Progress 01/01/05 to 12/31/05
Outputs
The major objective of this USAID MERC project was to provide training and resources so laboratories in the participating countries could develop new or improved molecular methods for detecting virus and viroids of citrus, cucumber, grapevine, potato, stone fruit and tomato. Also, another goal was to increase the scientific interaction among Israeli and Arab scientists. The countries involved in this project were: Jordan, Lebanon, Israel, Palestinian National Authority, Egypt, Tunisia, Morocco and two laboratories in USA. Faculty, staff and students received short term at Hebrew University of Jerusalem, Faculty of Sciences-Tunis, USDA-Laboratory, Beltsville, and University of Wisconsin-Madison. Molecular techniques used included: various ELISA methods, RT-PCR, Immunocapture-RT-PCR, dot blot hybridization with radio active and non-radioactive probes, squash blot and tissue blot hybridization methods, multiplex PCR, real time PCR, GroEL capture-RT-PCR method, and coat
protein expression systems. Methods were developed for Banana bunch top nanovirus, Citrus tristeza virus, Citrus exocortis viroid, Hop stunt viroid, Citrus viroid III, Citrus viroid IV, Cucurbit yellow stunting disorder virus, Cucumber mosaic virus, Australian grapevine viroid, Grapevine yellow speckle viroid, Grapevine fanleaf virus, Grapevine leafroll virus 1,3, Grapevine virus A, Grapevine leafroll associated virus, Potato leafroll virus, Potato virus X, Potato virus Y, Potato virus Y necrotic strain, Apple chlorotic leaf spot virus, Plum pox virus, Peach rosette mosaic virus, Prune dwarf virus, Peach latent mosaic viroid, Tomato yellow leaf curl virus, and Tomato yellow leaf curl Sardinia virus. Scientists participated in an annual meeting each year in Israel or an Arab country, when feasible, or otherwise in a politically neutral country. A handbook with 38 virus detection methods was prepared and these methods were provided to the Plant Protection Units for each country. At
least 20 post graduate degrees were given. Seven workshops were given on molecular methods for virus/viroid detection and at least 250 people participated in these workshops. Extensive manuals were prepared for each of these workshops. Forty-three research publications were published.
Impacts
This project provided the mechanism for effective communication among Israeli and Arab scientists. Five laboratories were equipped with the basic items for molecular detection methods of viruses/viroids. Over 38 virus detection methods were developed and technical training was provided for staff of the Plant Protection Units for each country.
Publications
- Abou-Jawdah, Y. 2005. Tissue-blot immunoassay a rapid method for virus detection in screening tomato lines for TYLCV resistance. Acta Horticult. 695:295-297.
- Anfoka, G. H., Abhary, M. K., Fattash, I., and Nakhla, M. K. 2005. Occurrence and distribution of Citrus tristeza virus (CTV) in Jordan Valley. Phytopath. Medit. 44:17-23.
- Djilani Khouadja F., Rouze-Jouan J., Guyader S., Marrakchi M., and Fakhfakh H. 2005. Biological and molecular characterization of Tunisian isolates of Potato leafroll virus. J. Plant Pathol. 87:91-99.
- Gorsane F., Gharsallah-Chouchane S., Nakhla M., Fekih-Hassen I., Maxwell D., Marrakchi M., and Fakhfakh H. 2005. Simultaneous and rapid differentiation of members of the tomato leaf curl virus complex by multiplex PCR. J. Plant Pathol. 87:39-44.
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Progress 04/15/98 to 09/30/05
Outputs
The major objective of this USAID MERC project was to provide training and resources so laboratories in the participating countries could develop new or improved molecular methods for detecting virus and viroids of citrus, cucumber, grapevine, potato, stone fruit and tomato. Also, another goal was to increase the scientific interaction among Israeli and Arab scientists. The countries involved in this project were: Jordan, Lebanon, Israel, Palestinian National Authority, Egypt, Tunisia, Morocco and two laboratories in USA. Faculty, staff and students received short term at Hebrew University of Jerusalem, Faculty of Sciences-Tunis, USDA-Laboratory, Beltsville, and University of Wisconsin-Madison. Molecular techniques used included: various ELISA methods, RT-PCR, Immunocapture-RT-PCR, dot blot hybridization with radio active and non-radioactive probes, squash blot and tissue blot hybridization methods, multiplex PCR, real time PCR, GroEL capture-RT-PCR method, and coat
protein expression systems. Methods were developed for Banana bunch top nanovirus, Citrus tristeza virus, Citrus exocortis viroid, Hop stunt viroid, Citrus viroid III, Citrus viroid IV, Cucurbit yellow stunting disorder virus, Cucumber mosaic virus, Australian grapevine viroid, Grapevine yellow speckle viroid, Grapevine fanleaf virus, Grapevine leafroll virus 1,3, Grapevine virus A, Grapevine leafroll associated virus, Potato leafroll virus, Potato virus X, Potato virus Y, Potato virus Y necrotic strain, Apple chlorotic leaf spot virus, Plum pox virus, Peach rosette mosaic virus, Prune dwarf virus, Peach latent mosaic viroid, Tomato yellow leaf curl virus, and Tomato yellow leaf curl Sardinia virus. Scientists participated in an annual meeting each year in Israel or an Arab country, when feasible, or otherwise in a politically neutral country. A handbook with 38 virus detection methods was prepared and these methods were provided to the Plant Protection Units for each country. At
least 20 post graduate degrees were given. Seven workshops were given on molecular methods for virus/viroid detection and at least 250 people participated in these workshops. Extensive manuals were prepared for each of these workshops. Forty-three research publications were published.
Impacts
This project provided the mechanism for effective communication among Israeli and Arab scientists. Five laboratories were equipped with the basic items for molecular detection methods of viruses/viroids. Over 38 virus detection methods were developed and technical training was provided for staff of the Plant Protection Units for each country.
Publications
- Abou-Jawdah, Y. 2005. Tissue-blot immunoassay a rapid method for virus detection in screening tomato lines for TYLCV resistance. Acta Horticult. 695:295-297.
- Anfoka, G. H., Abhary, M. K., Fattash, I., and Nakhla, M. K. 2005. Occurrence and distribution of Citrus tristeza virus (CTV) in Jordan Valley. Phytopath. Medit. 44:17-23.
- Djilani Khouadja F., Rouze-Jouan J., Guyader S., Marrakchi M., and Fakhfakh H. 2005. Biological and molecular characterization of Tunisian isolates of Potato leafroll virus. J. Plant Pathol. 87:91-99.
- Gorsane F., Gharsallah-Chouchane S., Nakhla M., Fekih-Hassen I., Maxwell D., Marrakchi M., and Fakhfakh H. 2005. Simultaneous and rapid differentiation of members of the tomato leaf curl virus complex by multiplex PCR. J. Plant Pathol. 87:39-44.
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Progress 01/01/04 to 12/31/04
Outputs
Eight research laboratories are involved in this project, which included Israel, the Palestinian National Authority, Jordan, Lebanon, Egypt, Tunisia, Morocco and USA, Research progress has continued on the development of new methods for virus detection for grapevine and citrus viroids, Citrus tristeza virus, Cucumber yellow stunting disorder virus, grapevine viruses, stone fruit viruses, and Tomato yellow leaf curl virus (TYLCV). Methods included RT-PCR, multiplex PCR, Real-Time PCR, non-radioactive hybridization, and serology. In most cases, PCR fragments were sequenced to confirm results. Virus detection protocols are being placed on the web (www.plantpath.wisc.edu/InVirLab/docs). These methods were used to detect grapevine viruses in Palestine and Tunisia. Citrus tristeza virus was characterized for the first time in Jordan. In Lebanon, the first serological detection assay for Cucumber yellow stunting disorder virus was developed. Australian grapevine viroid was
detected for the first time in the Mediterranean region in Tunisia. The mild strain of TYLCV was found in Jordan, and preliminary data indicate that TYLCSardiniaV is also present. The TYLCSardiniaV complex was found in both Tunisia and Morocco and mixed infections with TYLCV were prevalent. A new virus capture method involving the GroEL chaperon protein from whiteflies was studied. Efforts continued towards the development of serological detection methods using bacterial expressed viral coat proteins.
Impacts
Over 40 virus detection methods have been developed and are being tested in the various Middle East countries. These methods will be used in their respective certification and quarantine departments.
Publications
- Abou-Jawdah, Y., Sobh, H., Cordahi, N., Kawtharani, H., Nemer, G., Maxwell, D. P., and Nakhla, M. K. 2004. Immunodiagnosis of Prune dwarf virus using antiserum produced to its recombinant coat protein. J. Virol. Methods 121:31-38.
- Fekih-Hassan, I., F. Gorsan, F. Djilani, H. Fakhfakh, M. Nakhla, D. Maxwell, and M. Marrakchi. 2003. Detection of Tomato yellow leaf curl Sardinia virus in Tunisia. OEPP/EPPO Bulletin 33:347-350.
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Progress 01/01/03 to 12/31/03
Outputs
Nine research laboratories are involved in this project, which includes Israel, the Palestinian National Authority, Jordan, Lebanon, Egypt, Tunisia, Morocco and USA (two laboratories). The project with the USAID/NIH lasted the first three years. Morocco was the last country added in September 2001. Research funds have been totally spent for Israel, USA and Egypt. Research progress has continued on the development of new methods for virus detection for grapevine and citrus viroids, Citrus tristeza virus, Cucumber yellow stunting disorder virus, grapevine viruses, stone fruit viruses, and Tomato yellow leaf curl virus. Methods included RT-PCR, multiplex PCR, Real-Time PCR, non-radioactive hybridization, and serology. In most cases, PCR fragments were sequenced to confirm results. Six new virus detection protocols are being placed on the web (www.plantpath.wisc.edu/InVirLab/docs). These methods were used to detect grapevine viruses in Palestine and Tunisia. Citrus
tristeza virus was characterized for the first time in Jordan. In Lebanon, the first serological detection assay for Cucumber yellow stunting disorder virus was developed. Australian grapevine viroid was detected for the first time in the Mediterranean region in Tunisia. The mild strain of TYLCV was found in Jordan. The TYLCSardiniaV complex was found in both Tunisia and Morocco and mixed infections with TYLCV were prevalent. A new virus capture method involving the GroEL chaperon protein from whiteflies was studied. Efforts continued towards the development of serological detection methods using bacterial expressed viral coat proteins. In Palestine, the government has appointed the laboratory at Bethlehem University to assist them in virus detection for certification and quarantine issues. Each country will make an effort to transfer technology to the plant quarantine and certification units in their governments in 2004. Training involved sending two students to The Hebrew University
(Rehovot) from Bethlehem University and one student each from Jordan and Morocco to UW-Madison. Extremely successful workshops on Plum pox virus and on potato viruses were held in Morocco and attendance was from government, university, and private sector. Some MERC partners traveled to Morocco to view grapevine problems and tomato diseases. Also, some partners visited the group at UW and attended the American Phytopathological Society meeting. The MERC partners published nine articles or abstracts. The annual meeting of the team was held in Malaga, Spain in February 2003 and the next meeting will be in Crete in July 2004, which will be the last annual meeting for this project.
Impacts
Over 40 virus detection methods have been developed and are being tested in the various Middle East countries. These methods will be used in their respective certification and quarantine departments.
Publications
- Elleuch, A., M. Marrakchi, J.P. Perreault, and H. Fakhfakh. 2003. First report of Australian grapevine viroid from the Mediterranean region. J. of Plant Pathol. 85:53-57.
- Rezk A.A, A.A. Shalaby, M.K. Nakhla, S. El-Deeb, F. Abo El-Abbas, M. El-Hammady, H.M. Mazyad, and D.P. Maxwell. 2002. Molecular methods for detection of Banana Bunchy Top Virus (BBTV) from banana tissues and viruliferous banana aphids. Phytopathology 92:S75.
- Shalaby, A.A, M.K. Nakhla, A.M. Soliman, H.M. Mazyad, A. Hadidi, and D.P. Maxwell. 2002. Development of highly sensitive multiplex reverse transcription-polymerase chain reaction (m-RT-PCR) method for detection of three potato viruses in a single reaction and nested PCR. Arab J. Biotech, Vol. 5(2): 273-284.
- Soliman, A.M., A.A. Shalaby, B.N. Barsoum, G.G. Mohamed, M.K. Nakhla, H.M. Mazyad, and D.P. Maxwell. 2002. Molecular characterization and RT-PCR-ELISA detection of a potato virus X (PVX) isolate from Egypt. Annals Agric. Sci., Sp, Issue 4:1791-1804.
- Youssef, S.A., A.A. Shalaby, H.M. Mazyad, and A. Hadidi. 2002. Detection and identification of prune dwarf virus and plum pox virus by standard and multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA). J. of Plant Pathol. 84 (2):113-119.
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Progress 01/01/02 to 12/31/02
Outputs
The annual meeting of the group was held in Cyprus and a research plan was developed for 2002-2003. Over 30 virus detection protocols have been prepared and placed on the WEB at www.plantpath.wisc.edu/InVirLab/docs/virusindexing.htm. These include methods based on serology, hybridization, PCR, and ELISA-PCR. Protocols are available for viruses of bananas, grape vines, potatoes, tomatoes, stone fruits, and cucumbers. Next year, the different labs in the Middle East will verify these methods and then they will be distributed to quarantine and certification units in Jordan, Egypt, Lebanon, Israel, PNA, Tunisia and Morocco.
Impacts
This project has developed virus detection methods for bananas, grapes, tomatoes, stone fruits, potatoes and cucumbers. These methods will be used in quarantine and certification units in countries in the Middle East.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Virus detection methods for important viruses of potato, grapes, tomato, stone fruits,and banana were evaluated. Four countries, Lebanon, Tunisia, Jordan and Morocco, were added to this project. Methods were developed for detection of the necrotic strain of PVY and for Tomato yellow leaf curl Sardinia virus. ELISA PCR methods were evaluated in several laboratories for detection of PVX and PLRV. The coat protein of grapevine virus A was cloned and used in hybridization tests. The coat protein of prunus necrotic ringspot virus and prune dwarf virus were cloned. A new virus, apricot laten virus, was characterized from peach and detection methods developed. Both TYLCV and TYLCSarV were detected in tomato samples from southern Morocco. Non-radioactive hybridization methods for BBTV were evaluted. Several polyclonal kits are being evaluated for important viruses of potato and tomato. Molecular virus testing methods were introduced to Jordan and expanded in Lebanon, and the
Palestinian National Authority.
Impacts
Virus detection methods for important viruses of potato, grapes, tomato, stone fruits, and banana were evaluated. Methods were developed for detection of PVY, TYLCSarV, TYLCV, PVX, PLRV, grapevine virus A, BBTV, and several additional viruses of prune, peach, potato, and tomato. This project is important for numerous countries including Jordan, Lebanon, the Palestinian National Authority, Tunisia, and Morocco to gain an understanding of the viruses present and to develop detection and control methods to geminiviruses.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
Israel: Methods were developed for the purification of RNA from potato tubers and leaves. Primers were designed for the detection of potato virus Y and methods were developed for the identification of different strains of the virus. For the detection of PVY, PLRV, and PVX, dot blot hybridization, immunocapture RT-PCR and one step RT-PCR were developed. Agarose gel electrophoresis containing bisbenzimide-PEG technique was utilized to distinguish between PVY strains. For the detection of grapevine virus A (GVA) the coat protein of the virus was cloned and used as a probe in the dot blot hybridization method. Bethlehem University: For the first time molecular based techniques were used for the detection of plant viruses in the Palestinian Authority. Dot blot and squash blot hybridization methods were used. The Dig-DNA and Enhanced chemiluminescence non-radioactive labeling systems have been developed and applied successfully for the detection of TYLCV. Methods were
developed for the extraction of nucleic acids from grapes. Non-radioactive Dig-DNA labeling system was developed for the detection of GVA. For the detection of grapevine leaf roll virus, ELISA protocol was adapted using kits from Sanofi, France. Egypt: Production of Polyclonal Antisera Specific for PVY, PVX and PLRV: Using specific PCR primers based on the sequence of potato virus Y (PVY), potato virus X (PVX) and potato leaf roll virus (PLRV), the coat protein (CP) gene of each virus was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and subcloned into an affinity protein expression vector pCAL-n. The PVY, PVX and PLRV CPs were expressed as a fusion product of calmodulin-binding-peptide (CBP). This CBP-CP fusion protein reacted with PVY, PVX and PLRV-specific antisera in western blotting. After cell disruption, the CBP-CP fusion protein was purified by calmodulin resin affinity columns. Antisera obtained from rabbits, after injection with CPB-CP
protein, were specific to each virus (PVY, PVX or PLRV) in direct ELISA. The coat protein of TYLCV was cloned into the bacterial expression vector and the expressed protein was purified. Polyclonal antibodies against TYLCV were produced and used for the production of ELISA kits. This kit will be distributed to all collaborating institutions for evaluation. University of Wisconsin: Developed PCR-ELISA method for detection of potato leaf roll virus and PVX. USDA Lab: Detection and identification methods for purne dwarf virus and plum pox virus were developed with standard and multiplex PCR-ELISA.
Impacts
New molecular-based detection methods were developed for detection of viruses of tomato, potato, and grapes. These will be used in quarantine programs in Middle East countries.
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
Egypt: Virus Collections: The first year focused on collection and characterization of standard viruses for banana (banana bunchy top virus, cucumber mosaic virus), potato (potato virus X, potato virus Y, potato leaf roll virus), tomato (tomato yellow leaf curl virus, and tomato mosaic virus), and stone fruit (plum pox virus). These were stored either as dried tissue and or frozen. Production of Antisera: The coat protein gene for tomato yellow leaf curl virus was cloned into a bacterial expression vector, the protein was successfully expressed, and it was used for the production of antisera. Molecular Characterization of Banana Bunchy Top Virus: Mr. Adel Latif cloned partial DNA fragments from all six components and these were sequenced and submitted to GenBank: (AF102780, AF102781, AF102782, AF102783, AF102784, AF148139). Both non-radioactive and radioactive methods were compared for detection. This technology was then available for use in Egypt upon his return in
fall 1998. Israel: Evaluation of Virus Detection Methods: Squash blot hybridization methods were evaluated for the detection of tomato yellow leaf curl virus using radioactive methods. Squash blot-PCR methods and immunocapture-PCR methods were developed for the detection of tomato yellow leaf curl virus. Three methods were developed and evaluated for cucumber mosaic virus: RT-PCR, immunocapture followed by PCR, and ELISA. These technologies are critical for the success of this proposal and the protocols are being transferred the other collaborating laboratories. Bethlehem University: Establishment of a diagnostic laboratory: After reviewing the many options, the following equipment was purchased: laminar flow hood, ELISA reader, and hybridization oven. Techniques: These include PCR, print-PCR and non-radioactive hybridization. These techniques are now being evaluated in the laboratory at Bethlehem University. Recently, Ms. Tawil has been learning techniques for detection of grape
virus at Rehovot. University of Wisconsin: Characterization of Banana Bunchy Top Virus: Mr. Adel A. Rezk cloned the six components of an isolate of banana bunchy top virus from Egypt and found that it was nearly identical to the isolate characterized from Australia. He then developed both radioactive and non-radioactive methods for detection of this virus. In May, Mr. Ahmed Soliman started developing the ELISA-PCR methods for potato viruses. This ELISA-PCR method was original developed for stone fruit viroids by Dr. A. Hadidi, the USDA collaborator on this project. USDA Lab: Because funds were not available until summer 1999, Dr. Hadidi was restricted in his research activities. However, he did contribute the basic technology for ELISA-PCR, which was developed in his laboratory.
Impacts
Several standard methods for testing viruses of potato and tomato were developed in different laboratories. During the coming year, these virus-indexing methods will be evaluated in the different laboratories in the Middle East.
Publications
- Maxwell, D.P., Nakhla, M.K., Hadidi, A. Mazyad, H.M., Shalaby, A., Czosnek, H., Akad, F., Zeidan, M., Iraki, N. Tawil, J., and Dar-Issa, O.M. 1999. Development of a regional viral indexing and certification program for plant propagation materials in the Middle East -Middle East Research and Cooperation (MERC) Program-.7th International Plant Virus Epidemiology Symposium 11-16 April, 1999. Almeria, Spain.
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