Advertisement

European Journal of Plant Pathology

, Volume 153, Issue 1, pp 253–269 | Cite as

Identification and pathogenicity assessment of Colletotrichum isolates causing bitter rot of apple fruit in Belgium

  • Amelie Grammen
  • M. Wenneker
  • J. Van Campenhout
  • K. T. K. Pham
  • W. Van Hemelrijck
  • D. Bylemans
  • A. Geeraerd
  • W. Keulemans
Article

Abstract

Worldwide Colletotrichum spp. have been identified as a problem in the apple production. This is the first study executed and confirming the presence of Colletotrichum spp. causing the postharvest disease bitter rot on apple fruits in Belgium. The identification, genetic diversity of Colletotrichum isolates (present in Belgian apple orchards) their morphological traits and pathogenicity on two apple cultivars (cvs. Pinova and Nicoter) with a different level of susceptibility were studied. Based on sequence analysis of six different gene regions beta-tubuline (TUB2), histone H3 (HIS3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 gene (CHS-1), actin (ACT) and the Internal Transcriber Spacer (ITS) gene region, six different Colletotrichum spp., belonging to either the C. acutatum or C. gloeosporioides complexes, were isolated from twenty-one apple cultivars in three Belgian orchards: C. fioriniae, probably C. kahawae, C. salicis, C. rhombiforme, C. acutatum and C. godetiae. Colletotrichum godetiae was found to be the most present and pathogenic species in Belgian orchards. The species C. rhombiforme was found and identified on apple fruit for the first time. Reliable morphological discrimination between species, based on features such as in vitro growth rate, colony colour and spore measurements, is not possible. As such, molecular identification appears to outperform morphological analysis and was in this study the most ideal tool for identifying unknown isolates of Colletotrichum species. Inoculation assays on two apple cultivars revealed a significant difference in pathogenicity among isolates and among Colletotrichum species. The pathogenicity tests also showed that isolates coming from another host species, e.g. strawberry, are also pathogenic on apple fruits. Cultivar Pinova appeared to be more susceptible to bitter rot than cv. Nicoter. Given the difficulties with managing Colletotrichum infections, additional knowledge on the pathogen and the plant-pathogen interaction is essential for effective disease control.

Keywords

Postharvest fungal disease Apple bitter rot Molecular multilocus phylogeny Inoculation assays 

Notes

Acknowledgements

The authors thank the Fund for Scientific Research (FWO) Flanders for providing funding for this research (grant number 1S44116N). Thanks to Dalphy Harteveld for her scientific insight concerning this paper.

Funding

This study was funded by FWO (research foundation Flanders) Grant number: 1S44116N.

Compliance with ethical statement

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

Not applicable to this study, did not work with humans or animals.

Informed consent

Not applicable to this study, did not work with humans.

Supplementary material

10658_2018_1539_MOESM1_ESM.png (99 kb)
Supplementary Fig. 1 Multigene maximum-likelihood tree was constructed with concatenated sequences of ACT, GAPDH and HIS3 of Colletotrichum isolates causing bitter rot on apple fruits. Colletotrichum lindemuthianum data are used as an outgroup. Information about the names representing the isolates in this figure can be found in Table 1. The numbers at the branching of the tree represent the bootstrap values. (PNG 98 kb)
10658_2018_1539_MOESM2_ESM.png (668 kb)
Supplementary Fig. 2 Single gene maximum-likelihood tree was constructed with concatenated sequences of ACT of Colletotrichum isolates causing bitter rot on apple fruits. Colletotrichum lindemuthianum data was used as an outgroup. Information about the names representing the isolates in this figure can be found in Table 1. The numbers at the branching of the tree represent the bootstrap values. (PNG 667 kb)
10658_2018_1539_MOESM3_ESM.png (728 kb)
Supplementary Fig. 3 Single gene region maximum-likelihood tree was constructed with concatenated sequences of ITS of Colletotrichum isolates causing bitter rot on apple fruits. Colletotrichum lindemuthianum data was used as an outgroup. Information about the names representing the isolates in this figure can be found in Table 1. The numbers at the branching of the tree represent the bootstrap values. (PNG 728 kb)
10658_2018_1539_MOESM4_ESM.png (707 kb)
Supplementary Fig. 4 Single gene maximum-likelihood tree was constructed with concatenated sequences of TUB2 of Colletotrichum isolates causing bitter rot on apple fruits. Colletotrichum lindemuthianum data was used as an outgroup. Information about the names representing the isolates in this figure can be found in Table 1. The numbers at the branching of the tree represent the bootstrap values. (PNG 707 kb)
10658_2018_1539_MOESM5_ESM.png (597 kb)
Supplementary Fig. 5 Single gene maximum-likelihood tree was constructed with concatenated sequences of CHS-1 of Colletotrichum isolates causing bitter rot on apple fruits. Colletotrichum lindemuthianum data was used as an outgroup. Information about the names representing the isolates in this figure can be found in Table 1. The numbers at the branching of the tree represent the bootstrap values. (PNG 596 kb)
10658_2018_1539_MOESM6_ESM.png (700 kb)
Supplementary Fig. 6 Single gene maximum-likelihood tree was constructed with concatenated sequences of GAPDH of Colletotrichum isolates causing bitter rot on apple fruits. Colletotrichum lindemuthianum data was used as an outgroup. Information about the names representing the isolates in this figure can be found in Table 1. The numbers at the branching of the tree represent the bootstrap values. (PNG 700 kb)
10658_2018_1539_MOESM7_ESM.png (604 kb)
Supplementary Fig. 7 Single gene maximum-likelihood tree was constructed with concatenated sequences of HIS3 of Colletotrichum isolates causing bitter rot on apple fruits. Colletotrichum lindemuthianum data was used as an outgroup. Information about the names representing the isolates in this figure can be found in Table 1. The numbers at the branching of the tree represent the bootstrap values. (PNG 603 kb)
10658_2018_1539_MOESM8_ESM.docx (13 kb)
Supplementary table 1 (DOCX 12 kb)
10658_2018_1539_MOESM9_ESM.docx (19 kb)
Supplementary table 2 (DOCX 19 kb)

References

  1. Adaskaveg, J., & Hartin, R. (1997). Characterization of Colletotrichum acutatum isolates causing anthracnose of almond and peach in California. Phytopathology, 87, 979–987.CrossRefGoogle Scholar
  2. Agrios, G. N. (2005a). Anthracnose diseases caused by ascomycetes and Deureromycetes. In G. N. Agrios (Ed.), Plant pathology (pp. 483–501). Burlington: Elsevier Academic Press.Google Scholar
  3. Agrios, G. N. (2005b). Introduction. In G. N. Agrios (Ed.), Plant pathology (pp. 3–75). Burlington: Elsevier Academic Press.CrossRefGoogle Scholar
  4. Alaniz, S., Hernandez, L., & Mondino, P. (2015). Colletotrichum fructicola is the dominant and one of the most aggressive species causing bitter rot of apple in Uruguay. Tropical Plant Pathology, 40, 265–274.CrossRefGoogle Scholar
  5. Angay, O., Fleischmann, F., Recht, S., Herrmann, S., Matyssek, R., Oßwald, W., Buscot, F., & Grams, T. E. E. (2014). Sweets for the foe – Effects of non-structural carbohydrates on the susceptibility of Quercus robur against Phytophthora quercina. New Phytologist, 203, 1282–1290.CrossRefGoogle Scholar
  6. Bakkeren, G., Kronstad, J. W., & Lévesque, C. A. (2000). Comparison of AFLP fingerprints and ITS sequences as phylogenetic markers in Ustilaginomycetes. Mycologia, 92, 510–521.CrossRefGoogle Scholar
  7. Baroncelli, R., Sreenivasaprasad, S., Thon, M. R., & Sukno, S. A. (2014). First report of apple bitter rot caused by Colletotrichum godetiae in the United Kingdom. Plant Disease, 98, 1000.CrossRefGoogle Scholar
  8. Baroncelli, R., Zapparata, A., Sarrocco, S., Sukno, S. A., Lane, C.R., Thon, M.R., Vannacci, G., Holub, E., Sreenivasaprasad, S. (2015). Molecular Diversity of anthracnose pathogen populations associated with UK strawberry production suggests multiple introductions of three different Colletotrichum species. PLoS One Available on: doi: https://doi.org/10.1371/journal.pone.0129140, [13/08/2015].
  9. Bernstein, B., Zehr, E. I., & Dean, R. A. (1995). Characteristics of Colletotrichum from peach, apple, pecan and other hosts. Plant Disease, 79, 478–482.CrossRefGoogle Scholar
  10. Biggs, A. R., & Miller, S. S. (2001). RelativesSusceptibility of selected apple cultivars to Colletotrichum acutatum. Plant Disease, 85, 657–660.CrossRefGoogle Scholar
  11. Boratyn, G. M., Camacho, C., Cooper, P. S., Coulouris, G., Fong, A., Ma, N., Maden, T. L., Matten, W. T., McGinnis, S. D., Merezhuk, Y., Raytselis, Y., Sayers, E. W., Tao, T., Ye, J., & Zaretskaya, I. (2013). BLAST: A more efficient report with usability improvements. Nucleic Acids Research, 41, 29–33.CrossRefGoogle Scholar
  12. Børve, J., & Stensvand, A. (2015). Colletotrichum acutatum on apple in Norway. IOBC-WPRS Bulletin, 110, 151–157.Google Scholar
  13. Børve, J., & Stensvand, A. (2017). Colletotrichum acutatum occurs asymptomatically on apple leaves. European Journal of Plant Pathology, 147, 943–948.CrossRefGoogle Scholar
  14. Bragança, C. A. D., Damn, U., Baroncelli, R., Massola Jr., N. S., & Crous, P. W. (2016). Species of the Colletotrichum acutatum complex associated with anthracnose diseases of fruit in Brazil. Fungal Biology, 120, 547–561.CrossRefGoogle Scholar
  15. Cai, L., Hyde, K. D., Taylor, P. W. J., Weir, B. S., Waller, J. M., Abang, M. M., Zhang, J. Z., Yang, Y. L., Phoulivong, S., Liu, Z. Y., Prihastuti, H., Shivas, R. G., McKenzie, E. H. C., & Johnston, P. R. (2009). A polyphasic approach for studying Colletotricum. Fungal Diversity, 39, 183–204.Google Scholar
  16. Cannon, P. F., Damm, U., Johnston, P. R., & Weir, B. S. (2012). Colletotrichum – Current status and future directions. Studies in Mycology, 73, 181–213.CrossRefGoogle Scholar
  17. Carbone, I., & Kohn, L. M. (1999). A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia, 91, 553–556.CrossRefGoogle Scholar
  18. Corda, A. C. I. (1831). Die Pilze Deutschlands In: Sturm J (ed.) Deutschlands Flora in Abbildungen nach der Natur mit Beschreibungen, 3. Abtheilung, 12, 1–144.Google Scholar
  19. Crous, P. W., Groenewald, J. Z., Risede, J. M., & Hywel-Jones, N. L. (2004). Calonectria species and their Cylindrocladium anamorphs: Species with sphaeropedunculate vesicles. Studies in Mycology, 50, 415–430.Google Scholar
  20. Crous, P. W., Hawksworth, D. L., & Wingfield, M. J. (2015). Identifying and naming plant-pathogenix fungi: Past, present and future. Annual Review of Phytopathology, 53, 247–267.CrossRefGoogle Scholar
  21. Crusius, L. U., Forcelini, C. A., Sanhueza, R. M. V., & Fernandes, J. M. C. (2002). Epidemiology of apple leaf spot. Fitopatologia Brasileira, 27, 65–70.CrossRefGoogle Scholar
  22. Damm, U., Cannon, P. F., Woudenberg, J. H., & Crous, P. W. (2012). The Colletotrichum acutatum species complex. Studies in Mycology, 73, 37–113.CrossRefGoogle Scholar
  23. Dean, R., Van Kan, J. A. L., Pretorius, Z. A., Hammond-Kosack, K. E., & Di Pietro, A. (2012). The top 10 fungal pathogens in molecular plant pathology. Molecular Plant Pathology, 13, 414–430.CrossRefGoogle Scholar
  24. DeLong, J. M., Prange, R. K., & Harrison, P. A. (1999). Using the Streif index as a final harvest window for controlled-atmosphere storage of apples. Horticultural Science, 13, 1251–1255.Google Scholar
  25. Du, M., Schardl, C. L., Nuckles, E. M., & Vaillancourt, L. J. (2005). Using mating-type gene sequences for improved phylogenetic resolution of Colletotrichum species complexes. Mycologia, 97, 641–658.CrossRefGoogle Scholar
  26. Everett, K. R. (2014). Anthracnose and stem-end rots of tropical and subtropical fruit- new names for old foes. In D. Prusky & M. L. Gullino (Eds.), Post-harvest pathology. Plant pathology in the twenty-first century. Contributions to the 10th international congress, ICPP 2013 (Vol. 7, pp. 55–70). Switzerland: Springer International Publishing.Google Scholar
  27. Freeman, S., Katan, T., & Shabi, E. (1998). Characterization of Colletotrichum species responsible for anthracnose diseases of various fruits. Plant Disease, 82, 596–605.CrossRefGoogle Scholar
  28. Gariepy, T. D., Lévesque, C. A., de Jong, S. N., & Rahe, J. E. (2003). Species specific identification of the Neofabraea pathogen complex associated with pome fruits using PCR and multiplex DNA amplification. Mycological Research, 107, 528–536.CrossRefGoogle Scholar
  29. Glass, N. L., & Donaldson, G. C. (1995). Development of primer sets designed for the use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology, 61, 1323–1330.Google Scholar
  30. Gonzales, E., Sutton, T. B., & Correll, J. C. (2006). Clarification of the etiology of Glomerella leaf spot and bitter rot of apple caused by Colletotrichum spp. based on morphology and genetic, molecular, and pathogenicity tests. Phytopathology, 96, 982–992.CrossRefGoogle Scholar
  31. Guerber, J. C., Liu, B., Correll, J. C., & Johnston, P. R. (2003). Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs and mating compatibility. Mycologia, 95, 872–895.CrossRefGoogle Scholar
  32. Ismail, A. M., Cirvilleri, G., Yaseen, T., Epifani, F., Perrone, G., & Polizzi, G. (2015). Characterisation of Colletotrichum species causing anthracnose disease of mango in Italy. Journal of Plant Pathology, 97, 167–171.Google Scholar
  33. Ivic, D., Voncina, D., Sever, Z., Simon, S., & Pejic, I. (2013). Identification of Colletotrichum species causing bitter rot of apple and pear in Croatia. Journal of Phytopathology, 161, 284–286.CrossRefGoogle Scholar
  34. Kou, L., Gaskins, V. L., Luo, Y., & Jurick II, W. M. (2013). First report of Colletotrichum fioriniae causing postharvest decay on ‘Nittany’ apple fruit in the United States. Plant Disease, 98, 993.CrossRefGoogle Scholar
  35. Lattanzio, V., Venere, D. D., Linsalata, V., Bertolini, P., Ippolito, A., & Mario Salerno, M. (2001). Low temperature metabolism of apple phenolics and quiescence of Phlyctaena vagabunda. Journal of Agricultural and Food Chemistry, 49, 5817–5821.CrossRefGoogle Scholar
  36. Leyronas, C., Duffaud, M., & Nicot, P. C. (2012). Compared efficiency of the isolation methods for Botrytis cinerea. Mycology, 3, 221–225.Google Scholar
  37. Liu, F., Damm, U., Cai, L., & Crous, P. W. (2013). Species of the Colletotrichum gloeosporioides complex associated with anthracnose diseases of Proteacea. Fungal Diversity, 61, 89–105.CrossRefGoogle Scholar
  38. Liu, F., Weir, B. S., Damm, U., Crous, P. W., Wang, Y., Liu, B., Wang, M., Zhang, M., & Cai, L. (2015). Unravelling Colletotrichum species associated with Camellia: Employing ApMat and GS loc ito resolve species in the C. gloeosporioides complex. Persoonia, 35, 63–68.CrossRefGoogle Scholar
  39. Martin-Felix, Y., Groenewald, J. Z., Cai, L., Chen, Q., Marincowitz, S., Barnes, I., Benschn, K., Braun, U., Camporesi, E., Damm, U., de Beer, Z. W., Dissanayake, A., Edwards, J., Giraldo, A., Hernandez-Restrepo, M., Hyde, K. D., Jayawardena, R. S., Lombard, L., & Crous, P. W. (2017). Genera of phytopathogenic fungi: GOPHY 1. Studies in Mycology, 86, 99–216.CrossRefGoogle Scholar
  40. Munda, A. (2014). First report of Colletotrichum fioriniae and C. godetiae causing apple rot in Slovenia. Plant Disease, 98, 1282.CrossRefGoogle Scholar
  41. Munir, M., Amsden, B., Dixon, E., Vaillancourt, L., & Ward Gauthier, N. A. (2016). Characterization of Colletotrichum species causing bitter rot of apple in Kentucky orchards. Plant Disease, 100(11), 2194–2203.CrossRefGoogle Scholar
  42. Nodet, P., Baroncelli, R., Faugère, D., & Le Floch, G. (2016). First report of apple bitter rot caused by Colletotrichum fioriniae in Brittany, France. Plant Disease, 100, 1497.CrossRefGoogle Scholar
  43. Nour, V., Trandafir, I., & Ionica, M. A. (2010). Compositional characteristics of fruits of several apple (Malus domestica Borkh.) cultivars. Notulae Botanicae Horti Agrobotanici Cluj-Napoca., 38, 228–233.Google Scholar
  44. O’Donnell, K., & Cigelnik, E. (1997). Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetics and Evolution, 7, 103–116.CrossRefGoogle Scholar
  45. Peres, N., Souza, N., Zitko, S., & Timmer, L. (2002). Activity of benomyl for control of postbloom fruit drop of citrus caused by Colletotrichum acutatum. Plant Disease, 86, 620–624.CrossRefGoogle Scholar
  46. Peres, N., Souza, N., Peever, T., & Timmer, L. (2004). Benomyl sensitivity of isolates of Colletotrichum acutatum and C. gloeosporioides from citrus. Plant Disease, 88, 125–130.CrossRefGoogle Scholar
  47. Phoulivong, S., Cai, L., Chen, H., McKenzie, E. H. C., Abdelsalam, K., Chukeatirote, E., & Hyde, K. D. (2010). Colletotrichum gloeosporioides is not a common pathogen on tropical fruits. Fungal Diversity, 44, 33–43.CrossRefGoogle Scholar
  48. Phoulivong, S., McKenzie, E. H. C., & Hyde, K. D. (2012). Cross infection of Colletotrichum species; a case study with tropical fruits. Current Research in Environmental and Applied Mycology, 2, 99–111.CrossRefGoogle Scholar
  49. Prihastuti, H., Cai, L., Chen, H., McKenzie, E. H. C., & Hyde, K. D. (2009). Characterization of Colletotrichum species associated with coffee berries in northern Thailand. Fungal Diversity, 39, 89–109.Google Scholar
  50. Prusky, D., Alkan, N., Mengiste, T., & Fluhr, R. (2013). Quiescent and necrotrophic lifestyle choice during postharvest disease development. Annual Review of Phytopathology, 51, 55–76.CrossRefGoogle Scholar
  51. Raja, H. A., Miller, A. N., Pearce, C. J., & Oberlies, N. H. (2017). Fungal identification using molecular tools: A primer for the natural products research community. Journal of Natural Products, 80, 756–770.CrossRefGoogle Scholar
  52. Runion, G. B. (2003). Climate change and plant pathosystems-future disease prevention starts here. New Phytologist, 159, 531–538.CrossRefGoogle Scholar
  53. SAS Institute Inc. (2013). Using JMP 11. Cary, NC: SAS Institute Inc.Google Scholar
  54. Sutton, B. C. (1980). The coelomycetes. Fungi imperfecti with pycnidia, acervuli and stromata. CABI, Kew.Google Scholar
  55. Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729.CrossRefGoogle Scholar
  56. Valero, M., Garcıa-Martınez, S., Giner, M., Alonso, A., & Ruiz, J. (2010). Benomyl sensitivity assays and species-specific PCR reactions highlight association of two Colletotrichum gloeosporioides types and C. acutatum with rumple disease on Primofiori lemons. European Journal of Plant Pathology, 127, 399–405.CrossRefGoogle Scholar
  57. Velho, A., Stadnik, M., Casanova, L., Mondino, P., & Alaniz, S. (2014). First report of Colletotrichum nymphaeae causing apple bitter rot in southern Brazil. Plant Disease, 98, 567.CrossRefGoogle Scholar
  58. Velho, A. C., Alaniz, S., Casanova, L., Mondino, P., & Stadnik, M. J. (2015). New insights into the characterization of Colletotrichum species associated with apple diseases in southern Brazil and Uruguay. Fungal Biology, 119, 229–244.CrossRefGoogle Scholar
  59. von Arx, J. A. (1957). Die Arten der Gattung Colletotrichum Corde. Phytopathologische Zeitschrift, 29, 413–468.Google Scholar
  60. Weber, R. W. S., & Palm, G. (2010). Resistance against storage rot fungi Neofabraea perennans, N. alba, Glomerella acutata and Neonectria galligena against thiophanate-methyl in northern German apple production. Journal of Plant Diseases and Protection, 117, 185–191.CrossRefGoogle Scholar
  61. Weir, B. S., Johnston, P. R., & Damm, U. (2012). The Colletotrichum gloeosporioides species complex. Studies in Mycology, 73, 115–180.CrossRefGoogle Scholar
  62. Wenneker, M., Pham, K., Lemmers, M., de Boer, A., van der Lans, A., van Leeuwen, P., & Hollinger, T. (2016). First reports of Colletotrichum godetiae causing bitter rot on ‘golden delicious’ apples in the Netherlands. Plant Disease, 100, 218.CrossRefGoogle Scholar
  63. Yokosawa, S., Eguchi, N., Kondo, K., & Sato, T. (2017). Phylogenetic relationship and fungicide sensitivity of members of the Colletotrichum gloeosporioides species complex from apple. Journal of General Plant Pathology, 83, 291–298.  https://doi.org/10.1007/s10327-017-0732-9.CrossRefGoogle Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2018

Authors and Affiliations

  • Amelie Grammen
    • 1
  • M. Wenneker
    • 2
  • J. Van Campenhout
    • 3
  • K. T. K. Pham
    • 2
  • W. Van Hemelrijck
    • 3
  • D. Bylemans
    • 1
    • 3
  • A. Geeraerd
    • 4
  • W. Keulemans
    • 1
  1. 1.Laboratory for Fruit Breeding and Biotechnology, Department of BiosystemsKU LeuvenLeuvenBelgium
  2. 2.Wageningen University & ResearchWageningenThe Netherlands
  3. 3.Research Station of Fruit CultivationSint-TruidenBelgium
  4. 4.Division MeBioS, Department of BiosystemsKU LeuvenLeuvenBelgium

Personalised recommendations