Journal of Crop Protection

Molecular characterization of aster yellows phytoplasma associated with citrus varieties, using Multiplex PCR

10.48311/jcp.2015.1184
Volume 4, Issue 1
March 2015
Pages 19-27
Journal of Crop Protection

Authors

C. Moslemkhani 1
R. Shahbazi 1
S. Baghaii Ravari 2
F. Khelgatibana 2

1 Seed and Plant Certification and Registration Institute. P. O. Box: 31535-1516, Karaj, Iran.

2 Department of Crop Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, P. O. Box: 91775-1163, Mashhad, Iran.

Abstract
A survey was carried out in citrus nurseries in Mazandaran and Kerman provinces as the major citrus growing regions of Iran. Different varieties of orange and tangerine with abnormal symptoms were examined for the occurrence of Candidatus Liberobacter asiaticus, Spiroplasma citri and phytoplasma as the most important vascular limited pathogens in citrus. By visual inspection and molecular examination of 360 samples for the presence of fastidious prokaryotes only an occasional occurrence of phytoplasma and Spiroplasma citri were detected, using phytoplasma universal primer pairs P1/P7 and fU5/rU3 (in single tube nested PCR assay) and Spiroplasma specific primers P89-r/f and P58-6f/4r. PCR products of phytoplasmas were digested with MseI, AluI, BamHI and TaqI restriction enzymes and the digestion results showed phytoplasma related to aster yellows phytoplasma group. No amplicon was obtained for Candidatus Liberobacter asiaticus. The findings suggest when pathogen’s titer is low; symptoms of vascular limited diseases are almost confused with disorder and deficiency signs. So for sanitary selection and certification program, use of sensitive detection methods is recommended. Multiplex PCR assay used in this study successfully detected Candidatus Liberibacter asiaticus, Spiroplasma citri and phytoplasma in one reaction and is suggested for health certification programs.

Keywords

vascular limited
Citrus
Phytoplasma
Multiplex PCR
Alhudaib, K., Arocha, Y., Wilson, M. and Jones, P. 2009. Molecular identification, potential vectors and alternative hosts of the phytoplasma associated with a lime decline disease in Saudi Arabia. Crop Protection, 28: 13-18.
Berges, R., Rott, M. and Seemüller, E. 2000. Range of phytoplasma concentrations in various plant hosts as determined by competitive polymerase chain reaction. Phytopathology, 90: 1145-1152.
Bertolini, E., Olmos, A., Lopez, M. M. and Cambra, M. 2003. Multiplex nested reverse transcription-polymerase chain reaction in a single tube for sensitive and simultaneous detection for RNA viruses and Pseudomonas savastanoi pv. savastanoi in Olive trees, Phytopathology, 93: 286-292.
Bove´, J. M., Danet, J. L., Bananej, K., Hassanzadeh, N., Taghizadeh, M., Salehi, M. and Garnier, M. 2000. Witches’ broom disease of lime in Iran. Proceedings of the Fourteenth Conference of IOCV. IOCV, Riverside (US), pp. 207-212.
Bové. J. M. 2006. Huanglongbing: A destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology, 88: 7-37.
Chen, J., Pu, X., Deng, X., Liu, S., Li, H. and Civerolo, E. 2009. A phytoplasma related to Candidatus Phytoplasma asteris detected in citrus showing Huanglongbing (yellow shoot disease) symptoms in Guangdong, P. R. China. Phytopathology, 99: 236-242.
da Graça, J. V. 1991. Citrus greening disease. Annual Review of Phytopathology, 29: 109-136.
Deeley, J., Stevens, W. A. and Fox, R. T. V. 1979. Use of Diens Stain to detect plant diseases induced by mycoplasma-like organisms. Phytopathology, 69: 1169-1171.
Deng, S. and Hiruki, C. 1991. Amplification of 16S rRNA genes from culturable and nonculturable mollicutes. Journal Microbiology Methods, 14: 53-61.
Duduk, B., Paltrinieri, S., Lee, I-M.and Bertaccini, A., 2013. Nested PCR and RFLP analysis based on the 16S rRNA gene. Methods in Molecular Biology, 938: 159-171.
Frison, E. A. and Taher, M. N. 1991. FAO/IBPGR Technical Guidelines for the safe movement of citrus germplasm. Food and Agriculture Organization of the United Nations, Rome /International Board for Plant Genetic Resources, Rome.
Heinrich, M., Botti, S., Caprara, L., Arthofer, W., Strommer, S., Hanzer, V., Katinger, H., Bertaccini, A. and Machalo, M. L. C. 2001. Improved detection methods for fruit tree phytoplasmas. Plant Molecular Biology Reporter, 19: 169-179.
Hocquellet, A., Toorawa, P., Bove, J. M. and Garnier, M. 1999. Detection and identification of the two Candidatus Liberibacter sp. Associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of beta operon. Molecular and Cellular Probes. 13: 373-379.
Hung, T. H., Wu, M. L. and Su, H. J. 1999. Development of a rapid method for the diagnosis of citrys greening disease using the polymerase chain reaction. Journal of Phytopathology, 147: 599-604.
Kartte, S. and Seemüller, E. 1991. Histopathology of apple proliferation in Malus taxa and hybrids of different susceptibility. Journal of Phytopathology, 131: 149-160.
Lee, I. M., Gundersen, R. D. E., Davis, R. E. and Bartozyk, I. M. 1998 Revised classification scheme of phytoplasmas based on RFLP analysis of 16S rRNA and ribosomal protein gene sequences. International Journal of Systematic Bacteriology, 48: 1153-1169.
Lorenz, K. H., Schneider, B., Ahrens, U., Seemuller, E. 1995. Detection of the apple proliferation and pear decline phytoplasmas by PCR amplification of ribosomal and nonribosomal DNA. Phytopathology, 85: 771-776
Mannan, S., Ibrahim, M., Nadeem Chohan, S. and Holford, P. 2010. Detection of phytoplasma in citrus orchards of Pakistan Journal of pathology, 2: 49-54.
Marcone, C. 2010. Movement of Phytoplasmas and the Development of Disease in the Plant In: Weintraub, P. and Jones, P. (Eds.), Phytoplasmas: genomes, plant hosts and vectors. Allingford, UK, CABI Publishing.
Mohkami, A., Satari, R., Lori, Z., Ehsani, A. and Nazemi, A. 2011. First report of citrus huanglongbing in the Orzooiyeh region in Kerman province. Iranian Journal of Plant Pathology, 47: 29 (In Persian with English summary).
Musetti, R. 2013. Phytoplasma. Methods in Molecular Biology, 938: 109-113.
Nejat, N., Salehi, M., Fayyazi, M. and Izadpanah, K. 2007. Survey of sweet orange cultivars for stubborn disease resistance in Iran. Bulletin of Insectology, 60: 305-306.
Nejat, N. Vadamalai, G. Sijam, K. and Dickinson, M. 2011. First report of Spiroplasma citri associated with periwinkle lethal yellows in Southeast Asia. Plant Disease, 95: 1312-1320.
OEPP/EPPO, 2006. Candidatus Phytoplasma aurantifoliae. OEPP/EPPO Bulletin. 36: 117-119.
Raju, B. C., Nyland, G., Backus, E. A. and McLean, D. L. 1981. Association of Spiroplasma with brittle root of horseradish. Phytopathology, 71: 1067-1072.
Salehi, M., Izadpanah, K., Siampour, M., Bagheri, A. and Faghihi, S. M. 2007. Transmission of Candidatus Phytoplasma aurantifolia to Bakraee (Citrus reticulata hybrid) by feral leafhopper Hishimonus phycitis in Iran. Plant Disease, 91: 466.
Seemuller, E., Garnier, M. and Schneider, B. 2002. Mycoplasmas of plants and insects. In: Razin, S. and Herrmann R. (Eds.), Molecular Biology and Pathology of Mycoplasmas. Academic/Plenum Kluwer, London, pp 91-116.
Silva-Stenico, M. E., Pacheco, F. T. H., Pereira-Filho, E. R., Rodrigues, J. L. M., Souza, A. N., Etchegaray, A. D., Gomes, J. E. and Tsai, S. M. 2009. Nutritional deficiency in citrus with symptoms of citrus variegated chlorosis disease. Brazilian Journal of Biology, 69 (3): 859-864.
Teixeira, D. C., Wulff, N. A., Martins, E. C., Kitajima, E. W, Ayres, A. J., Eveillard, S., Saillard, C. and Bove, J. M. 2008. A phytoplasma closely related to the pigeon pea witches’ broom phytoplasma (16SrIX) is associated with citrus huanglongbing symptoms in the state of São Paulo, Brazil. Phytopathology, 98: 977-984.
Weintraub, P. and Jones. P, eds, 2010. Phytoplasmas: genomes, plant hosts and vectors. Allingford, UK, CABI Publishing.
Yokomi, R. K., Mello, A. F. S., Saponari, M. and Fletcher, J. 2008. Polymerase chain reaction based detection of Spiroplasma citri associated with citrus stubborn disease. Plant Disease, 92: 253-260.

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