Genetic characterization of Moniliophthora perniciosa from Ecuador and in vitro sensitivity to compost tea


Moniliophthora perniciosa is the causal agent of the witches’ broom disease (WBD) in cacao but the morphological and molecular diversity of M. perniciosa in Ecuador—the top fine flavor cacao producer worldwide—are poorly understood and the pathogen’s sensitivity to compost teas is unknown. A total of 90 isolates of M. perniciosa were obtained from symptomatic samples of cocoa branches and pods collected from four cocoa-producing regions of Ecuador. Growth of each isolate was assed and fitted to a Gompertz model, and the molecular variability was evaluated by sequencing the ITS1, 5 .8s, and ITS2 rDNA regions as well as by inter-retrotransposon amplified polymorphism (IRAP), retrotransposon microsatellite amplified polymorphism (REMAP), and simple sequence repeats (SSR) analyses. Sensitivity of M. perniciosa to a compost tea was evaluated at concentrations of 0.5, 1, 1.5, 2, 2.75, and 3.5% in PDA. Results showed morphological and growth rate homogeneity across isolates. Mycelial growth fitted to Gompertz model gave similar parameter estimates for isolates from the different sampling sites. No molecular variability was observed based on ITS and SSR analysis but IRAP and REMAP tests showed high polymorphism. The total genetic diversity based on IRAP and REMAP tests was 0.28 and the genetic diversity within population was 0.24, with Nei’s diversity, Shannon’s Information, and percent polymorphism values of 0.315, 0.48, and 95.24, respectively for the whole sampled population; isolates from Los Rios province being the most polymorphic. Compost tea sensitivity analysis performed on 85 of the isolates showed a significant inhibitory effect at concentrations of 2.75% or above but concentrations below 1.5% significantly enhanced the growth of most isolates of the pathogen. Results confirmed the high genetic diversity of M. perniciosa and provided insights into growth rate and compost tea sensitivity of the pathogen in Ecuador.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. Addinsoft (2019). XLSTAT statistical and data analysis solution. Long Island, NY, USA. Accessed 11 March 2019.

  2. Aime, M. C., & Phillips-Mora, W. (2005). The causal agents of witches’ broom and frosty pod rot of cacao (chocolate, Theobroma cacao ) form a new lineage of Marasmiaceae. Mycologia, 97(5), 1012–1022.

  3. Aragundi, J. A. (1982). Culture and histology of Crinipellis perniciosa (Stahel) singer. Gainesville, FL: University of Florida

  4. Artero, A. S., Silva, J. Q., Albuquerque, P. S. B., Bressan, E. A., Leal, G. A., Sebbenn, A. M., et al. (2016). Spatial genetic structure and dispersal of the cacao pathogen Moniliophthora perniciosa in the Brazilian Amazon. Plant Pathology, 66, 1–12.

  5. Burdon, J. J. (1993). Genetic variation in pathogen populations and its implications for adaptation to host resistance. In: T. Jacob & J. E. Parlevliet (Eds.), Durability of disease resistance. Current Plant Science and Biotechnology in Agriculture (pp. 41–56). Dordrecht: Springer.

  6. Cenis, J. L. (1992). Rapid extraction of fungal DNA for PCR amplification. Nucleic Acids Research, 20(9), 2380.

  7. de Arruda, M. C., Ferreira, M. A., Miller, R. N., Resende, M. L., & Felipe, M. S. (2003). Nuclear and mitochondrial rDNA variability in Crinipellis perniciosa from different geographic origins and hosts. Mycological Research, 107, 25–37.

  8. de Queiroz, C. B., Santana, M. F., da Silva, G. F., Mizubuti, E. S. G., de Araújo, E. F., & de Queiroz, M. V. (2014). Use of the IRAP marker to study genetic variability in Pseudocercospora fijiensis populations. Current Microbiology, 68(3), 358–364.

  9. Evans, H. C. (1978). Witches’ broom disease of cocoa Crinipellis perniciosa) in Ecuador. Annals of Applied Biology, 89(2), 185–192.

  10. Evans, H. C. (1980). Pleomorphism in Crinipellis perniciosa, causal agent of witches’ broom disease of cocoa. Transactions of the British Mycological Society, 74(3), 515–523.

  11. Ferreira, L. F., Duarte, K. M., Gomes, L. H., Carvalho, R. S., Leal Junior, G. A., Aguiar, M. M., et al. (2012). Genetic diversity of polysporic isolates of Moniliophthora perniciosa (Tricholomataceae). Genetics and Molecular Research: GMR, 11(3), 2559–2568.

  12. Furtek, T. A. (1994). Random amplified polymorphic DNA (RAPD) analysis of Crinipellis perniciosa isolates from different hosts. Plant Pathology, 43(6), 1020–1027.

  13. Garcia-Vallve, S., Palau, J., & Romeu, A. (1999). Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis. Molecular Biology and Evolution, 16(9), 1125–1134.

  14. Garzon, C. D., & Flores, F. J. (2013). Hormesis: Biphasic dose-responses to fungicides in plant pathogens and their potential threat to agriculture. In: M. Nita (Ed.), Fungicides - Showcases of Integrated Plant Disease Management from Around the World. London: IntechOpen.

  15. Gramacho, K. P., Risterucci, A. M., Lanaud, C., Lima, L. S., & Lopes, U. V. (2007). Characterization of microsatellites in the fungal plant pathogen Crinipellis perniciosa. Molecular Ecology Notes, 7(1), 153–155.

  16. Griffith, G. W., Nicholson, J., Nenninger, A., Birch, R. N., & Hedger, J. N. (2003). Witches’ brooms and frosty pods: two major pathogens of cacao. New Zealand Journal of Botany, 41(March 2013), 423–435.

  17. Henríquez, P., & Li-Pun, H. (2013). Innovaciones de impacto: Lecciones de la agricultura familiar en América Latina y el Caribe. San Jose, Costa Rica: Instituto Interamericano de Cooperacion para la Agricultura.

  18. Hernández-Villegas, J. J. (2016). Incidencia De La Escoba De Bruja (Crinipellis perniciosa) Sobre El Rendimiento De Dos Agroecosistemas De Cacao Con Diferentes Condiciones De Manejo. Bioagro, 28(1), 59–64.

  19. Holliday, P. (1970). Crinipellis perniciosa. CMI Descriptions of Pahogenic Fungi and Bacteria, (223), 1–2.

  20. Jimenez, M. I. (2008). Effect of the nutritional status of Banana (Musa Spp.) on leaf disease infestation by Mycosphaerella Fijiensis Morelet in Ecuador. Ku Leuven, (1), 161.

  21. Jimenez, M., Van der Veken, L., Neirynck, H., Rodríguez, H., Ruiz, O., & Swennen, R. (2007). Organic banana production in Ecuador: its implications on black Sigatoka development and plant–soil nutritional status. Renewable Agriculture and Food Systems, 22(04), 297–306.

  22. Lana, T. G., Azevedo, J. L., Pomella, A. W., Monteiro, R. T., Silva, C. B., & Araújo, W. L. (2011). Endophytic and pathogenic isolates of the cacao fungal pathogen Moniliophthora perniciosa (Tricholomataceae) are indistinguishable based on genetic and physiological analysis. Genetics and Molecular Research: GMR, 10(1), 326–334.

  23. Lima, L. S., Gramacho, K. P., Gesteira, A. S., Lopes, U. V., Gaiotto, F. A., Zaidan, H. A., Pires, J. L., Cascardo, J. C. M., & Micheli, F. (2008). Characterization of microsatellites from cacao-Moniliophthora perniciosa interaction expressed sequence tags. Molecular Breeding, 22(2), 315–318.

  24. Magdama, F. (2010). Estudio del efecto de Bioles y cepas de Trichoderma sp. aisladas de zona cacaotera, como alternativa de control de Moniliophthora roreri, en condiciones in vitro. Guayaquil, Ecuador: ESPOL

  25. Mamede, R., Montini, D. C., Raimundo, J., Passos, D. S., & Ferreira, A. (2006). Digital monitoring of mycelium growth kinetics and vigor of Shiitake (Lentinula edodes (berk.) Pegler) on agar medium. Brazilian Journal of Microbiology, 37, 90–95.

  26. Maridueña-Zavala, M. G., Villavicencio-Vásquez, M. E., Cevallos-Cevallos, J. M., & Peralta, E. L. (2016). Molecular and morphological characterization of Moniliophthora roreri isolates from cacao in Ecuador. Canadian Journal of Plant Pathology, 38(4), 460–469.

  27. Medeiros, F. H. V., Pomella, A. W. V., de Souza, J. T., Niella, G. R., Valle, R., Bateman, R. P., et al. (2010). A novel, integrated method for management of witches’ broom disease in Cacao in Bahia, Brazil. Crop Protection, 29(7), 704–711.

  28. Meinhardt, L. W., Gilson Lacerda Costa, G., Thomazella, D. P., José Teixeira, P. P., Falsarella Carazzolle, M., Schuster, S. C., et al. (2014). Genome and secretome analysis of the hemibiotrophic fungal pathogen, Moniliophthora roreri, which causes frosty pod rot disease of cacao: mechanisms of the biotrophic and necrotrophic phases. BMC Genomics, 15, 1–25.

  29. Melo, B. L. B., de Souza, J. T., Santos, R. M. F., Rehner, S. a., Solis, K. H., Suarez, C., et al. (2014). Development of microsatellites for the cacao frosty pod rot pathogen,\n Moniliophthora roreri. Forest Pathology, 44(4), 320–324.

  30. Mengesha, W. K., Gill, W. M., Powell, S. M., Evans, K. J., & Barry, K. M. (2017). A study of selected factors affecting efficacy of compost tea against several fungal pathogens of potato. Journal of Applied Microbiology, 123, 732–747.

  31. Midi, H., Sarkar, S. K., & Rana, S. (2010). Collinearity diagnostics of binary logistic regression model. Journal of Interdisciplinary Mathematics, 13(3), 253–267.

  32. Milton, B., Alfonso, V., Andrea, M., José, C. C., Arroyo, S. C., Morales, D., et al. (2016). Comportamiento Agroproductivo De 31 Clones De Cacao Nacional (Theobroma Cacao L.) Con La Aplicación De Un Biocontrolador Para Moniliasis (Moniliophthora Roreri). Investigación y Saberes, V(1), 39–54.

  33. Naturland (2015). How to grow organic cocoa. An illustrated hanbook on organic principles of cocoa production. Resource document. Association for Organic Agriculture. Accessed 21 Sept 2018.

  34. Osorio-Solano, C., Orozco-Castaño, C. A., López-Gartner, G. A., & Rivera-Páez, F. A. (2012). Genetic variability of Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora, comb. nov. (Agaricales – Marasmiaceae) in varieties of cocoa (Theobroma cacao L.). Acta Agronómica, 61(2), 85–93.

  35. Páez, O., Valverde, R., Gómez, L., & Brenes, A. (2005). Diversidad genetica de aislamientos de Phytophthora infestans en plantaciones de papa en Costa Rica con el uslo de RAPDS 1. Agronomía Costarricense, 29(1), 41–55.

  36. Pane, C., Celano, G., Villecco, D., & Zaccardelli, M. (2012). Control of Botrytis cinerea, Alternaria alternata and Pyrenochaeta lycopersici on tomato with whey compost-tea applications. Crop Protection, 38, 80–86.

  37. Peever, T. L., Zeigler, R. S., Dorrance, A. E., & Correa-Victoria, F. J. (2000). Pathogen population genetics and breeding for disease resistance. APSnet Feature Articles

  38. Pereira, J. F., Almeida, A. P. M. M., Cota, J., Pamphile, J. A., da Silva, G. F., de Araújo, E. F., et al. (2013). Boto, a class II transposon in Moniliophthora perniciosa, is the first representative of the PIF/ Harbinger superfamily in a phytopathogenic fungus. Microbiology (United Kingdom), 159(1), 112–125.

  39. Pereira, J. F., Araújo, E. F., Brommonschenkel, S. H., Queiroz, C. B., Costa, G. G. L., Carazzolle, M. F., Pereira, G. A. G., & Queiroz, M. V. (2015). MpSaci is a widespread gypsy-Ty3 retrotransposon highly represented by non-autonomous copies in the Moniliophthora perniciosa genome. Current Genetics, 61, 185–202.

  40. Phillips-Mora, W., Aime, M. C., & Wilkinson, M. J. (2007). Biodiversity and biogeography of the cacao (Theobroma cacao) pathogen Moniliophthora roreri in tropical America. Plant Pathology, 56(6), 911–922.

  41. Ploetz, R. C., Schnell, R. J., Ying, Z., Zheng, Q., Olano, C. T., Johnson, E., & Motamayor, J. C. (2005). Molecular diversity in Crinipellis perniciosa with AFLPs. European Journal of Plant Pathology, 111, 317–326.

  42. Purdy, L., & Schmidt, R. (1996). STATUS OF CACAO WITCHES’BROOM: biology, epidemiology, and management. Annual Review of Phytopathology, 34, 573–594.

  43. Quingaísa, E. (2007). Estudio de caso: denominación de origen “cacao arriba”. Quito, Ecuador: FAO

  44. Rincones, J., MAZOTT, G. D., Griffith, G. W., POMEL, A., FIGUEIRA, A., Leal, G. A., Jr., et al. (2006). Genetic variability and chromosome-length polymorphisms of the witches’ broom pathogen Crinipellis perniciosa from various plant hosts in South America. Mycological Research, 110(7), 821–832.

  45. Santana, M. F., de Araújo, E. F., de Souza, J. T., Mizubuti, E. S. G., & de Queiroz, M. V. (2012). Development of molecular markers based on retrotransposons for the analysis of genetic variability in Moniliophthora perniciosa. European Journal of Plant Pathology, 134(3), 497–507.

  46. Scheuerell, S., & Mahaffee, W. (2002). Compost tea: Principles and prospects for plant disease control. Compost Science & Utilization, 10(4), 313–338.

  47. Senasica. (2016). Escoba de Bruja del Cacao Moniliophthora perniciosa. Ficha técnica N o 4. Resource document. SENASICA. Accessed 21 Sept 2018.

  48. Silva, D. V., Araújo, I. S., Branco, S. M. J., Aguilar-Vildoso, C. I., Lopes, U. V., Marelli, J. P., Motamayor, J. C., Royaert, S., Rebouças, R. C., & Corrêa, R. X. (2014). Analysis of resistance to witches’ broom disease (Moniliophthora perniciosa) in flower cushions of Theobroma cacao in a segregating population. Plant Pathology, 63, 1264–1271.

  49. Stukenbrock, E. H., & McDonald, B. A. (2008). The origins of plant pathogens in agro-ecosystems. Annual Review of Phytopathology, 46(1), 75–100.

  50. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28(10), 2731–2739.

  51. Tirado-Gallego, P. A., Lopera-álvarez, A., & Ríos-osorio, L. A. (2016). Estrategias de control de Moniliophthora roreri y Moniliophthora perniciosa en Theobroma cacao L.: revisión sistemática. Revista Corpoica - Ciencia y Tecnología Agropecuaria, 17(3), 417–430.

  52. Tjørve, K. M. C., & Tjørve, E. (2017). The use of Gompertz models in growth analyses, and new Gompertz-model approach: an addition to the unified-Richards family. PLoS One, 12(6), e0178691.

  53. Winsor, C. P. (1932). The Gompertz curve as a growth curve. Proceedings of the National Academy of Sciences, 18(1), 1–8.

  54. Yeh, F. C., & Boyle, T. J. B. (1997). Population genetic analysis of codominant and dominant markers and quantitative traits. Belgian Journal of Botany, 129, 157–163.

  55. Yoo, W., Mayberry, R., Bae, S., Singh, K., He, Q., & Lillard, J., Jr. (2014). A study of effects of multicollinearity in the multivariable analysis. International Journal of Applied Science and Technology, 4(5), 9–19.

Download references


This research was financially supported by VLIR NETWORK Ecuador grant to JMC.


This study was funded by VLIR NETWORK Ecuador grant to JMC.

Author information

Correspondence to Juan Manuel Cevallos-Cevallos.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Online Resource 1

(PDF 526 kb)

Online Resource 2

(PDF 525 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Maridueña-Zavala, M.G., Freire-Peñaherrera, A., Espinoza-Lozano, R.F. et al. Genetic characterization of Moniliophthora perniciosa from Ecuador and in vitro sensitivity to compost tea. Eur J Plant Pathol 154, 943–959 (2019) doi:10.1007/s10658-019-01714-1

Download citation


  • Witches’ broom disease
  • IRAP
  • Microsatellites