Skip to main content
SpringerLink
Log in

Analysis of protease activity in Aspergillus flavus and A. parasiticus on peanut seed infection and aflatoxin contamination

  • Published:
European Journal of Plant Pathology Aims and scope Submit manuscript

Abstract

Aspergillus flavus and A. parasiticus are aflatoxin-producing fungi that can infect peanut seeds in field crops. An association between A. parasiticus proteolytic enzyme activities and peanut fungal infection was examined. For this study, a model of inductive and non-inductive culture media to produce A. parasiticus extracellular protease before infection was used. These A. parasiticus cultures were used to infect peanut seeds of cultivars resistant and susceptible to aflatoxin contamination. Peanut seeds of both cultivars exposed to fungi grown on casein medium (inductive medium) showed higher internal and external infection and a higher fungal protease content than those observed on potato dextrose agar (PDA) and sucrose medium (non-inductive media). A further study showed higher fungal colonisation and aflatoxin contamination in seeds of the resistant cultivar pre-incubated with Aspergillus extracellular proteases than in those incubated without proteases. Moreover, protease activities affected the viability of non-infected resistant cultivar seeds, inhibiting germination and radicle elongation and enhancing seed tissue injury. The results strongly suggest that protease production by A. parasiticus is involved in peanut seed infection and aflatoxin contamination resulting in seed tissue damage, affecting seed viability and facilitating the access of fungi through the testa. The analysis of fungal extracellular proteases formed on peanut seed during infection showed that A. flavus and A. parasiticus produced metallo and serine proteases; however, there were differences in the molecular masses of the enzymes between both species. The greatest activity in both species was by serine protease, that could be classified as subtilase.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Abnet, C. C. (2007). Carcinogenic food contaminants. Cancer Investigation, 25, 189–196. doi:10.1080/07357900701208733.

    Article  PubMed  CAS  Google Scholar 

  • Asis, R., Barrionuevo, D. L., Giorda, L. M., Nores, M. L., & Aldao, M. A. (2005). Aflatoxin Production in six peanut (Arachis hypogaea L.) genotypes infected with Aspergillus flavus and A. parasiticus, isolated from peanut production areas of Cordoba, Argentina. Journal of Agricultural and Food Chemistry, 53, 9274−9280. doi:10.1021/jf051259+.

    Article  PubMed  CAS  Google Scholar 

  • Bindschedler, L. V., Sanchez, P., Dunn, S., Mikan, J., Thangavelu, M., Clarkson, J. M., et al. (2003). Deletion of the SNP1 trypsin protease from Stagonospora nodorum reveals another major protease expressed during infection. Fungal Genetics and Biology, 38, 43−53. doi:10.1016/S1087-1845(02)00517-0.

    Article  PubMed  Google Scholar 

  • Brown, R. L., Chen, Z. Y., Cleveland, T. E., Cotty, P. J., & Cary, J. W. (2001). Variation in vitro α-amylase and protease activity is related to the virulence of Aspergillus flavus isolates. Journal of Food Protection, 64, 401–404.

    PubMed  CAS  Google Scholar 

  • Brown, R. L., Cleveland, T. E., Cotty, P. J., & Mellon, J. E. (1992). Spread of Aspergillus flavus in cotton bolls, decay of intercarpellary membranes, and production of fungal pectinases. Phytopathology, 82, 462–467. doi:10.1094/Phyto-82-462.

    Article  CAS  Google Scholar 

  • Carlile, A. J., Bindschedler, L. V., Bailey, A. M., Bowyer, P., Clarkson, J. M., & Cooper, R. M. (2000). Characterization of SNP1, a cell wall-degrading trypsin, produced during infection by Stagonospora nodorum. Molecular Plant-Microbe Interactions, 13, 538–550. doi:10.1094/MPMI.2000.13.5.538.

    Article  PubMed  CAS  Google Scholar 

  • Chen, Z.-Y., Brown, R. L., Damann, K. E., & Cleveland, T. E. (2007). Identification of maize kernel endosperm proteins associated with resistance to aflatoxin contamination by Aspergillus flavus. Phytopathology, 97, 1094–1103. doi:10.1094/PHYTO-97-9-1094.

    Article  PubMed  CAS  Google Scholar 

  • Di Pietro, A., Huertas Gonzales, M. D., Gutierrez-Corona, J. F., Martinez-Cadena, G., Meglecz, E., & Roncero, M. I. G. (2001). Molecular characterization of a subtilase from the vascular wilt fungus Fusarium oxysporum. Molecular Plant-Microbe Interactions, 14, 653–662. doi:10.1094/MPMI.2001.14.5.653.

    Article  PubMed  Google Scholar 

  • Dowell, F. E., Dorner, J. W., Cole, R. J., & Davidson Jr., J. I. (1990). Aflatoxin reduction by screening farmers stock peanuts. Peanut Science, 17, 6–8.

    CAS  Google Scholar 

  • Guo, B. Z., Russin, J. S., Cleveland, T. E., Brown, R. L., & Damman, K. E. (1996). Evidence for cutinase production by Aspergillus flavus and its possible role in infection of corn kernels. Phytopathology, 86, 824–829. doi:10.1094/Phyto-86-824.

    Article  CAS  Google Scholar 

  • Holbrook, C. K., Rucker, D. M., Wilson, J. E., Hook, J. E., & Matheron, M. E. (2000). Preharvest aflatoxin contamination in drought-tolerant and drought-intolerant peanut genotypes. Peanut Science, 27, 45–48.

    Article  CAS  Google Scholar 

  • Horn, B. W. (2006). Relationship between soil densities of Aspergillus species and colonization of wounded peanut seeds. Canadian Journal of Microbiology, 52, 951–960. doi:10.1139/W06-050.

    Article  PubMed  CAS  Google Scholar 

  • Kolattukudy, P. E., Lee, J. D., Rogers, L. M., Zimmerman, P., Ceselski, S., Fox, B., Stein, B., & Copelan, E. A. (1993). Evidence for possible involvement of an elastolytic serine protease in Aspergillosis. Infection and Immunity, 61, 2357–2368.

    PubMed  CAS  Google Scholar 

  • Liang, X. Q., Luo, M., & Guo, B. Z. (2006). Resistance mechanisms to Aspergillus flavus infection and aflatoxin contamination in Peanut (Arachis hypogaea). The Plant Pathology Journal, 5, 115–124.

    Article  Google Scholar 

  • Luo, M., Liang, X. Q., Dang, P., Holbrook, C. C., Bausher, M. G., Lee, R. D., & Guo, B. Z. (2005). Microarray-based screening of differentially expressed genes in peanut in response to Aspergillus parasiticus infection and drought stress. Plant Science, 169, 695–703. doi:10.1016/j.plantsci.2005.05.020.

    Article  CAS  Google Scholar 

  • Mellon, J. E., Cotty, P. J., & Dowd, M. K. (2007). Aspergillus flavus hydrolases: their roles in pathogenesis and substrate utilization. Applied Microbiology and Biotechnology, 77, 497–504. doi:10.1007/s00253-007-1201-8.

    Article  PubMed  CAS  Google Scholar 

  • Middleton, K. J., Pande, S., Sharma, S. B., & Smith, D. H. (1994). Groundnut Crop: A scientific basis for improvement. In (Ed.), Diseases (pp. 336–78). London: Chapman & Hall.

    Google Scholar 

  • Monod, M., Capoccia, S., Léchenne, B., Zaugg, C., Holdom, M., & Jousson, O. (2002). Secreted proteases from pathogenic fungi. International Journal of Medical Microbiology, 292(5–6), 405–419. doi:10.1078/1438-4221-00223.

    Article  PubMed  CAS  Google Scholar 

  • Movahedi, S., & Heale, J. B. (1990). The roles of aspartic proteinase and endo-pectin lyase enzymes in the primary stages of infection and pathogenesis of the various host tissues by different isolates of Botrytis cinerea. Physiological and Molecular Plant Pathology, 36, 303–324. doi:10.1016/0885-5765(90)90061-2.

    Article  CAS  Google Scholar 

  • Olivieri, F., Zanetti, M. E., Oliva, C. R., Covarrubias, A. A., & Casalongue, C. A. (2002). Characterization of an extracellular serine protease of Fusarium eumartii and its action on pathogenesis related proteins. European Journal of Plant Pathology, 108, 63–72. doi:10.1023/A:1013920929965.

    Article  CAS  Google Scholar 

  • Pekkarinen, A. I., & Jones, B. (2002). Trypsin-like proteinase produced by Fusarium culmorum grown on grain proteins. Journal of Agricultural and Food Chemistry, 50, 3849–3855. doi:10.1021/jf020027x.

    Article  PubMed  CAS  Google Scholar 

  • Plummer, K. M., Clark, S. J., Ellis, L. M., Loganathan, A., Al-Samarrai, T. H., Rikkerink, E. H. A., et al. (2004). Analysis of a secreted aspartic peptidase disruption mutant of Glomerella cingulata. European Journal of Plant Pathology, 110, 265–274. doi:10.1023/B:EJPP.0000019796.78598.8c.

    Article  CAS  Google Scholar 

  • Ramesh, M. V., Sirakova, T., & Kolattukudy, P. E. (1994). Isolation, characterization, and cloning of the cDNA and the gene for an elastinolytic serine proteinase from Aspergillus flavus. Infection and Immunity, 62, 79–85.

    PubMed  CAS  Google Scholar 

  • Ramesh, M. V., Sirakova, T., & Kolattukudy, P. E. (1995). Cloning and characterization of the cDNAs and genes (mep20) encoding homologous metalloproteinases from Aspergillus flavus and A. fumigatus. Gene, 165, 121–125. doi:10.1016/0378-1119(95)00434-8.

    Article  PubMed  CAS  Google Scholar 

  • Reed, C. E., & Kita, H. (2004). The role of protease activation of inflammation in allergic respiratory diseases. The Journal of Allergy and Clinical Immunology, 114, 997–1008. doi:10.1016/j.jaci.2004.07.060.

    Article  PubMed  CAS  Google Scholar 

  • Reichard, U., Buttner, S., Eiffert, H., Staib, F., & Ruchel, R. (1990). Purification and characterisation of an extracellular serine proteinase from Aspergillus fumigatus and its detection in tissue. Journal of Medical Microbiology, 33, 243–251.

    PubMed  CAS  Google Scholar 

  • Rhodes, J. C., Amlung, T. W., & Muller, M. S. (1990). Isolation and characterization of an elastinolytic proteinase from Aspergillus flavus. Infection and Immunity, 58, 2529–2534.

    PubMed  CAS  Google Scholar 

  • Roncero, M. I. G., Hera, C., Ruiz-Rubio, M., García Maceira, F. I., Madrid, M. P., Caracuel, Z., et al. (2003). Fusarium as a model for studying virulence in soilborne plant pathogens. Physiological and Molecular Plant Pathology, 62, 87–98. doi:10.1016/S0885-5765(03)00043-2.

    Article  Google Scholar 

  • Sarath, G., De La Motte, R. S., & Wagner, F. W. (1989). Proteolytic Enzymes: A Practical Approach. In (Ed.), Protease Assay Methods (pp. 25–55). Oxford: IRL Press.

    Google Scholar 

  • Sieber, P., Schorderet, M., Ryser, U., Buchala, A., Kolattukudy, P., Metraux, J. P., et al. (2000). Transgenic Arabidopsis plants expressing a fungal cutinase show alterations in the structure and properties of the cuticle and postgenital organ fusions. The Plant Cell, 12, 721–738.

    Article  PubMed  CAS  Google Scholar 

  • Sreedhar, L., Kobayashi, D. Y., Bunting, T. E., Hillman, B. I., & Belanger, F. C. (1999). Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host. Gene, 235, 121–129. doi:10.1016/S0378-1119(99)00201-2.

    Article  PubMed  CAS  Google Scholar 

  • St Leger, R. J., Joshi, L., & Roberts, D. W. (1997). Adaptation of proteases and carbohydrates of saprophytic, phytopathogenic and entomopathogenic fungi to the requirements of their ecological niches. Microbiology, 143, 1983–1992.

    Article  PubMed  CAS  Google Scholar 

  • Tubajika, K. M., & Damann, K. E. (2001). Sources of resistance to aflatoxin production in maize. Journal of Agricultural and Food Chemistry, 49, 2652–2656. doi:10.1021/jf001333i.

    Article  PubMed  CAS  Google Scholar 

  • Williams, J. H., Phillips, T. D., Jolly, P. E., Stiles, J. K., Jolly, C. M., & Aggarwal, D. (2004). Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. The American Journal of Clinical Nutrition, 80, 1106–1122.

    PubMed  CAS  Google Scholar 

  • Xu, H., Annis, S., Linz, J., & Trail, F. (2000). Infection and colonization of peanut pods by Aspergillus parasiticus and the expression of the aflatoxin biosynthetic gene, nor-1, in infection hyphae. Physiological and Molecular Plant Pathology, 56, 185–196. doi:10.1006/pmpp.2000.0265.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors wish to acknowledge the critical review and constructive comments of Dr. L.M. Giorda in the preparation of this manuscript. This work was supported by a grant from Agencia Cordoba Ciencia and Agencia Nacional de Promocion Cientifica y Tecnologica, Argentina. Ramon Asis and Virginia Muller are members and fellow, respectively, of CONICET (Argentina). The authors thank Dr. Olga Ortiz Carranza and Silvia Caceres for her technical assistance in a review of the English.

Author information

Authors and Affiliations

  1. Departamento de Bioquímica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre interseccion Medina Allende, Ciudad Universitaria, CP5000, Córdoba, Argentine

    R. Asis, V. Muller, D. L. Barrionuevo, S. A. Araujo & M. A. Aldao

Authors
  1. R. Asis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. V. Muller
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D. L. Barrionuevo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. S. A. Araujo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. A. Aldao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to R. Asis.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Asis, R., Muller, V., Barrionuevo, D.L. et al. Analysis of protease activity in Aspergillus flavus and A. parasiticus on peanut seed infection and aflatoxin contamination. Eur J Plant Pathol 124, 391–403 (2009). https://doi.org/10.1007/s10658-008-9426-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10658-008-9426-7

Keywords

Search

Navigation