Bio-Control of Alternaria alternata during Banana Storage by Purified AFP Using Isoelectric Focusing Technique

Hassan Barakat

Department of Food Science, Faculty of Agriculture, Benha University, 13736 Moshtohor, Kaliuobia, Egypt.

DOI: 10.4236/fns.2014.515161                

Abstract

Interestingly, antifungal protein AFP was purified from Aspergillus giganteus supernatants with modified isoelectric focusing procedure after adaptation of the secretion conditions. Subsequently, the antifungal activity as well as the mode of action against Alternaria alternata was tested in vitro. Moreover, different concentrations of AFP were applied to banana fruits for 15 days at 20°C in vivo. Obtained results illustrated that A. giganteus was able to secrete about 39.78 ± 2.39 mg AFP·l^-1 Olson medium. The employed ammonium sulfate (AS 75%) precipitation procedure followed by dialysis steps yielded about 16 - 22 mg AFP·l^-1 culture supernatant with general mean of 18.67 ± 1.98 mg·l^-1. The lost amount of AFP during purification using AS and 3KDa cut-off dialysis membrane is about 50% thus, purification procedure must be further improved. Indeed, concluded results from MIC and hyphal extension inhibition test noticed that AFP was efficiently affected by either growth or hyphae form of A. alternata in vitro. The MIC of AFP against A. alternata was 2μg·ml^-1. However,short, thick and highly septated hyphae with damaged constricted apical regions extruding from condensed mycelium aggregates in treated hyphae compared to the untreated culture was remarkably shown. The mode of action of in vitro experiment manifested that AFP was effective to act the fungal cell and permeabilize the cell membrane of A. alternata. Furthermore, the in vivo experiment showed that AFP could reduce post-harvest decay on banana caused by A. alternata. AFP at concentration of 15 and 25 μg·ml^-1 exhibit Alternaria decayed reduction by 45.45% and 77.27%, respectively. While no Alternaria decayed area was observed when 50 μg·ml^-1 was applied during the storage time. Quantification of DNA by species-specific PCR could exude a positive correlation between the DNA amount and decayed area. In conclusion, AFP can be efficiently used as a bio-preservative agent during storage and handling of banana fruits, and considered as an excellent biological alternative to combat secondary growth of filamentous fungi.

Keywords

Antifungal Protein (AFP), Alternaria alternata, Isoelectric Focusing, Antifungal Activity,

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Ng, T.B. (2013) Chapter 23—Antifungal and Antibacterial Peptides of Fungal Origin. In: Kastin, A.J., Ed., Handbook of Biologically Active Peptides, 2nd Edition, Academic Press, Boston, 157-161. http://dx.doi.org/10.1016/B978-0-12-385095-9.00023-3
[2]	Liu, R., Huang, H.W., Yang, Q. and Liu, W. (2002) Purification of α-Sarcin and an Antifungal Protein from Mold (Aspergillus giganteus) by Chitin Affinity Chromatography. Protein Expression and Purification, 25, 50-58. http://dx.doi.org/10.1006/prep.2001.1608
[3]	Olmo, N., Turnay, J., de Buitrago, G.G., de Silanes, I.L., Gavilanes, J.G. and Lizarbe, M.A. (2001) Cytotoxic Mechanism of the Ribotoxin α-Sarcin. European Journal of Biochemistry, 268, 2113-2123. http://dx.doi.org/10.1046/j.1432-1327.2001.02086.x
[4]	Szappanos, H., Szigeti, G.P., Pál, B., Rusznák, Z., Szucs, G., Rajnavölgyi, é., Balla, J., Balla, G., Nagy, E., Leiter, é., Pócsi, I., Hagen, S., Meyer, V. and Csernoch, L. (2006) The Antifungal Protein AFP Secreted by Aspergillus giganteus Does Not Cause Detrimental Effects on Certain Mammalian Cells. Peptides, 27, 1717-1725. http://dx.doi.org/10.1016/j.peptides.2006.01.009
[5]	Lacadena, J., Martinez del Pozo, A., Gasset, M., Patino, B., Campos-Olivas, R., Vazquez, C., Martinez-Ruiz, A., Mancheno, J.M., Onaderra, M. and Gavilanes, J.G. (1995) Characterization of the Antifungal Protein Secreted by the Mould Aspergillus giganteus. Archive of Biochemistry and Biophysics, 324, 273-281. http://dx.doi.org/10.1006/abbi.1995.0040
[6]	Hao, J.-J., Xu, Y.-Z., Geng, C.-D., Liu, W.-Y., Wang, E.-D., Gong, Z.-Z. and Ulbrich, N. (1998) Purification of α-Sarcin and an Antifungal Protein from Aspergillus giganteus by Blue Sepharose CL-6B Affinity Chromatography. Protein Expression and Purification, 14, 295-301. http://dx.doi.org/10.1006/prep.1998.0957
[7]	Theis, T. (2003) Target Site and Mode of Action of the Antifungal Protein from Aspergillus giganteus. Ph.D. Thesis, Technische Universität, Berlin.
[8]	Barakat, H., Spielvogel, A., Hassan, M., El-Desouky, A., El-Mansy, H., Rath, F., Meyer, V. and Stahl, U. (2010) The Antifungal Protein AFP from Aspergillus giganteus Prevents Secondary Growth of Different Fusarium Species on Barley. Applied Microbiology and Biotechnology, 87, 617-624. http://dx.doi.org/10.1007/s00253-010-2508-4
[9]	Ghosh, M. (2009) Purification of a Lectin-Like Antifungal Protein from the Medicinal Herb, Withania somnifera. Fitoterapia, 80, 91-95. http://dx.doi.org/10.1016/j.fitote.2008.10.004
[10]	Mhetras, N.C., Bastawde, K.B. and Gokhale, D.V. (2009) Purification and Characterization of Acidic Lipase from Aspergillus niger NCIM 1207. Bioresource Technology, 100, 1486-1490. http://dx.doi.org/10.1016/j.biortech.2008.08.016
[11]	Iijima, S., Shiba, K., Inoue, J., Yoshida, T. and Kimura, M. (1997) Simultaneous Analysis of Microheterogeneity of Immunoglobulins and Serum Protein Fraction Using High-Voltage Isoelectric Focusing on Six Cellulose Acetate Membranes. Journal of Clinical Laboratory Analysis, 11, 220-224. http://dx.doi.org/10.1002/(SICI)1098-2825(1997)11:4<220::AID-JCLA8>3.0.CO;2-C
[12]	Theis, T., Marx, F., Salvenmoser, W., Stahl, U. and Meyer, V. (2005) New Insights into the Target Site and Mode of Action of the Antifungal Protein of Aspergillus giganteus. Research in Microbiology, 156, 47-56. http://dx.doi.org/10.1016/j.resmic.2004.08.006
[13]	Theis, T., Wedde, M., Meyer, V. and Stahl, U. (2003) The Antifungal Protein from Aspergillus giganteus Causes Membrane Permeabilization. Antimicrobial Agents and Chemotherapy, 47, 588-593. http://dx.doi.org/10.1128/AAC.47.2.588-593.2003
[14]	Moreno, A.B., Penas, G., Rufat, M., Bravo, J.M., Estopa, M., Messeguer, J. and San Segundo, B. (2005) Pathogen-Induced Production of the Antifungal AFP Protein from Aspergillus giganteus Confers Resistance to the Blast Fungus Magnaporthe grisea in Transgenic Rice. Molecular Plant-Microbe Interaction, 18, 960-972. http://dx.doi.org/10.1094/MPMI-18-0960
[15]	Shai, Y. (2002) Mode of Action of Membrane Active Antimicrobial Peptides. Biopolymers, 66, 236-248. http://dx.doi.org/10.1002/bip.10260
[16]	Ferket, K.K.A., Levery, S.B., Park, C., Cammue, B.P.A. and Thevissen, K. (2003) Isolation and Characterization of Neurospora crassa Mutants Resistant to Antifungal Plant Defensins. Fungal Genetics and Biology, 40, 176-185. http://dx.doi.org/10.1016/S1087-1845(03)00085-9
[17]	Park, C., Bennion, B., Francois, I.E., Ferket, K.K., Cammue, B.P., Thevissen, K. and Levery, S.B. (2005) Neutral Glycolipids of the Filamentous Fungus Neurospora crassa: Altered Expression in Plant Defensin-Resistant Mutants. Journal of Lipid Research, 46, 759-768. http://dx.doi.org/10.1194/jlr.M400457-JLR200
[18]	Hagen, S., Marx, F., Ram, A.F. and Meyer, V. (2007) The Antifungal Protein AFP from Aspergillus giganteus Inhibits Chitin Synthesis in Sensitive Fungi. Applied and Environmental Microbiology, 73, 2128-2134. http://dx.doi.org/10.1128/AEM.02497-06
[19]	Liu, H., Kauffman, S., Becker, J.M. and Szaniszlo, P.J. (2004) Wangiella (Exophiala) dermatitidis WdChs5p, a Class V Chitin Synthase, Is Essential for Sustained Cell Growth at Temperature of Infection. Eukaryotic Cell, 3, 40-51. http://dx.doi.org/10.1128/EC.3.1.40-51.2004
[20]	Parkunan, V., Li, S., Fonsah, E.G. and Ji, P. (2013) First Report of Alternaria Leaf Spot of Banana Caused by Alternaria alternata in the United States. Plant Disease, 97, 1116. http://dx.doi.org/10.1094/PDIS-01-13-0007-PDN
[21]	Wiest, P.M., Wiese, K., Jacobs, M.R., Morrissey, A.B., Abelson, T.I., Witt, W. and Lederman, M.M. (1987) Alternaria Infection in a Patient with Acquired Immunodeficiency Syndrome: Case Report and Review of Invasive Alternaria Infections. Reviews of Infectious Diseases, 9, 799-803.
[22]	Ostry, V. (2008) Alternaria Mycotoxins: An Overview of Chemical Characterization, Producers, Toxicity, Analysis and Occurrence in Foodstuffs. World Mycotoxin Journal, 1, 175-188. http://dx.doi.org/10.3920/WMJ2008.x013
[23]	Barakat, H., Hassan, M., El-Desouky, A.I., Stahl, U. and El-Mansy, H. (2012) The Antifungal Protein AFP from Aspergillus giganteus Prevents Altarnaria Spoilage on Tomato and Mango Fruits during Storage. The 1st International Conference on Biotechnology Applications in Agriculture, Benha University, Moshtohor and Hurghada, 18-22 February 2012, 29-38.
[24]	Meyer, V. (2008) A Small Protein That Fights Fungi: AFP as a New Promising Antifungal Agent of Biotechnological Value. Applied Microbiology and Biotechnology, 78, 17-28. http://dx.doi.org/10.1007/s00253-007-1291-3
[25]	Konstantinova, P., Bonants, P.J.M., Van Gent-Pelzer, M.P.E., Van Der Zouwen, P. and Van Den Bulk, R. (2002) Development of Specific Primers for Detection and Identification of Altevnavia spp. in Carrot Material by PCR and Comparison with Blotter and Plating Assays. Mycological Research, 106, 23-33. http://dx.doi.org/10.1017/S0953756201005160
[26]	Merril, C.R. and Harrington, M.G. (1984) “Ultrasensitive” Silver Stains: Their Use Exemplified in the Study of Normal Human Cerebrospinal Fluid Proteins Separated by Two-Dimensional Electrophoresis. Clinical Chemistry, 30, 1938-1942.
[27]	Bradford, M.M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 72, 248-254. http://dx.doi.org/10.1016/0003-2697(76)90527-3
[28]	Roberts, W.K. and Selitrennikoff, C.P. (1986) Isolation and Partial Characterization of Two Antifungal Proteins from Barley. Biochimica et Biophysica Acta (BBA)-General Subjects, 880, 161-170. http://dx.doi.org/10.1016/0304-4165(86)90076-0
[29]	Vila, L., Lacadena, V., Fontanet, P., del Pozo, A.M. and Segundo, B.S. (2001) A Protein from the Mold Aspergillus giganteus Is a Potent Inhibitor of Fungal Plant Pathogens. Molecular Plant-Microbe Interactions, 14, 1327-1331. http://dx.doi.org/10.1094/MPMI.2001.14.11.1327
[30]	Hu, X. and Reddy, A.S. (1997) Cloning and Expression of a PR5-Like Protein from Arabidopsis: Inhibition of Fungal Growth by Bacterially Expressed Protein. Plant Molecular Biology, 34, 949-959.
[31]	Thevissen, K., Terras, F.R. and Broekaert, W.F. (1999) Permeabilization of Fungal Membranes by Plant Defensins Inhibits Fungal Growth. Applied and Environmental Microbiology, 65, 5451-5458.
[32]	Fredlund, E., Gidlund, A., Olsen, M., Börjesson, T., Spliid, N.H.H. and Simonsson, M. (2008) Method Evaluation of Fusarium DNA Extraction from Mycelia and Wheat for Down-Stream Real-Time PCR Quantification and Correlation to Mycotoxin Levels. Journal of Microbiological Methods, 73, 33-40. http://dx.doi.org/10.1016/j.mimet.2008.01.007
[33]	Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.
[34]	Hagen, S. (2006) Identification of Fungal Constituents that Determine the Sensitivity of Fungi towards the Antifungal Protein (AFP) of Aspergillus giganteus. Ph.D. Thesis, Technische Universität, Berlin.
[35]	Barakat, H., Hassan, M., El-Desouky, A.I., El-Mansy, H. and Stahl, U. (2012) Purification of Antifungal Protein (AFP) from Aspergillus giganteus and Its Antifungal Activity. The 1st International Conference on Biotechnology Applications in Agriculture, Benha University, Moshtohor and Hurghada, 18-22 February 2012, 15-28.
[36]	Moreno, A.B., Del Pozo, A.M., Borja, M. and Segundo, B.S. (2003) Activity of the Antifungal Protein from Aspergillus giganteus against Botrytis cinerea. Phytopathology, 93, 1344-1353. http://dx.doi.org/10.1094/PHYTO.2003.93.11.1344
[37]	Martinez Del Pozo, A., Lacadena, V., Mancheno, J.M., Olmo, N., Onaderra, M. and Gavilanes, J.G. (2002) The Antifungal Protein AFP of Aspergillus giganteus Is an Oligonucleotide/Oligosaccharide Binding (OB) Fold-Containing Protein that Produces Condensation of DNA. The Journal of Biological Chemistry, 277, 46179-46183. http://dx.doi.org/10.1074/jbc.M207472200
[38]	Marx, F., Salvenmoser, W., Kaiserer, L., Graessle, S., Weiler-Görz, R., Zadra, I. and Oberparleiter, C. (2005) Proper Folding of the Antifungal Protein PAF Is Required for Optimal Activity. Research in Microbiology, 156, 35-46. http://dx.doi.org/10.1016/j.resmic.2004.07.007
[39]	Takeshita, N., Yamashita, S., Ohta, A. and Horiuchi, H. (2006) Aspergillus nidulans Class V and VI Chitin Synthases CsmA and CsmB, Each with a Myosin Motor-Like Domain, Perform Compensatory Functions That Are Essential for Hyphal Tip Growth. Molecular Microbiology, 59, 1380-1394. http://dx.doi.org/10.1111/j.1365-2958.2006.05030.x
[40]	Moreno, A.B., Martinez Del Pozo, A. and San Segundo, B. (2006) Biotechnologically Relevant Enzymes and Proteins. Antifungal Mechanism of the Aspergillus giganteus AFP against the Rice Blast Fungus Magnaporthe grisea. Applied Microbiology and Biotechnology, 72, 883-895. http://dx.doi.org/10.1007/s00253-006-0362-1
[41]	Huang, H.W. (2000) Action of Antimicrobial Peptides: Two-State Model. Biochemistry, 39, 8347-8352. http://dx.doi.org/10.1021/bi000946l
[42]	Thevissen, K., Ferket, K.K.A., François, I.E.J.A. and Cammue, B.P.A. (2003) Interactions of Antifungal Plant Defensins with Fungal Membrane Components. Peptides, 24, 1705-1712. http://dx.doi.org/10.1016/j.peptides.2003.09.014
[43]	Thevissen, K., Osborn, R.W., Acland, D.P. and Broekaert, W.F. (2000) Specific Binding Sites for an Antifungal Plant Defensin from Dahlia (Dahlia merckii) on Fungal Cells Are Required for Antifungal Activity. Molecular Plant-Microbe Interactions, 13, 54-61. http://dx.doi.org/10.1094/MPMI.2000.13.1.54
[44]	Kobiler, I., Shalom, Y., Roth, I., Akerman, M., Vinokur, Y., Fuchs, Y. and Prusky, D. (2001) Effect of 2,4-Dichlorophenoxyacetic Acid on the Incidence of Side and Stem End Rots in Mango Fruits. Postharvest Biology and Technology, 23, 23-32. http://dx.doi.org/10.1016/S0925-5214(01)00092-8