ-PHP-NIS) CONTACT: Vessela Mavrodieva ([email protected], 301-504-7100)

viral RNA were amplified and sequenced (6). Sequence analysis of isolate 44188 putative protein showed highest similarity with PPV D isolates from Canada and USA.

Surprisingly, isolate 44191 predicted protein sequence showed significant similarity of 96% with PPV W3174 (AAX99418) isolate from Canada (fig.2). Isolate 44189 se-quence was identical with that of isolate 44191.

PPV W3174 (3) was found in one only residential location in Canada and it constitutes a new strain of PPV (4) based on significant sequence differences with the previously de-scribed strains of PPV D, M, ElAmar, Cherry and Rec. There were a couple of striking similarities between PPV W3174 and 44189/44191 isolates in addition to the se-quence similarity: i) they were found on plum; ii) there were some data that the PPV W3174 germplasm originated from Eastern Europe and all accessions tested in this work origi-nated from Ukraine. Further characterization of the PPV-W like isolates could shed light on the origin of the PPV W strain and pathways for virus spread. Therefore we contin-ued working on molecular and serological characterization of PPV 44189 isolate while trying to preserve it. We have successfully transferred PPV 44189 onto N. bentamiana tobacco (a good experimental host for PPV) and we are maintaining it on prunus. We have obtained more than 90% of the full-length sequence of 44189. The PPV W specific primers designed by Dr. D. James (4) did not work well with 44189 PPV isolate. Detailed analysis showed significant mismatch in the 3’end of the forward primer. This finding demonstrates the importance of characterization of newly found isolates and strains in order to improve the existing diagnostics.

Plum pox virus (PPV) is an important regulatory pathogen that causes significant loses of the stone fruit production world-wide. In addition to the already well-known strains of PPV D, M, ElAmar and Cherry, new strains were identified and described in recent years such as PPV-W in Canada (3) and PPV Rec in Europe (2). Therefore existing diagnos-tic methods must be constantly updated for reliable detec-tion of the newly described strains and isolates. This work project focuses on improvement of the existing diagnostic tools for the PPQ PPV Program.

Although PPV is already present in the U.S. (5) and Can-ada (7) prevention of secondary introduction of new exotic isolates is of extreme importance for the success of the on-going eradication programs in both countries. To prevent disease introduction through infected plant material in this country all imported prunus germplasm undergoes exten-sive 2-year testing (indexing).

In 2004 PPV-like symptoms were observed in GF-305 seedlings bud-grafted with plum accessions from Ukraine while undergoing indexing in the PGQO-ARS facility in Beltsville, MD. All seventeen accessions from the same region (Donetsk, Ukraine) were hand-carried to the U.S. without an official permit. PPV presence was confirmed in three plum accessions by ELISA and immunocapture re-verse transcription - polymerase chain reaction (IC-RT-PCR).

Using PPV strain specific RT-PCR (1) we confirmed PPV D in the 44188 plum accession. Isolates 44189 and 44191 did not amplify with either primer pair (fig.1). We conducted RFLP analysis of the coat protein (CP) RT-PCR products of the three Ukrainian isolates with RsaI and Alu I endonu-cleases that also confirmed PPV D presence in 44188. CP RT-PCR products of 44189 and 44191 did not posses ei-ther site: RsaI or AluI (results not shown). These finding suggested that PPV isolates from accessions 44189 and 44191 did not belong to either PPV D or PPV M strains.

To further characterize these isolates 1.4bp fragments en-compassing the 3’-end of the polymerase gene, the entire coat protein (CP) gene and the 3’non-coding region of the


VIRAL PLANT PATHOGENS


cherry producing areas in Bulgaria and Romania, where PPV Cherry has been previously reported. Such isolates (if found) could be very useful for studying susceptibility of USA cherry cultivars to PPV. References 1. Candresse, T., et al. (1998) Phytopathology 88:198-204. 2. Glasa, M., et al. (2004) J. Gen. Virol. 85, 2671–2681. 3. James, D., et al. (2003). Plant Dis. 87:1119-1124. 4. James, D., and A. Varga (2004) Acta Horticulturae 657: 177-182 5. Levy, L., et al. (2000). Plant Dis., 84:202. 6. Nemchinov, L., et al. (1996) Phytopathology 86:1215-1221. 7. Thomson, D., et al. (2001). Plant Dis. 85:97.

We anticipate completion of the full-length sequencing for PPV 44189 in 2010. In addition we have obtained strain-specific antibodies and controls to complete serological determination of this isolate.

We have established contacts and signed a cooperative agreement with two Ukrainian research institutions in 2008 to conduct further surveys on PPV in Ukraine in attempt to find more PPV-W like isolates. Our cooperators have col-lected samples from several major prunus producing ar-eas. PPV was detected in numerous samples from plum, peach and apricot. These isolates are being further tested for strain specificity. We also expect to receive grafting plant material of some of them and continue our work on molecular and serological characterization of some of the Ukrainian isolates.

We have signed cooperative agreements with researches in Bulgaria and Romania focused on PPV cherry isolates. Despite existing sequencing data, the few cherry isolates found in Europe have been lost. Our goal is to survey

Fig. 2. Phylogenetic tree of CP amino acid sequence of iso-late 44191 and representative from the major PPV groups of strains.

Fig. 1 Agarose gel (1.5%) electrophoresis with PPV D strain specific primers (A) and PPV M strain specific prim-ers (B) of Ukrainian isolates of PPV and some controls.

BIOTECHNOLOGY PLATFORMS EVALUATION/ ADAPTATION


Adaptation of CANARY Biosensors for Rapid Detection of Regulated Plant Pathogens CPHST STAFF: Zhaowei Liu (lead); Kate Rappaport CHAMPIONS: Russ Bulluck (National Science Program Leader, USDA, APHIS, PPQ, CPHST) CONTACT: Zhaowei Liu ([email protected], 301- 504-7100)

detector. Since it depends on antibody/antigen interaction, the CANARY system is as specific as other immunoassays, but is almost as sensitive as PCR and is much faster than other established identification methods. CPHST scientists started collaboration with researchers in Lincoln Laboratory from the early stage of technology devel-opment. Three plant-pathogen-specific B cell lines were developed for detecting Potyvirus (virus), Ralstonia spp. (bacterium), and Phytophthora spp. (oomycetes), respec-tively. The eventual goal is to implement CANARY in USDA diagnostic laboratories and deploy biosensors as detectors at ports of entry.

Introduction Early plant pathogen detection is critical in preventing vari-ous plant pathogens from entry, establishment or spread to the US agricultural and natural ecosystem. Similarly, rapid identification of plant pathogens is important for disease control and management. It reduces crop loss and pesti-cide usage. Early detection requires technologies to be simple, fast, specific, sensitive and inexpensive. Currently, immunoassays and nucleic acid based poly-merase chain reaction (PCR) methods are two major bio-chemical and molecular technologies used for plant patho-gen identification. Although an immunoassay such as an ELISA utilizes a simple procedure and instrumentation, it has a limit of sensitivity in the range of thousands of patho-gen agents, takes 15 minutes to several hours and may produce high assay background and false positives. PCR is most specific since it detects nucleic acids and most sensitive because of extreme amplification. However, a typical PCR reaction may take at least 30 minutes, which does not take into account the time for nucleic acid extrac-tion. In addition, PCR protocols usually demand a clean environment, specialized instruments and trained techni-cians. CANARY biosensors using pathogen-specific B cell lines offer a highly sensitive assay that can be completed in a few minutes. It uses a protocol requiring simple instrumen-tation and training. The technology can be used in a labo-ratory diagnostic setting, in plant inspection stations and field surveys. The method was invented by scientists in Lincoln Laboratory at Massachusetts Institute of Technol-ogy and was initially developed for rapid detection of bio-terror agents such as anthrax. CANARY biosensors (Fig. 1) are mouse B cells engi-neered with a bioluminescent gene and antibody genes. Antibodies are expressed and anchored on the outer mem-brane of the cell. Upon cross linking of antigens of a spe-cific pathogen to the antibodies, B cells produce an ele-vated level of calcium. The process triggers a conformation change on the bioluminescent protein and leads to light emission. The emitted light is detected by a photon-

Figure 1. CANARY Biosensor

BIOTECHNOLOGY PLATFORMS EVALUATION / ADAPTATION


curve. To call a sample positive, the signal intensity must exceed at least three times of that generated by the background and a typical kinetic curve must be produced. This experiment demonstrated the extreme sensitivity and rapid speed of the technology. The data generated by 10-fold serial dilutions of bacteria indi-cated that B cells reacted to pathogens in a dose-response fashion (Fig. 2), i.e. the signal intensity in-creases incrementally from low- to high-doses of anti-gens.

2. Testing diagnostic samples: We tested Ralstonia in-

fected Maudeville spp. roots which had been character-ized by a serological method. The results showed that all six samples contained high amount of bacteria (Fig. 3). As a comparison, a positive control was included in the test, which was prepared with 100,000 CFU R. so-lanacearum from pure culture. To test a single sample, the entire process, including sample prep, can be com-pleted in less than 10 minutes.

Approaches and Methods To develop the CANARY system, we setup the facility for animal cell culture and received all three B cell lines from MIT. We established procedures and protocols for B cell storage and thawing, routine cell care and feeding, and preparation of B cells for CANARY assays. We carried out experiments to determine the analytical sensitivity of CANARY with Ralstonia. R. solanacearum was grown on petri-dishes. A colony was selected to make serial dilutions with assay buffer for both CFU (colony form-ing unit) counting and CANARY assays. We then per-formed testing with some “real-world” Ralstonia-infected plant tissues. A small piece of diseased tissue was taken and soaked in assay buffer. During soaking, the bacteria stream out of the plant tissue and are suspended in buffer. A short, low speed spin was used to precipitate the root tissue but not to pellet the bacteria. One-hundred µl super-natant was then transferred for the CANARY assay. Second, we conducted Phytophthora detection by CA-NARY. This assay requires a more complex sample prepa-ration procedure. When infecting plant tissues, Phy-tophthora mycelia usually tend to tangle and intertwine with matrix of plant tissues. The grinding of the diseased tissue results in abundant small plant debris which cannot be eas-ily separated from Phytophthora mycelia. The debris inter-feres with the CANARY assay by blocking the light detec-tion and blocking the access to mycelia by B cells. There-fore, a second Phytophthora antibody, which is different than the B cell antibodies, is produced by a hybridoma cell line and used to coat magnetic beads. The antibody coated beads are applied to capture mycelia for the CANARY as-say. As a first step of development, we conducted CA-NARY tests with Phytophthora mycelia prepared from pure cultures. A size of approximately 5 x 10 mm2 of growing Phy-tophthora mycelia was sliced out from a culture of 20 - 30 days of growth, and placed in a centrifuge tube containing 400 µl assay buffer and Lysing Matrix A. The mycelia were homogenized using FastPrep-24 (MP Biomedicals) for 40 seconds. Serial dilutions were then made from the mycelial suspension for CANARY tests. Results and Discussion 1. Analytical sensitivity: As shown in Fig. 2, the CANARY

assay was capable of detecting as few as 3 CFU of Ralstonia/test. The response triggered by the plant pathogen usually produced a kinetic curve that peaked within a minute, while the negative control using only the assay buffer generated a wavy but low signal

Figure 2. Analytical sensitivity

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BIOTECHNOLOGY PLATFORMS EVALUATION / ADAPTATION


3. Phytophthora dose response curve: It is difficult, if not

impossible, to determine the limit of detection for anti-gens consisted of as part of the mycelial components. We, therefore, performed a dosage-based test with ho-mogenized Phytophthora mycelia. As revealed in the experiment for Ralstonia tests (Fig. 2), the Phytophthora-specific B-cell line also showed a dose-dependent re-sponse to P. infestans (Fig. 4), which would provide as a reliable dosage reference if a positive control is included when testing a diagnostic sample.

4. Tests with 10 species of Phytophthora prepared from

pure cultures: We also tested 9 other species of Phy-tophthora using homogenized mycelial dilutions prepared from pure cultures (Fig. 5). They all responded to the Phytophthora B cell line positively with different degrees of sensitivity, which is not unexpected. The experiment indicated a possible broad detection range for species in the Phytophthora genus.

Future Work For Ralstonia work, we will optimize the protocols for highly sensitive and specific detection. We will also test exten-sively with “real-world” samples. For Phytophthora work, we will produce the monoclonal antibodies from the hybri-doma line and attach them to magnetic beads. The MAb coated beads will be used to capture Phytophthora mycelia from both pure cultures and infected plants for CANARY analysis. We will continue experimenting for potyvirus de-tection, which is currently under development, but has so far not yet produced satisfactory results. Reference: Rider et al. (2003). B Cell-based sensor for rapid identification of pathogens. Science, 301:213-215.

Figure 4. Phytophthora dose response curve

Figure 5. Testing pure Phytophthora spp. cultures

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METHODS VALIDATION

Validation of diagnostic qPCR assays for detection and diagnosis of Phytophthora kernoviae, an exotic pathogen of Beech, Oak and other Hosts CPHST STAFF: Kurt A. Zeller (lead) CHAMPIONS: Jonathan Jones CONTACT: Kurt. A. Zeller ([email protected], 301-504-7100)

Problem: Phytophthora kernoviae is a recently described species of Phytophthora identified as causing trunk and stem cankers on Beech and Oaks, and stem cankers on understory plants. P. kernoviae was initially discovered during surveys for Phytophthora ramorum in Great Britain, and shows some similarities in host range and pathogenic potential (Brasier et a., 2005). Among known hosts of potential concern for US agriculture and Forests are Oaks, Beeches, Rhododendrons and members of the plant ge-nus Vaccinium (which includes cultivated cranberry and blueberry) (Beales et al, 2008). Shortly after the descrip-tion of P. kernoviae DNA sequence matches to were iden-tified between the reference sequences deposited in online databases, and incompletely characterized isolates that had been recovered in New Zealand. The current known distribution of P. kernoviae is restricted to scattered locations in Great Britain, and from orchard and forest locations in New Zealand.

Although not known within the US, P. kernoviae has a host range similar to that of P. ramorum on orna-mental plants. Because of this host range similarity, P. kernoviae could be introduced to the US, and spread within the country by means similar to that of P. ramorum. P. kernoviae could have similar damaging effects on both the nursery industry, and forest ecosystems in the US to those observed for P. ramorum.

Approach: We have pursued two general directions in developing and validating diagnostic methods for P. kerno-viae. First, we have adopted two established Real-time qPCR assays developed in Great Britain, have minimally modified those assays to work in our standard Real-time PCR platform, and have added internal amplification qual-ity control assays targeting Phytophthora and Plant DNA targets to these established assays. Both of these qPCR assays are capable of detecting femptogram (10-9 gram) quantities of P. kernoviae target DNA even when in a background of contaminating plant DNA. Also, these qPCR assays do not cross-react with DNAs from any other tested Phytophthora spp, including that of the recently described close relative, P. morindae (Nelson & Abad, 2010).

Figure 1: AC-18 Locus Conventional PCR products amplified from 6 DNAs of P. kernoviae isolates from New Zealand (NZ) and 1 from Great Britain (GB)

Our second area of focus has been to develop other diag-nostic assays targeting novel genetic loci that, in addition to providing a ‘yes’ or ‘no’ diagnostic answer as to whether P. kernoviae is present, can allow us to differenti-ate among genotypes. Preliminary testing of isolates from Great Britain and New Zealand with PCR primers to am-plify these novel loci can differentiate genotypes of P. kernoviae from one another, and that the limited set of isolates from these two geographic sources may have different sets of genotypes (Figure 1).


Collectively, deployment of these molecular assays will give us the ability to both reliably detect, and potentially determine likely origin, of this exotic plant pathogen. Technology transfer: During 2009 over 20 NPDN, Uni-versity and State diagnosticians were given hands-on training using the 2 validated qPCR assays for diagnos-ing P. kernoviae. These were provided in a total of four (4) separate sessions during February and March 2009. A further training session with the goal of introducing these methods to PPQ scientists who would be involved in initial identifications has been scheduled for January 2010. Brasier et al. (2005), Mycological Research, 109: 853-859. Beales et al. (2008). http://www.bspp.org.uk/publications/new-disease-reports/ndr.php?id=017026 Nelson & Abad (2010), Mycologia, 102: 122–134

METHODS VALIDATION

Implementation of a conventional PCR test and development of a Real-Time PCR test for rapid on-site detection of potato wart (Synchytrium endobioticum) CPHST STAFF: Kurt A. Zeller (lead) CHAMPIONS: Osama El-Lissy, Lynn Goldner CONTACT: Kurt. A. Zeller ([email protected], 301-504-7100)

Problem: Potato Wart (PW) is caused by the fungus Syn-chytrium endobioticum and is a significant quarantine dis-ease of potato. Once established in a field, Synchytrium produces a resting spore infective in the soil for decades. Establishment of PW within potato growing regions of the US would bring significant trade consequences and eco-nomic losses to the US potato market, similar to those observed for producers on Prince Edward Island (PEI) in Canada since 2000.

Although not known within the US, PW is found in both PEI, and in Newfoundland in Canada. PW is wide-spread in Europe, but with a fragmentary distribution, and is known from Asia, South Africa, and several countries in South America. All of these regions could serve as sources for introduction of PW into the US and could have large scale impacts on the value and marketability of US potatoes. Preventing the establishment of this select agro-bioterrorism agent in U.S. potato growing regions requires vigilance, rapid detection and unambiguous diagnoses.

Approach: Our focus has been to validate diagnostic pro-tocols and procedures for Synchytrium- that have already been developed for diagnostics use in Canada and Europe. Several such alternative methods using both con-ventional PCR and Real-time PCR techniques have been developed and published. During 2009, we worked to complete validation of two Real-time qPCR (quantitative PCR) assays developed for use in Canada. These two assays target different segments within the genomic ribosomal DNA of S. endobioticum. When run on dilution series of target DNAs from S. endo-bioticum, each of these assays can reliably detect as few as 200 copies of this DNA (equivalent to a few spores – see Figure 1). Both qPCR assays, including time for sam-ple preparation and DNA isolation, can be completed by a single analyst in less than a working day, and both assays include an internal amplification quality control reaction that targets plant DNA. We have also tested both diag-nostic assays against an array of other Synchytrium DNAs provided to us by collaborators in Canada to test for poten-

Figure 1: Standard Curve on a dilution series for the ITS-targeting qPCR assay to detect S. endobioticum.

tial sources of false positive reactions should these as-says need to be deployed. After testing against 14 other Synchytrium spp., we have determined that the diagnostic assays are >1000 times less sensitive to non-target Synchytrium DNAs than to that of S. endobioticum.

Other work begun in 2009 includes beginning development of a reliable inoculation protocol for potatoes under controlled conditions. Once we have established a reliable inoculation protocol, we can continue on with de-velopment of other novel diagnostic assays and proce-dures, and can begin to work out methods for testing and sampling for PW from infested soils.


Technology transfer: During 2009 14 NPDN, University and State diagnosticians were given hands-on training using the 2 validated qPCR assays for diagnosing S. endobioticum. These were provided in a total of three (3) separate sessions during September and October 2009. Further training sessions with the goal of introduc-ing these methods to PPQ scientists who would be in-volved in initial identifications of suspect PW has been scheduled for January of 2010.

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PROFICIENCY TEST PANEL DEVELOPMENT FOR THE PPQ NATIONAL PLANT PROTECTION LABORATORY

ACCREDITATION PROGRAM (NPPLAP)


Development of proficiency testing reagents and administrating of Proficiency Testing Programs for high consequence plant pathogens CPHST STAFF: Vessela Mavrodieva (lead) Sarika Negi CHAMPIONS: John Paine, Patrick Gomes, Jonathan Jones CONTACT: Vessela Mavrodieva ([email protected], 301-504-7100)

The Proficiency Testing (PT) Program started in 2005 as a part of the Provisional Approval Program for P. ramorum (the causal agent of Sudden Oak Death disease). A Fed-eral Order was issued in December 2004 for molecular testing and certification of all potential P. ramorum host nursery plants shipped outside the states of CA, OR and WA. This order would consequently overwhelm the NPGBL (National Plant Germplasm and Biotechnology Laboratory in Beltsville, MD) with thousands of P. ramorum diagnostic samples. In order to expedite and facilitate the molecular diagnostic process, the decision was made to involve the NPDN (National Plant Diagnostic Network), Federal and State Laboratories in the molecular testing following facility inspection and proficiency testing of their personnel. Over the past five years the Provisional Approval program has evolved into the National Plant Protection Laboratory Accreditation Program (NPPLAP). NPPLAP is a voluntary program based on inspection, proficiency testing and use of validated methods to perform molecular diagnostic test-ing of regulatory plant pathogens. In order for a laboratory to participate in the program, the facilities, equipment, diag-nostician’s qualifications, samples management, record keeping and data control are inspected by a visiting team of NPPLAP inspectors.

After the laboratory is found complaint with the NPPLAP standards diagnosticians can chose to participate in the PT programs to obtain certification for molecular diagnostic of a particular pathogen. The NPGBL develops and produces the PT panels and manages the PT programs. We also pro-vide work instructions of PPQ approved validated diagnostic methods. In addition to the P. ramorum PT program, a second pro-gram for Huanglongbing (HLB), also known as citrus green-ing, started in 2007. In 2009, the NPGBL PT team pro-duced and distributed the P. ramorum PT panel from De-cember 2008 to April 2009 and the HLB PT panel from June 2009 to January 2010. The following table (1) provides an overview of each of the PT programs and the number of participants receiving certification to perform diagnostic test-ing for the specific pathogen at their laboratory. Although HLB PT panel and P. ramorum PT panels have been developed for testing the participants’ ability to diag-nose two very different pathogens, the molecular biology tools used for diagnostic are very similar (DNA extraction from plant material, Conventional PCR and Real-time PCR). For this reason, composition, development and validation of both PT panels are similar.

Table 1.

Panels pro-duced in 2009

Total # of Participants

# of Partici-pants ap-proved at

first attempt

# of Partici-pants ap-proved at second at-

tempt

Failure # of Partici-pants not

submitting the results

# of Labora-tories with Certified

Participants

HLB PT09 17 14 2 0 1 10

PRAM PT09 24 20 2 1 1 14


ACCREDITATION PROGRAM (NPPLAP) (CONT)


Each panel is a randomized set of twelve blind samples: lyophilized tissue samples (six to eight) and DNA sam-ples (six to eight). PT panels consist of healthy and in-fected samples at different level of infection (range of pathogen titer). Successful DNA extraction is a critical part of reliable molecular diagnostics therefore we have included tissue samples in our PT panels to fully evalu-ate diagnosticians’ proficiency. DNA is extracted from the lyophilized tissue samples prior to initiating the re-quired molecular diagnostic tests. Each of the lyophi-lized tissue samples is produced by processing a batch of freshly chopped leaf tissue and dispensing an exact weight of the processed tissue in numerous glass vials. Tissue samples are lyophilized in a freeze dryer (Figs. 4 & 5) for preserving the tissue and the pathogen. We also provide corresponding healthy and positive PCR con-trols with each of the PT panels. Panel Validation: Through validation we establish and confirm that each batch of tissue is homogeneous e.g. individual samples of each batch produce consistent results when tested by various analysts. We test 12%-15% of samples from each batch produced. The valida-tion process of each tissue batch involves DNA extrac-tion, PCR testing (conventional PCR and real-time PCR), and data analysis.

For the validation of DNA panel set only PCR testing (conventional and real-time PCR) is performed. In our validation testing we evaluate each batch (tissue or DNA) for sample to sample variability, analyst to ana-lyst variability and sample stability.

Sample to sample variability: Figures 1 and 2 show the real-time PCR results of two different tissue batches prepared to be used in the HLB PT09 panel. Twenty randomly selected samples from each batch were tested by three NPGBL analysts. The upper line in the graph refers to the variability of the level of pathogen infection and the lower line refers to the level of plant DNA detected (internal control for extraction method) in the 20 samples tested by real-time PCR. The samples from batch # HLB09-02 (Fig. 1) showed very stable and consistent infection level and plant DNA level therefore this batch was used as one of the tissue samples for HLB PT09. The samples from batch # HLB09-05 (Fig. 2) produce consistent values for internal control plant DNA level but pathogen infection level was inconsistent between the samples including a false negative This batch was excluded from the panel due to high sample to sample variability.

Figure 1. Real-time PCR results for Batch # HLB09-02 Figure 2. Real-time PCR results for Batch # HLB09-05


ACCREDITATION PROGRAM (NPPLAP) (CONT)


Evaluation of analyst to analyst variability: is achieved by having three individual NPGBL scientists test equal number of samples from each batch. This is done to mimic the variable skill levels of PT panel participants. Sample stability: In order to monitor the stability of each of tissue and DNA panel samples overtime, we perform quality control (QC) testing periodically, at least over the duration of panel distribution. Figure 3 graph shows the results of a tissue batch prepared in November of 2006 to be included in HLB PT07 panel. The stability of this in-fected tissue batch was monitored from November 2006-July 2008. The HLB infection level (indicated by the upper line) and the plant DNA level (internal control, indicated by the lower line) are both stable over the two year’s testing period.

Following validation, panels are assembled and distributed to participating labs according to a schedule. Initial evalua-tion of the PT results is conducted by the NPGBL with final certification by the NPPLAP. As a result of the PT programs for P. ramorum and HLB total number of samples sent to the PPQ PHP Molecular Diagnostic Lab has decreased several folds. Samples turn-around time is much faster that allow for timely regulatory decisions. The PT programs have also helped considerably to improve NPDN and State diagnosticians’ skills in molecu-lar testing, and to expand laboratory network capacity, and improved preparedness to potential plant pathogen emer-gencies. We would like to acknowledge our collaborators on this project. We have a cooperative agreement with Dr Paul Tooley’s laboratory at USDA-ARS-FDWSR Unit in Ft. Detrick, MD. Dr Tooley’s laboratory provides us with healthy and infected plant tissue for P. ramorum PT panel. For the HLB PT panel we receive healthy tissue from Dr John Hartung’s laboratory at ARS (Agriculture Research Service) in Beltsville and Dr John Bash from CCPP (Citrus Clonal Protection Program) at University of California, River-side, CA. Infected HLB field tissue is provided to us by Hilda Gomez at USDA-APHIS-PPQ-CHRP in Plantation, FL.

Figure 3. Infected tissue batch QC tested from Nov 2006 to Jul 2008.

Figure 4. Glass vials containing the processed tissue placed inside freeze dryer for lyophilization.

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QUALITY MANAGEMENT/QUALITY ASSURANCE


CPHST STAFF: Renee DeVries (lead) CHAMPIONS: EDP Prgram managers (Jonathan Jones, Osama El-Lissy, Don Albright, Pat Gomes), PHP-PGQP ( Joseph Foster, Margarita Licha, Jorge Abad, Clarissa Maroon-Lango) and the NPDN CONTACT: Renee.M. [email protected], (301) 504-7100)

New or Up-dated (Group)

Pathogen detection by PCR Conventional PCR assay

Real-time PCR assay

Release Dates Original or (Revision)

NEW (Germplasm) Blackcurrant reversion virus (BRV) WI-B-T-G-18 WI-B-T-G-14 04-15-2009; 05-19-2009

NEW (Germplasm) Colombian datura virus (CDV) WI-B-T-G-12 WI-B-T-G-9 02-13-2009; 03-30-2009;

NEW (Germplasm) Potato yellow vein virus (PYVV) WI-B-T-G-15 WI-B-T-G-13 04-15-2009; (06-29-2009); 07-10-2009

NEW (Germplasm) Sweet potato chlorotic fleck virus (SPCFV)

WI-B-T-G-21 WI-B-T-G-16 06-01-2009; 07-10-2009

NEW (Germplasm) Sweet potato chlorotic stunt virus (SPCSV) –West African (WA) group

WI-B-T-G-19 WI-B-T-G-17 06-17-2009; 07-10-2009

NEW (Germplasm) Sweet potato mild speckling virus (SPMSV)

WI-B-T-G-11 WI-B-T-G-7 01-15-2009; (02-17-2009); 03-30-2009

NEW (Select Agent) Citrus Variegated Chlorosis (CVC) strain of Xylella fastidiosa

WI-B-T-G-16 WI-B-T-G-15 05-19-2009; 06-01-2009

Updated (Detection and Con-firmation) and NEW Espanol

Citrus Greening in PLANTS (HLB, Huanglongbing) Candidatus Liberi-bacter asiaticus

WI-B-T-C-2 WI-B-T-D-2; ESPANOL WI-B-T-D-2; ESPANOL WI-B-T-C-2

(04-08-2009); (04-27-2009); Rel Es 8-19-2009 Rel Es 8-27-2009

Updated (Detection and Con-firmation) and NEW Espanol

Citrus Greening in PSYLLIDS (HLB, Huanglongbing) Candidatus Liberi-bacter asiaticus

WI-B-T-D-1; Espanol WI-B-T-D-1

(04-08-2009); Rel Es 8-19-2009

Updated (Detection and Con-firmation)

Phytophthora ramorum by Elicitin Target

WI-B-T-1-7 (02-24-2009)

NEW (Detection) Phytophthora kernoviae WI-B-T-1-18 02-09-2009

NEW (Approved for Train-ing only-Detect)

Potato Wart (Synchytrium endobioticum)

WI-B-T-1-22 09-14-2009

NEW and Updated (NPPLAP Test Pan-els)

Proficiency Panel Work Instruction P. ramorum _PCR_PT08 Panel

PT-B-T-0-11 PT-B-T-1-6 New PT-B-T-1-7

(12-03-2008); (12-03-2008); 12-03-2008

QUALITY MANAGEMENT/QUALITY ASSURANCE (CONTINUED)


Non-PCR Procedure

Document

Title Release Dates Original or (Revision)

Updated

WI-B-T-1-16 Psyllid Sample Extraction for use in Citrus Greening or HLB (Huanglongbing) Molecular Diagnostic Assays

(10-3-2008); (10-08-2008); (04-08-2009)

Updated WI-B-T-1-17 Plant Sample Extraction for use in Citrus Greening or HLB (Huanglongbing) Molecular Diagnostic Assays

(04/27/2009)

NEW WI-B-T-2-9 Extraction of DNA from Potatoes for Detection of Potato Wart by PCR 09-14-2009

NEW and Updated

WI-B-T-G-1 RNA Extraction for Potyvirus that Infect Germplasm 10-16-2008; (06-12-2009)

NEW and Updated

WI-B-T-G-8 with Attach-ment A (primer list)

Preparation of cDNA from Total RNA, for use in the Molecular Detection of Potyviruses in Plant Germplasm

04-27-2009; At 04-27-2009; At (06-01-2009); At (06-17-2009); At (07-10-2009)

Updated (NPPLAP –Test Panels)

PT-B-T-2-3 Proficiency Panel Work Instruction

Extraction of DNA from Lyophilized PT09 Panel Samples for Phy-tophthora spp. PCR analysis

(12-03-2008)

New and Updated

Attachments A, B, C, D, E

Proficiency Panel Flow Chart Attachments A: Control Evaluation_Real-time PCR for PT panel B: Sample Evaluation_Real-time PCR for PT panel

C: Control Evaluation_Conventional PCR for PT panel D: Sample Evaluation_Conventional PCR for PT panel E: Determinations using Real-time and Conv results

A (12-02-2008) B (12-02-2008) C (12-02-2008) New D 12-02-2008 New E 12-02-2008

NEW Espa-nol

ESP-WI-B-T-1-16

Psyllid Sample Extraction for use in Citrus Greening or HLB (Huanglongbing) Molecular Diagnostic Assays

08-19-2009

Updated Expanol

ESP-WI-B-T-1-17

Plant Sample Extraction for use in Citrus Greening or HLB (Huanglongbing) Molecular Diagnostic Assays

(08-18-2009)

NEW WI-B-T-NV-11 Plant Sample Extraction for use in Citrus Variegated Chlorosis (CVC) strain of Xylella fastidiosa Molecular Diagnostic Assays

09-02-2009

NEW WI-B-T-NV-12 Culture and Isolation of Xylella fastidiosa from citrus plants 09-02-2009

NEW WI-B-T-NC-13 DAS-ELISA for detection of Xylella fastidiosa 09-02-2009

NEW WI-B-T-NC-14 Pathogenicity testing Xylella fastidiosa in Sweet Orange citrus plants 09-02-2009

PLANT PATHOGENS DETECTION ACTIVITIES


Pathogen Detections Diagnosed by CPHST- NPGBL in 2009

Pathogen tested Dates # samples Host Source Parallel tested with PHP-MDL

Results

Citrus Variegated Chlorosis strain of Xylella fastidiosa

6/11 & 12/2009 3 Citrus Georgia Negative

1/23/2009 3 Citrus Guam yes Inconclusive

9/15/2009 5 Psyllids Tijauana yes Negative

11/6/2008 9 Psyllids California no Negative

11/5/2008 2 Psyllids Georgia no Negative


10/28/2008 1 Psyllids Alabama no Negative



10/24/2008 12 Psyllids Puerto Rico no Negative




10/20/2008 6 Psyllids Alabama no Negative

10/17/2008 98 Psyllids Puerto Rico no One Inconclusive;

97 negative

10/17/2008 39 Zanthophyllum Forwarded from Florida in March,

2008 no Negative





10/1/2008 7 Psyllids Louisiana no Negative


10/1/2008 3 Psyllids South Carolina no Negative

Huanglongbing

Candidatus

Liberibacter

asiaticus

PLANT PATHOGENS DETECTION ACTIVITIES (CONTINUED)


Pathogen Detections Diagnosed by CPHST- NPGBL in 2009

Pathogen tested

Dates # samples Host Source Parallel tested with PHP-

Results

Potato Cyst

Nematode

(Globodera

pallida)

10/3/2008 4 potato Idaho yes Negative

12/5/2008 4 potato Idaho yes Positive for G. pallida

5/27/2009 3 potato Idaho yes Negative (Determined to be G. tabacum)

6/01/2009 1 potato Idaho no

Negative (Determined to be G. tabacum)

Plum Pox

Virus

(PPV)

3/4/2009 20 with 8-10 sticks each

Prunus bud-wood (forced)

California no Negative

3/6/2009 106 Prunus Washington no Negative

6/16/2009 9 Prunus New York no 1 POSITIVE


6/26 & 30/2009 6 Prunus New York no 2 not tested & 4 negative



7/21/2009 3 Prunus Michigan no Negative

7/28/2009 4 Prunus Michigan no Negative

7/30 & 31/2009 3 Prunus Michigan no Quality Control samples (POSITIVE)

7/31/2009 3 Prunus New York no

Quality Control samples (POSITIVE)


8/13/2009 1 Prunus New York no Negative

9/22/2009 3 prunus Michigan no Negative

3/3/2009 3 Geranium Virginia no Negative for Rs

3/10/2009 22 Rinsate from Geranium

Wisconsin no Negative for Rs

7/2/2009 5 Mandevilla Michigan no Negative for Rs Race 3 Biovar 2 (Determined to

be Rs Biovar 3)

Ralstonia so-lanacearum

and Rs Race3, Biovar 2

TECHNOLOGY TRANSFER

PAGE 32 2009 ANNUAL REPORT CPHST NPGBL, MD

Training Workshops Provided by NPGBL staff:

Title: Molecular detection of Blackcurrant reversion nepovirus (BRV) using conventional multiplex RT-PCR and real-time multiplex RT-PCR. Instructors: Mark Nakhla and Kristina Owens Date: September 21, 2009 Location: Beltsville, MD

Title: Molecular detection of Sweet potato chlorotic stunt crinivirus (SPCSV) using conventional multiplex RT-PCR and real-time multiplexed RT-PCR. Instructors: Mark Nakhla and Gang Wei Date September 3, 2009 Location: Beltsville, MD

Title: Detection and quantification of Candidatus Liberibacter species associated with citrus huanglongbing by conven-tional and real-time PCR. Instructors: Wenbin Li and Elizabeth Twieg Date: Aug. 24-27, 2009. Location: Beltsville, MD

Title: Molecular detection of Plum pox virus (PPV). Instructor: Vessela Mavrodieva Date: June, 2009 Location: Beltsville, MD

Title: Molecular detection of Blackcurrant reversion nepovirus (BRV) using conventional multiplex RT-PCR and real-time multiplex RT-PCR. Instructors: Mark Nakhla and Kristina Owens Date: June 22, 2009 Location: Beltsville, MD

Title: Molecular detection of Sweet potato chlorotic stunt crinivirus (SPCSV) using real-time multiplexed RT-PCR. Instructors: Mark Nakhla and Gang Wei Date June 15, 2009 Location: Beltsville, MD

Title: Molecular detection of exotic plant pathogens - Biosecurity Issues and Technologies course. Instructors: Laurene Levy and Mark Nakhla Date: April 8-9, 2009. Location, Rutgers University, NJ.

Title: Detection and quantification of Candidatus Liberibacter species associated with citrus huanglongbing by conven-tional and real-time PCR. Instructors: Wenbin Li and Elizabeth Twieg Date: Feb. 2-5, 2009. Location: Beltsville, MD

Title: Detection and quantification of Candidatus Liberibacter species associated with citrus huanglongbing by conven-tional and real-time PCR. Instructors: Wenbin Li and Elizabeth Twieg Date: Jan. 2-5, 2009. Location: Beltsville, MD

TECHNOLOGY TRANSFER

2009 ANNUAL REPORT CPHST NPGBL, MD PAGE 33

Training Workshops Provided by NPGBL staff (cont.):

Title: Detection and quantification of Candidatus Liberibacter species associated with citrus huanglongbing by conven-tional and real-time PCR. Instructors: Wenbin Li and Elizabeth Twieg Date: Dec 8-11, 2008 Location: Beltsville, MD

Title: Detection and quantification of Candidatus Liberibacter species associated with citrus huanglongbing by conven-tional and real-time PCR Instructors: Wenbin Li Date: Nov. 2-8, 2008. Location: Queretaro, Mexico.

Title: Detection and identification of Potato Cyst Nematode using conventional and real-time PCR. Instructors: Mark Nakhla, Kristina Owens and Gang Wei. Date: October 21-23, 2008. Location: Beltsville, MD

Title: Detection and identification of Potato Cyst Nematode using conventional and real-time PCR. Instructors: Mark Nakhla, Kristina Owens and Gang Wei. Date: October 8-10, 2008. Location: Beltsville, MD Invited Presentations Provided by NPGBL staff

L. Levy and P. Berger. 2009. Regulatory Diagnostics in the U.S., Challenges and Opportunities. EPPO Conference on Diagnostics, York, GB May 10-15, 2009.

Li W and Levy L (2009) Citrus huanglongbing diagnosis based on molecular detection of associated Liberibacter species. Proc. Int. Workshop on Citrus Quarantine Pests, Villahermosa, Tabasco, Mexico.

Li W and Levy L (2009) Genomewide search for candidate genes for detection and identification of ‘Candidatus’ Liberibac-ter species. Citrus Huanglongbing and Potato Zebra Chip Joint Conference, McAllen, TX, November.

Nakhla, M.K., and Levy, L. 2009. One step ahead of a pathogen. The University of Delaware, DE. Presentations Provided by NPGBL staff in professional meetings Li W, Abad JA, and Levy L (2008) ‘Candidatus Liberibacter solanacearum’ associated with zebra chip of potato is not as-sociated with citrus huanglongbing and is absent in Asian citrus psyllids. Proc Int Research Conf on Huanglongbing: 168, Orlando, FL. (oral)

Li W, Duan Y, Brlansky RH, Twieg E, and Levy L (2008) Incidence and population of ‘Candidatus Liberibacter asiaticus’ in Asian citrus psyllids (Diaphorina citri) on citrus plants affected by huanglongbing in Florida. Proc Int Research Conf on Huanglongbing: 231, Orlando, FL.(poster)

Z. Liu, K. Rappaport and L. Levy. 2009. Adaptation of CANARY Biosensors for Rapid Detection of Plant Pathogens. Poster. The American Phytopathological Society Meeting, Portland, OR. August 1-5, 2009. Phytopathology. Vol. 99 (No. 6, Supplement).

L. Levy, V. Mavrodieva, and P. Shiel. Activities at the USDA PPQ CPHST Laboratory in Beltsville, MD for diagnostics test development, validation, training, and proficiency testing. EPPO Conference on Diagnostics, York, GB May 10-15, 2009. (Poster Presentation).

TECHNOLOGY TRANSFER

PAGE 34 2009 ANNUAL REPORT CPHST NPGBL, MD

Other meetings attended by NPGBL staff : The American Phytopathological Society Meeting, Portland, OR. August 1-5, 2009. (Mark Nakhla, Zhaowei Liu, Sarika Negi, Derik Picton, Kristina Owens and Kate Rappaport). The 21st International Conference on Virus and Other Graft Transmissible Diseases of Fruit Crops (ICVF), Neustadt, Ger-many. July 5-10, 2009. (Vessela Mavrodieva). Fourth Sudden Oak Death Science Symposium, Santa Cruz, CA, June 15-18, 2009 (Kurt Zeller). Foreign Plant Germplasm ARS and APHIS Annual Meeting, ARS, BARC-West, Beltsville, MD, May 4, 2009 (Laurene Levy and Mark Nakhla). The Integrated Consortium of Laboratory Networks, Atlanta, GA. 2 days. March, 26-27, 2009 Network Methods Technical Working Group, (Laurene Levy) Network Coordinating Group Quality Assurance Technical Working Group, (Renee DeVries) Network Training Working Group, (Mark Nakhla) National Select Agent Workshop. APHIS. Riverdale, MD. 1 days. December, 09-2008 (Laurene Levy and Renee DeVries) The Integrated Consortium of Laboratory Networks, Training Group, Ft. Detrick, MD, October 16, 2008 (Mark Nakhla) Publications Duan Y, Zhou L, Hall DG, Li W, Doddapaneni H, Lin H, Liu L, Gariel DW, Vahling CM, Williams K, Dickerman A, and Gottwald T (2009) Complete genome sequence of citrus huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’ obtained through metagenomics. Mol Plant-Microbe Interact 22:1011-1020. Li W, Li D, Twieg E, Hartung JS, and Levy L (2008) Optimized quantification of unculturable ‘Candidatus Liberibacter sp.’ in host plants by real-time PCR. Plant Dis 92: 854-861. Li W, Abad JA, French-Monar RD, Rascoe J, Wen A, Gudmestad NC, Secor GA, Lee I.-M, Duan Y, Levy L (2009) Multi-plex real-time PCR for detection, identification and quantification of ‘Candidatus Liberibacter solanacearum’ in potato plants with zebra chip. J Microbiol Methods 78: 59-65. Li W, Levy L, and Hartung JS (2009) Quantitative distribution of ‘Candidatus Liberibacter asiaticus’ in citrus plants with citrus huanglongbing. Phytopathology 99: 139-144. Wang N, Li W, Irey M, Albrigo G, Bo K, Kim J-S (2009) Citrus huanglongbing – an invited mini-review. Tree and Forestry Science and Biotechnology, pp. 66-72, Global Science Books. Wen A, Mallik I, Alvarado VY, Pasche JS, Wang X, Li W, Levy L, Lin H, Scholthof HB, Mirkov TE, Rush CM, and Gudme-stad NC (2009) Detection, distribution and genetic variations of ‘Candidatus Liberibacter sp’ associated with zebra com-plex of potato in North America. Plant Dis 93:1102-1115

STAFF DIRECTORYSTAFF DIRECTORYSTAFF DIRECTORY


Laurene Levy Lab Director (301) 504-7100 [email protected] Renee DeVries Quality Manager (301) 504-7100 [email protected] Hazel Goodwin Lab Support Assistant (301) 504-7100 [email protected] Wenbin Li Citrus Pathogen Detection Program Leader (301) 504-7100 [email protected]

Zhaowei Liu Biotechnology Evaluation Program Leader (301) 504-7100 [email protected] Vessela Mavrodieva Proficiency Test Program Manger & PPV Detection Program Leader (301) 504-7100 [email protected] Mark Nakhla Foreign Germplasm Pathogen Detection Program Leader (301) 504-7100 [email protected] Kurt Zeller Fungal Pathogen Detection Program Leader (301) 504-7100 [email protected]

STAFF DIRECTORY STAFF DIRECTORY STAFF DIRECTORY (((CONTCONTCONT.).).)


Sarika Negi Proficiency Test Panel Development Manager (301) 504-7100 [email protected] Kristina Owens Foreign Germplasm Pathogen Detection Program (301) 504-7100 [email protected] Deric Picton Foreign Germplasm Pathogen Detection Program (301) 504-7100 [email protected] Kate Rappaport Biotechnology Evaluation Program (301) 504-7100 [email protected]

Elizabeth Twieg Citrus Pathogen Detection Program (301) 504-7100 [email protected] Gang Wei Foreign Germplasm Pathogen Detection Program (301) 504-7100 [email protected] Karen Williams Proficiency Test & PPV Detection Program (301) 504-7100 [email protected] Ping Yang Fungal Pathogen Detection Program (301) 504-7100 [email protected]

USDA-APHIS-PPQ-CPHST-NPGBL BARC-East, Bldg-580, Powder Mill Road, Beltsville, MD 20705-2350 PHONE: (301) 504-7100 FAX: (301) 504-8539

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