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Activity 4.1. Develop PCR-based diagnostic assay for banana xanthomonas wilt (BXW)
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- Activity 4.2. Develop, test and deploy assays for detection of whitefly- and aphid-transmitted viruses.
Activity 4.1. Develop PCR-based diagnostic assay for banana xanthomonas wilt (BXW).Work conducted to develop a PCR assay for BXW is in press at this writing: Lewis Ivey, M. L., Tusiime, G. and Miller, S. A. 2009. A PCR assay for the detection of Xanthomonas campestris pv. musacearum in bananas. Plant Disease 93 (in press).
The IPDN is working closely with the Insect-Transmitted Virus global theme to identify critical virus problems and improve virus detection/diagnosis capability, primarily in Central America and West Africa. Evaluation of the AgDia Plant Sap Collection and Testing Kit for Detection of DNA and RNA viruses. The method that we have been using for preparing samples for testing for infection with whitefly-transmitted begomoviruses is the squash blot, in which plant tissues are squashed directly onto nylon membranes. These nylon membranes are then returned to the laboratory where they can be treated and hybridized with begomovirus DNA probes. Alternatively, the squashed tissues on membranes can be used as a source of DNA for PCR (squash blot-PCR). This method has worked well and has been used for the characterization of begomoviruses from West Africa, Latin America and Asia (see publications Zhou et al., 2008; Kon et al., 2009 at the beginning of the report).More recently, AgDia has developed a new method for collection of plant sap for various purposes, including testing for the presence of virus infection. This method involves the generation of plant sap and application onto adsorption strips, which are thin strips or sticks that have a fibrous binding matrix (pad) at the base, on which the sap is applied. This method shows promise for the detection of DNA viruses such as begomoviruses, as well as viruses with an RNA genome. Detection of begomovirus infection in samples from West Africa. To assess this method for begomovirus detection, we were provided a number of these adsorption strips for evaluation for detection of begomovirus in samples collected in West Africa in September 2009. The samples tested were tomatoes and okra from various locations in Mali that showed begomovirus-like symptoms (Table 1). Sap was prepared and samples were applied to the adsorption strips following the procedure provided by Agdia, except that the sap was prepared using buffer and bags that were provided with AgDia immunostrips. This modification was used based on our preliminary tests performed with RNA viruses. The sap was prepared in a hotel room and put onto the pads of the adsporption strips. The strips were allowed to air-dry and then taped onto index cards. The strips were then transported back to UC Davis where DNA was extracted from the pad using a modified Dellaporta procedure. The DNA extract was then used in the PCR with degenerate begomovirus primers (UPV1/1000C) and primers for detection of betasatellites (the small satellite DNA molecules that are commonly associated with monopartite begomoviruses). The expected size (~1.6 kb) begomovirus fragment was amplified from all three tomato samples with different begomovirus symptoms and from four of five okra samples with begomovirus symptoms (Table 1). The expected size ~1.4 kb betasatellite fragment was amplified from the same four okra samples that the begomovirus fragment was amplified from, but not from any of the tomato samples (Table 1). The begomovirus fragments amplified from two tomato samples (one with symptoms typical of infection with Tomato yellow leaf crumple virus [ToYLCrV] and the other with a severe symptom [broccoli] phenotype) and three okra samples with okra leaf curl symptoms were sequenced. From both tomato samples, the sequence of the amplified fragment was 97-98% identical to ToYLCrV, one of the known tomato-infecting begomoviruses in West Africa (Zhou et al., 2008). Furthermore, this result was consistent with one of the samples having symptoms typical of ToYLCrV infection. The severe symptom phenotype can be due to mixed infection of begomoviruses (including ToYLCrV) or a begomovirus and a betasatellite (Chen et al., 2009). The symptoms in this particular plants were probably due to mixed begomovirus infection (i.e., ToYLCrV and another begomovirus, possibly Tomato leaf curl Mali virus) based upon the finding of no betasatellite in this plant. In the case of the okra, the sequence of all three of the PCR-amplified fragments was 95-96% identical to Okra yellow crinkle virus (OYCrV), a begomovirus known to be associated with okra leaf curl in Mali (Kon et al., 2009). Furthermore, the detection of the betasatellite in all the samples in which OYCrV was detected was consistent with OLCD being caused by a complex of begomoviruses and a betasatellite (Kon et al., 2009). These results indicate that the adsorption strips can be used for sampling of plants with suspected begomovirus symptoms for subsequent PCR analyses. This could be combined with a new PCR detection method that requires no DNA extraction step. Further testing needs to be done with additional plant species from other locations, but these results are very encouraging and this method should compliment the squash blot method. Note that the squash blot method represents an alternative to PCR-based methods as one can use hybridization with DNA probes to detect begomovirus nucleic acids. Furthermore, the fact that the AgDia Plant Sap Collection and Testing Kit method worked with okra, a malvaceous host with mucilaginous sap that can be difficult to use in the SB-PCR method, indicates that the utility of this method for a wide variety of host plants. Detection of RNA viruses. We also assessed the technique for detection of RNA viruses. In initial experiments, we assessed the method for detecting a known virus, Tomato spotted wilt virus (TSWV). Sap was prepared using the immunostrip bags and buffer, and applied to the adsorption strips. RNA was extracted according to a method provided by AgDia for extraction of RNA from immunostrips and used in RT-PCR with TSWV primers (tswvN1 and tswvN777). From all three known TSWV-infected samples, the expected ~700-800 bp fragment was amplified. No fragment was amplified from a healthy tomato control. This suggested that this method may be suitable for preparation of sap for detection of RNA viruses. Further, we tested the method for the detection of cucurbit-infecting viruses in samples collected in West Africa in September 2009. Samples were collected from a cucurbit field in Baguineda showing three different symptom types: 1) a typical Zucchini yellow mosaic virus (ZYMV)-type symptom (strong leaf distortion, strap-leaf symptoms and mosaic), 2) light green interveinal mosaic symptom (potyvirus-like) and 3) a spotting/yellowing symptom that appeared more similar to symptoms induced by the crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). Sap was prepared as described above and applied to the adsorption sticks in Mali (in a hotel room), and the sticks were brought to UC Davis. RNA was extracted as described above and two samples of each of the three symptom types were tested for infection with potyviruses or CYSDV by RT-PCR with degenerate potyvirus primer pairs (HcPro primer pair: DegHcProFrw/DegHcProRev and the CI primer pair: DegCIFrw/DegCIRev) or a primer pair that amplifies the CYSDV capsid protein sequence (CY-13/CY-14). The expected-sized potyvirus fragments were amplified from extracts of samples with the ZYMV-like symptoms and the light green interveinal mosaic. Sequence analysis of these fragments revealed >90% identity with ZYMV sequences, indicating that both of these symptoms were probably caused by infection with ZYMV. No fragments were amplified from the sample with the spotting/yellowing symptoms with RT-PCR and either the potyvirus or CYSDV primers; thus, it is not clear what is causing these symptoms. However, these results reveal the promise of this sample preparation method for detection of RNA viruses. We also were able to use this method to successfully detect Rice yellow mottle virus (RYMV) infection in rice from samples collected in Mali. Table 1. Samples prepared using the Agdia Plant Sap Collection and Testing Kit and tested for begomovirus and betasatellite infection using PCR. PCR amplification of expected-size fragment Host Location Symptoms Begomovirus Betasatellite Sequence Tomato Kati typical ToYLCrV + - ToYLCrV (97%) Tomato Kati typical leaf curl + - ND Tomato Kati severe symptom + - ToYLCrV (98%) phenotype Okra Sikasso leaf curl, crumple + + OYCrV (96%) Okra Baguineda leaf curl, crumple + + OYCrV (96%) Okra Baguineda leaf curl, crumple - - Okra Kati leaf curl, crumple + + OYCrV (95%) Okra Kati leaf curl, crumple + + ND Abbreviations: ToYLCrV=Tomato yellow leaf crumple virus; OYCrV=Okra yellow crinkle virus; and ND=not determined. Appendix A Certificate of Achievement for KARI NARL Diagnostics Training 
APPENDIX B Participants in the 3rd Central American IPDN Diagnsotics Workshop, Guatemala City (Universidad de Rafael Landivar) September 17-18, 2009 NAME INSTITUTION EMAIL ADDRESS 1- Julio García ANALAB, Guatemala julio.rgm@anacafe.org 2- Javier Díaz FHIA, Honduras fjdiaz@fhia.org.hn 3- José Melgar FHIA, Honduras jmelgar@fhia.org.hn 4- Reina Flor de Serrano CENTA, El Salvador reinafserrano@hotmail.com 5- Claudia Toledo UVG, Guatemala palmieri@uvg.edu.gt 6- Mónica Orozco UVG, Guatemala palmieri@uvg.edu.gt 7- Margarita Palmieri UVG, Guatemala palmieri@uvg.edu.gt 8- Cristina Bailey URL, Guatemala acbailey@url.edu.gt 9- Charles MacVean URL, Guatemala cmacvean@url.edu.gt 10- Eduardo García URL, Guatemala megarcia@url.edu.gt 11- Nelson García Santos MAGA, Guatemala erodriguez341@yahoo.com 12- Edil Rodríguez MAGA, Guatemala erodriguez341@yahoo.com 13- Marvin Morales AGROEXPERTOS marvin.morales@agroexpertos.com 14- Mario Arévalo AGROEXPERTOS info@agroexpertos.com 15- Marco Arévalo AGROEXPERTOS marco.arevalo@agroexpertos.com 16- Isabel Arias AGROEXPERTOS isabel.arias@agroexpertos.com 17- Julio del Cid Soluciones Analíticas delcidjr@solucionesanaliticas.com 18- Edin Orozco FAUSAC edin@hotmail.com 19- Alex Pacheco FAUSAC edin@hotmail.com 20- Gabriela Calderón FAUSAC edin@hotmail.com APPENDIX C Agenda for Stakeholder Meeting and Participatory Appraisal Schedule for the IPDN Training Workshop IER, Bamako, Mali Day 1: 16 February, 2009 09:00
10:30
13:00 – 14:00 Lunch Break 14:00 – 17:00
Day 2: 17 February 2009 08:30
13:00 – 14:00 Lunch Break 14:00 – 17:00
Day 3: 18 February 2009 08:30 – 13:00
13:00 – 14:00 Lunch Break 14:00 – 17:00
Evening Social Gathering and Dinner Day 4: 19 February 2009 08:00 – 13:00 Field trip to Kati: participants collect disease samples for practical exercises 13:00 – 14:00 Lunch Break 14:00 Lab exercise with samples taken from the field Day 5: 20 February 2009 Evening Dinner at Hotel Plaza 08:30 – 13:00
13:00 – 14:00 Lunch Break 14:00
Short notes by Bandyopadhyay Short notes by Don Mullins Short notes by Dr. Ekow Akyeampong Notes by Adama Traoré APPENDIX D List of Participants in the West Africa IPDN Workshop
APPENDIX E Plant Disease and Pest Diagnostics Training Workshop “New and Emerging Problems and Diagnostic Techniques” Bishkek, Kyrgyzstan Wednesday June 3 – Thursday, June 4, 2009 Training Leader: Dr. Sally Miller, The Ohio State University, Wooster, Ohio USA Trainers: Dr. George Bird, Michigan State University Dr. Barry Jacobsen, Montana State University Dr. Doug Landis, Michigan State University Dr. Frank Zalom, University of California – Davis Dr. Mustafa Bohssini, ICARDA
What to expect – training topics Introduction to the International Plant Diagnostics Network (IPDN) Dr. Sally Miller, Ohio State University 8:45 – 9:45 Emerging and endemic diseases caused by fungi – what is new in diagnostics Dr. Barry Jacobsen, Montana State University 9:45– 10:05 Coffee/Tea Break 10:05 – 11:05 Diagnosing diseases caused by bacteria, phytoplasmas and viruses – molecular and serological approaches Dr. Sally Miller, Ohio State University 11:05 - 12:05 Diagnosing diseases caused by nematodes Dr. George Bird, Michigan State University 12:05 – 13:05 Insect pests of wheat Dr. Mustafa Bohssini, ICARDA 13:05 -14:05 Lunch 14:05 – 17:00 Field diagnostics Dr. Barry Jacobsen, Dr. Murat Aitmatov, Dr. Mustafa Bohssini, leaders Dr. Sally Miller, Dr. Frank Zalom, Dr. Doug Landis, Dr. George Bird
• Field-usable immunoassays for disease diagnosis • Taking good digital images • Collect samples for Friday lab Thursday June 4 8:30 – 13:00 Laboratory (~2 hr segments) Station 1a: Nematode diagnostics – basic for students Station 1b: Nematode diagnostics – advanced for professionals Station 2: Immunoassays for pathogen diagnosis Dot /squash blots, lateral flow devices, ELISA (2, 2 hr segments) Station 3: Sample triage, simple diagnostic steps for bacterial and fungal identification, record-keeping (2, 2 hr segments)
(2, 2 hr segments) (Coffee/Tea break at 10:00 – 10:15) 13:00 – 14:00 Lunch 14:00 – 14:30 Developing a multidisciplinary diagnostic laboratory: the Michigan State University experience Dr. George Bird, Michigan State University 14:30 – 15:00 Diagnostic networking: digital diagnostics and information management Dr. Sally Miller, Ohio State University 15:00 – 16:00 Discussion – priorities for the IPM CRSP Phase 2 proposal 16:00 – 16:30 Closing remarks, acknowledgements and distribution of certificates Yüklə 152,36 Kb. Dostları ilə paylaş:
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