Comparación del método de reacción almidón – iodo con la Amplificación por Polimerasa y Recombinasa para el diagnóstico del Huanglongbing (HLB) de los cítricos

Luis Roberto González Segnana; Darío Rafael Acosta Aveiro; Cesar Darío Pino Quintana; Diego Dionisio González Espínola

doi:10.18004/investig.agrar.2020.diciembre.2202586

Authors

Luis Roberto González Segnana Universidad Nacional de Asunción, Facultad de Ciencias Agrarias. San Lorenzo, Paraguay. https://orcid.org/0000-0003-2545-1820
Darío Rafael Acosta Aveiro Universidad Nacional de Asunción, Facultad de Ciencias Agrarias. San Lorenzo, Paraguay.
Cesar Darío Pino Quintana Universidad Nacional de Asunción, Facultad de Ciencias Agrarias. San Lorenzo, Paraguay.
Diego Dionisio González Espínola Universidad Nacional de Asunción, Facultad de Ciencias Agrarias. San Lorenzo, Paraguay. https://orcid.org/0000-0002-3639-4190

DOI:

https://doi.org/10.18004/investig.agrar.2020.diciembre.2202586%20

Keywords:

HLB, citrus, detection

Abstract

Huanglongbing (HLB) is one of the most important citrus diseases worldwide. A field level diagnosis is difficult when it is based on visual symptoms, since they can be confused with similar symptoms of other diseases or nutritional deficiencies of the plant. Molecular methods are used for the diagnosis of the disease, which are laborious, time-consuming and costly, in addition to not allowing rapid detection in the field. In relation to this problem, the method of starch-iodine reaction (RAI) was proposed as a cheap alternative for the diagnosis of HLB by various authors, however, the method has not been widely used, possibly due to lack of diffusion. In the present work the efficacy of the RAI diagnostic method was demonstrated, by comparison with the molecular method of Amplification by recombinase and polymerase (ARP); a molecular method that allows the detection of HLB in the field, but with higher cost. A total of 825 leaf samples were taken from 33 sweet orange (Citrus sinensis L.) plants from orchards of the Department of Caazapá, Paraguay. The samples were analyzed by RAI method, staining and intensity of coloration were observed by optical microscopy. Among these, 33 samples were selected with positive and negative reactions to be analyzed by the ARP method. Results revealed a correlation of 93.6% between both methods. The RAI method, in addition to differentiating HLB symptoms from other diseases and nutritional deficiencies in the plant, is a simple, fast, effective, and economical method for diagnosis if the disease.

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References

Aidoo, O. F., Tanga, C. M., Mohamed, S. A., Rasowo, B. A., Khamis, F. M., Rwomushana, I., … Borgemeister, C. (2019). Distribution, degree of damage and risk of spread of Trioza erytreae (Hemiptera: Triozidae) in Kenya. Journal of Applied Entomology, 143(8), 822-833.

Albrecht, U. & Bowman, K. D. (2012). Transcriptional response of susceptible and tolerant citrus to infection with Candidatus Liberibacter asiaticus. Plant Science 185-186, 118-130. 10.1016/j.plantsci.2011.09.008

Bassanezi, R. B., Montesino, L. H., Gasparoto, M. C. G., Bergamin Filho, A. & Amorim, L. (2011). Yield loss caused by huanglongbing in different sweet orange cultivars in São Paulo, Brazil. European Journal of Plant Pathology 130 (4), 577-586. https://doi.org/10.1007/s10658-011-9779-1

Bové, J. M. (2006). Huanglongbing: A Destructive, Newly-Emerging, Century-Old Disease of Citrus. Journal of Plant Pathology 88 (1), 7-37. Recuperado de: http://jstor.org/stable/41998278

Burckhardt, D. & Ouvrard, D. (2012). A revised classification of the jumping plant-lice (Hemiptera: Psylloidea). Zootaxa 3509 (1) : 1-34.

Ding, F., Duan, Y., Yuan, Q., Shao, J. & Hartung, J. S. (2016). Serological detection of ‘Candidatus Liberibacter asiaticus’ in citrus, and identification by GeLC-MS/MS of a chaperone protein responding to cellular pathogens. Scientific Reports 6 (1), 1-11.

Duan, Y. P., Gottwald, T., Zhou, L. J. & Gabriel, D. W. (2008). First report of dodder transmission of ‘Candidatus Liberibacter asiaticus’ to tomato (Lycopersicon esculentum). Plant Disease 92 (5), 831.

Ghosh, D. K., Bhose, S., Warghane, A., Motghare, M., Sharma, A. K., Dhar, A. K. & Gowda, S. (2016). Loop-mediated isothermal amplification (LAMP) based method for rapid and sensitive detection of ‘Candidatus Liberibacter asiaticus’ in citrus and the psyllid vector, Diaphorina citri Kuwayama. Journal of Plant Biochemistry and Biotechnology 25 (2), 219-223.

González, L. R. & Tullo, C. C. (2019). Cultivo de cítricos. Guía técnica. Facultad de Ciencias Agrarias. UNA. 80 p.

González, P., Etxeberria, E., Achor, D., Dawson, W., Spann, T., Yates, J. D. & Albrigo, G. (2007). Uso de la reacción almidón - yodo para la selección de hojas sospechosas con HLB: Distribución anatómica de niveles anormalmente altos de almidón en arboles de Naranja Valencia positivos al HLB. Horticultural Sciences Department, University of Florida. Consultado 5 mayo 2016. Disponible en: Disponible en: http://calcitrusquality.org/wp-content/uploads/2009/05/Pedro-Gonzalez-Uso-de-la-Reaccion-Ioco-Almidon-Articulo-Completo.pdf

Gopal, K., Gopi, V., Palanivel, S. & Sreenivasulu, Y. (2007). Molecular detection of greening disease in citrus by PCR: tissue source and time of detection. Annals of Plant Protection Sciences 15 (2), 384-390.

Gottwald, T. R., da Graça, J. V da & Bassanezi, R. B. (2007). Citrus huanglongbing: the pathogen and its impact. Plant Health Progress 6 (1), 1-18.

Hall, D. G. & Hentz, M. G. (2010). Sticky Trap and Stem-Tap Sampling Protocols for the Asian Citrus Psyllid (Hemiptera: Psyllidae). Journal of Economic Entomology, 103(2), 541-549. https://doi.org/10.1603/EC09360.

Hall, D. G. (2008). Biological control of Diaphorina citri. In Proceedings of the International Workshop on Huanglongbing of citrus, 1-7.

Jagoueix, S., Bove, J. M. & Garnier, M. (1994). The phloem-limited bacterium of greening disease of citrus is a member of the α subdivision of the Proteobacteria. International Journal of Systematic Bacteriology. https://doi.org/10.1099/00207713-44-3-379.

Kim, J. & Easley, C. J. (2011). Isothermal DNA amplification in bioanalysis: strategies and applications. Bioanalysis 3 (2), 227-239. https://doi.org/10.4155/bio.10.172.

Li, W., Levy, L. & Hartung, J. S. (2009). Quantitative distribution of ‘Candidatus Liberibacter asiaticus’ in citrus plants with citrus huanglongbing. Phytopathology 99 (2), 139-144.

Manjunath, K. L., Halbert, S. E., Ramadugu, C., Webb, S. & Lee, R. F. (2008). Detection of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and Its Importance in the Management of Citrus Huanglongbing in Florida. Phytopathology 98(4), 387-396. https://doi.org/10.1094/PHYTO-98-4-0387.

Onuki, M., Truc, N. T. N., Nesumi, H., Hong, L. T. T. & Kobayashi, H. (2002). Useful histological method for distinguishing citrus yellowing leaves infected with huanglongbing from those caused by other factors. In: Proceedings of the 2002 annual workshop of JIRCAS Mekong Delta Project, 114-118.

Pandey, S. S. & Wang, N. (2019). Targeted Early Detection of Citrus Huanglongbing Causal Agent ‘Candidatus Liberibacter asiaticus’ before Symptom Expression. Phytopathology 109 (6), 952-959. https://doi.org/10.1094/PHYTO-11-18-0432-R.

Piepenburg, O., Williams, C. H., Stemple, D. L. & Armes, N. A. (2006). DNA detection using recombination proteins. PLoS Biology 4(7), 1115-1121. https://doi.org/10.1371/journal.pbio.0040204.

Russell, P. F., McOwen, N., Bohannon, S., Amato, M. A. & Bohannon, R. (2015). Rapid On-Site Detection of the Huanglongbing/Citrus Greening Causal Agent ‘Candidatus Liberibacter asiaticus’ by AmplifyRP®, a Novel Rapid Isothermal Nucleic Acid Amplification Platform. Acta Horticulturae 1065, 905-912. [

Sagaram, U. S., DeAngelis, K. M., Trivedi, P., Andersen, G. L., Lu, S. E. & Wang, N. (2009). Bacterial Diversity Analysis of Huanglongbing Pathogen-Infected Citrus, Using PhyloChip Arrays and 16S rRNA Gene Clone Library Sequencing. Applied and Environmental Microbiology 75 (6), 1566-1574. https://doi.org/10.1128/AEM.02404-08.

Schaffer, A. A., Liu, K.-C., Goldschmidt, E. E., Boyer, C. D. & Goren, R. (1986). Citrus Leaf Chlorosis Induced by Sink Removal: Starch, Nitrogen, and Chloroplast Ultrastructure. Journal of Plant Physiology 124 (1), 111-121. https://doi.org/10.1016/S0176-1617(86)80183-3.

SENAVE (Servicio Nacional de Calidad y Sanidad Vegetal y de Semillas, Paraguay). (2017). HLB EN PARAGUAY. Experiencias en le Gestión Nacional. Consultado 7 mar. 2019. Disponible en: Disponible en: http://web.senave.gov.py:8081/docs/libros/Libro%20HLB%202017.pdf

Smith, P. F. (1975). Zinc accumulation in the wood of citrus trees affected with blight. Proceedings of the Florida State Horticultural Society 1974, 87, 91-95.

Teixeira, D. C., Wulff, N. A., Martins, E. C., Kitajima, E. W., Bassanezi, R., Ayres, A. J., … Bové, J. M. (2008). A Phytoplasma Closely Related to the Pigeon Pea Witches’-Broom Phytoplasma (16Sr IX) Is Associated with Citrus Huanglongbing Symptoms in the State of São Paulo, Brazil. Phytopathology, 98 (9), 977-984. https://doi.org/10.1094/PHYTO-98-9-0977.

Texeira, D. C., Ayres, J., Kitajima, E. W., Danet, L., Jagoueix-Eveillard, S., Saillard, C. & Bové, J. M. (2005). First Report of a Huanglongbing-Like Disease of Citrus in Sao Paulo State, Brazil and Association of a New Liberibacter Species, “Candidatus Liberibacter americanus”, with the Disease. Plant Disease 89(1), 107. https://doi.org/10.1094/PD-89-0107A.

Wulff, N. A., Fassini, C. G., Marques, V. V., Martins, E. C., Coletti, D. A. B., Teixeira, D. do C., … Bové, J. M. (2019). Molecular characterization and detection of 16SrIII group phytoplasma associated with huanglongbing symptoms. Phytopathology 109 (3), 366-374. https://doi.org/10.1094/PHYTO-03-18-0081-R.

Yelenosky, G. & Guy, C. L. (1977). Carbohydrate accumulation in leaves and stems of’Valencia’orange at progressively colder temperatures. Botanical Gazette, 138 (1), 13-17.

Zaghloul, H. & El-shahat, M. (2014). Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis. World Journal of Hepatology, 6(12), 916.