Skip to main content
Log in

Identification of Alternaria spp. associated with tomato early blight in Iran and investigating some of their virulence factors

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

Abstract

Tomato is one of the major crops grown in Iran, which is attacked by a large number of pathogens. Early blight is among the most important and harmful diseases of this plant caused by Alternaria spp. This study was conducted to identify Alternaria species causing early blight disease and to evaluate different virulence factors of this fungal pathogen. The samples were collected in Khorassan-Razavi province in Iran from tomato plants showing characteristic symptoms of the disease. Morphological identification of the isolates was done on PCA medium and under controlled conditions. Six Alternaria species, including A. alternata, A. tenuissima, A. arborescens, A. mimicula, A. interrupta and A. infectoria were identified. The ITS1 and ITS4 primers were used for molecular identification of the isolates via sequence analysis. The highest frequency was observed for A. alternata and the least frequent was A. infectoria. All isolates obtained in this study were pathogenic on tomato cultivar Peto Early Ch. The results showed significant differences in pathogenicity of the isolates on tomato plants. A. alternata and A. tenuissima had the highest and A. mimicula and A. infectoria had the lowest level of pathogenicity. In the qualitative analysis of cell wall degrading enzymes (CWDEs), all tested isolates were able to produce pectinase, cellulase, amylase, protease and lipase, but production of pectinase and cellulase had direct correlation with pathogenicity of the isolates.

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

Access this article

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

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Abdel-Razik AA (1970) The parasitism of white Sclerotium cepivorum Berk., the incitant of white rot of onion. PhD thesis, Fac Agric, Assiut University, Assiut, Egypt

  • Aminian H, Zad J, Sharifi Tehrani A, Okhovat S, Talebi Jahromi K (2004) A study of tomato stem canker in Busher province. Iranian J Agric Sci 35:245–252

    Google Scholar 

  • Anand T, Bhaskaran R, Gandhi KT, Rajesh M, Senthilraja G (2008) Production of cell wall degrading enzymes and toxins by Colletotrichum capsici and Alternaria alternata causing fruit rot of chillies. J Plant Protec Res 48:437–451

    Article  CAS  Google Scholar 

  • Andualema B, Gessesse A (2012) Microbial lipases and their industrial applications. Biotechnology 11:100–118

    Article  CAS  Google Scholar 

  • Archer DB, Wood DA (1995) Fungal exoenzymes. In: The growing fungus. Springer, Dordrecht, pp 137–162

    Chapter  Google Scholar 

  • Bashir U, Mushtaq S, Akhtar N (2014) First report of Alternaria metachromatica from Pakistan causing leaf spot of tomato. Pak J Agric Sci 51:305–308

    Google Scholar 

  • Berto P, Belingheri L, Dehorter B (1997) Production and purification of a novel extracellular lipase from Alternaria brassicicola. Biotechnol Lett 19:533–536

    Article  CAS  Google Scholar 

  • Berto P, Comménil P, Belingheri L, Dehorter B (1999) Occurrence of a lipase in spores of Alternaria brassicicola with a crucial role in the infection of cauliflower leaves. FEMS Microbiol Lett 180:183–189

    Article  CAS  PubMed  Google Scholar 

  • Bezerra JDP, Santos MGS, Svedese VM, Lima DMM, Fernandes MJS, Paiva LM, Souza-Motta CM (2012) Richness of endophytic fungi isolated from Opuntia ficus-indica mill. (Cactaceae) and preliminary screening for enzyme production. World J Microbiol Biotechnol 28:1989–1995

    Article  CAS  PubMed  Google Scholar 

  • Blancard D (2012) Tomato diseases: identification, biology and control: a colour handbook. CRC Press, Boca Raton

    Book  Google Scholar 

  • Chaerani R, Groenwold R, Stam P, Voorrips RE (2007) Assessment of early blight (Alternaria solani) resistance in tomato using a droplet inoculation method. J Gen Plant Pathol 73:96–103

    Article  Google Scholar 

  • Chandrasekaran M, Sathiyabama M (2014) Production, partial purification and characterization of protease from a phytopathogenic fungi Alternaria solani (Ell. and Mart.) Sorauer. J Basic Microbiol 54:763–774

    Article  CAS  PubMed  Google Scholar 

  • Cho Y, Davis JW, Kim KH, Wang J, Sun QH, Cramer J, Lawrence CB (2006) A high throughput targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element (LME) constructs. Mol Plant-Microbe Interact 19:7–15

    Article  CAS  PubMed  Google Scholar 

  • Cihangir N, Sarikaya E (2004) Investigation of lipase production by a new isolate of Aspergillus sp. World J Microbiol Biotechnol 20:193–197

    Article  CAS  Google Scholar 

  • Colowich SP (1995) Methods in enzymology. Academic Prees INC, London

    Google Scholar 

  • Dale C, Vergnolle N (2008) Protease signaling to G protein-coupled receptors: implications for inflammation and pain. J Recept Signal Transduc 28:29–37

    Article  CAS  Google Scholar 

  • Dang HX, Pryor B, Peever T, Lawrence CB (2015) The Alternaria genomes database: a comprehensive resource for a fungal genus comprised of saprophytes, plant pathogens, and allergenic species. BMC Genomics 16:239

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Deep S, Sharma P, Behera N (2014) Optimization of extracellular cellulase enzyme production from Alternaria brassicicola. Int J Curr Microbiol App Sci 3:127–139

    CAS  Google Scholar 

  • Esfandiari E (1948) Troixieme liste de fungi ramasses en Iran. Entomol Phytopathol Appl 8:1–12

    Google Scholar 

  • Eshel D, Miyara I, Ailing T, Dinoor A, Prusky D (2002) pH regulates endoglucanase expression and virulence of Alternaria alternata in persimmon fruit. Mol Plant-Microbe Interact 15:774–779

    Article  CAS  PubMed  Google Scholar 

  • García-Calvo L, Ullán RV, Fernández-Aguado M, García-Lino AM, Balaña-Fouce R, Barreiro C (2018) Secreted protein extract analyses present the plant pathogen Alternaria alternata as a suitable industrial enzyme toolbox. J Proteome 177:48–64

    Article  CAS  Google Scholar 

  • Gibson DM, King BC, Hayes ML, Bergstrom GC (2011) Plant pathogens as a source of diverse enzymes for lignocellulose digestion. Curr Opin Microbiol 14:264–270

    Article  CAS  PubMed  Google Scholar 

  • Gräfenhan T, Patrick SK, Roscoe M, Trelka R, Gaba D, Chan JM, McKendry T, Clear RM, and Tittlemier SA (2013) Fusarium damage in cereal grains from Western Canada. 1. Phylogenetic analysis of moniliformin-producing Fusarium species and their natural occurrence in mycotoxin-contaminated wheat, oats, and rye. J Agric Food Chem 61(23):5425–5437

  • Griebeler N, Polloni AE, Remonatto D, Arbter F, Vardanega R, Cechet JL, Di Luccio M, de Oliveira D, Treichel H, Cansian RL, Rigo E (2011) Isolation and screening of lipase-producing fungi with hydrolytic activity. Food Bioproc Technol 4:578–586

    Article  CAS  Google Scholar 

  • Grogan RG, Kimble KA, Misaghi I (1975) A stem canker disease of tomato caused by Alternaria alternata f. sp lycopersici. Phytopathology 65:880–886

    Article  Google Scholar 

  • Hajianfar R, Zarbakhsh A (2006) Identification of causal organism of early blight and stem canker diseases on tomato in major production regions of country. Proceedings of the 17th Iranian Plant Protection Congress. Karaj, Iran

  • Hankin L, Zucker M, Sands DC (1971) Improved solid medium for the detection and enumeration of pectolytic bacteria. Appl Microbiol 22:205–209

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hong J, Tamaki H, Akiba S, Yamamoto K, Kumagai H (2001) Cloning of a gene encoding a highly stable endo-β-1, 4-glucanase from Aspergillus niger and its expression in yeast. J Biosci Bioeng 92:434–441

    Article  CAS  PubMed  Google Scholar 

  • Hong S, Kim W, Choi H, Lee Y, Shim H (2011) June. Occurrence of early blight on black nightshade caused by Alternaria tomatophila in Korea. Phytopathology 101:S74

    Google Scholar 

  • Hubballi M, Sornakili A, Nakkeeran S, Anand T, Raguchander T (2011) Virulence of Alternaria alternata infecting noni associated with production of cell wall degrading enzymes. J Plant Protec Res 51:87–92

    Article  Google Scholar 

  • Iftikhar T, Abdullah R, Iqtedar M, Kaleem A, Aftab M, Niaz M, Sidra BT, Majeed H (2015) Production of lipases by Alternaria sp. (mbl 2810) through optimization of environmental conditions using submerged fermentation technique. Int J Biosci 7:178–186

    Article  CAS  Google Scholar 

  • Isshiki A, Akimitsu K, Yamamoto M, Yamamoto H (2001) Endopolygalacturonase is essential for citrus black rot caused by Alternaria citri but not brown spot caused by Alternaria alternata. Mol Plant-Microbe Interac 14:749–757

    Article  CAS  Google Scholar 

  • Jain V, Dhawan K (2008) Major cell wall degrading enzymes in two contrasting cultivars of Brassica juncea infected with Alternaria brassicae. Crucifers Newslett 27:20–21

    Google Scholar 

  • Kaur M, Aggarwal NK (2017) Screening of Alternaria pathogens associated with Parthenium hysterophorus for the production of lignocellulolytic enzymes. Bioengin Biosci 5:14–23

    CAS  Google Scholar 

  • Khaledi N, Taheri P, Falahati Rastegar M (2017) Identification, virulence factors characterization, pathogenicity and aggressiveness analysis of Fusarium spp., causing wheat head blight in Iran. Eur J Plant Pathol 147:897–918

    Article  CAS  Google Scholar 

  • Kikot GE, Hours RA, Alconada TM (2009) Contribution of cell wall degrading enzymes to pathogenesis of Fusarium graminearum: a review. J Basic Microbiol 49:231–241

    Article  CAS  PubMed  Google Scholar 

  • Kokaeva LY, Belosokhov AF, Doeva LY, Skolotneva ES, Elansky SN (2015) Distribution of Alternaria species on blighted potato and tomato leaves in Russia. J Plant Dis Protec 125:205–212

    Google Scholar 

  • Kumar V, Haldar S, Pandey KK, Singh RP, Singh AK, Singh PC (2008) Cultural, morphological, pathogenic and molecular variability amongst tomato isolates of Alternaria solani in India. World J Microbiol Biotechnol 24:1003–1009

    Article  CAS  Google Scholar 

  • Kusaba M, Tsuge T (1994) Nuclear ribosomal DNA variation and pathogenic specialization in Alternaria fungi known to produce host-specific toxins. Appl Environ Microbiol 60:3055–3062

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kusaba M, Tsuge T (1995) Phologeny of Alternaria fungi known to produce host-specific toxins on the basis of variation in internal transcribed spacers of ribosomal DNA. Curr Genet 28:491–498

    Article  CAS  PubMed  Google Scholar 

  • Li KN, Rouse DI, German TL (1994) PCR primers that allow intergeneric differentiation of ascomycetes and their application to Verticillium spp. Appl Environ Microbiol 60:4324–4331

    CAS  PubMed  PubMed Central  Google Scholar 

  • MacMillan JD, Voughin RH (1964) Purification and properties of a polyglacturonic acid- transeliminase produced by Clastridium multiformentans. Biochemistry 3:564–572

    Article  CAS  PubMed  Google Scholar 

  • Martinez MJ, Alconada MT, Guillén F, Vázquez C, Reyes F (1991) Pectic activities from Fusarium oxysporum f. sp. melonis: purification and characterization of an exopolygalacturonase. FEMS Microbiol Lett 81:145–149

    Article  CAS  Google Scholar 

  • Morris PF, Connolly MS, Clair DAST (2000) Genetic diversity of Alternaria alternata isolated from tomato in California assessed using RAPDs. Mycol Res 104:286–292

    Article  CAS  Google Scholar 

  • Murphy DJ (2001) The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog Lipid Res 40:325–438

    Article  CAS  PubMed  Google Scholar 

  • Nees von Esenbeck CG (1816) Das System der Pilze and Schwame. XXVIII – XLIV

  • Niture SK, Kumar AR, Pant A (2006) Role of glucose in production and repression of polygalacturonase and pectate lyase from phytopathogenic fungus Fusarium moniliforme NCIM 1276. World J Microbiol Biotechnol 22:893–899

    Article  CAS  Google Scholar 

  • Ortega LM, Kikot GE, Astoreca AL, Alconada TM (2013) Screening of Fusarium graminearum isolates for enzymes extracellular and deoxynivalenol production. J Mycol Article ID 358140:7. https://doi.org/10.1155/2013/358140

  • Panda T, Nair SR, Kumar MP (2004) Regulation of synthesis of the pectolytic enzymes of Aspergillus Niger. Enz Microb Technol 34:466–473

    Article  CAS  Google Scholar 

  • Pavon MÁ, Luna A, de la Cruz S, González I, Martín R, García T (2012) PCR-based assay for the detection of Alternaria species and correlation with HPLC determination of altenuene, alternariol and alternariol monomethyl ether production in tomato products. Food Cont 25:45–52

    Article  CAS  Google Scholar 

  • Perkins DD (1962) Preservation of Neurospora stock cultures with anhydrous silica gel. Can J Microbiol 8:592–594

    Article  Google Scholar 

  • Pryor BM, Michailides TJ (2002) Morphological, pathogenic, and molecular characterization of Alternaria isolates associated with Alternaria late blight of pistachio. Phytopathology 92:406–416

    Article  CAS  PubMed  Google Scholar 

  • Rathod SR, Chavan AM (2010) Incidence of Alternaria species on different cereals, pulses and oil seeds. J Ecobiotechnol 2:63–65

    Google Scholar 

  • Riou C, Freyssinet G, Fevre M (1991) Production of cell wall-degrading enzymes by the phytopathogenic fungus Sclerotinia sclerotiorum. Appl Environ Microbiol 57:1478–1484

    CAS  PubMed  PubMed Central  Google Scholar 

  • Roberts RG, Reymond ST, Andersen B (2000) RAPD fragment pattern analysis and morphological segregation of small-spored Alternaria species and species groups. Mycol Res 104:151–160

    Article  CAS  Google Scholar 

  • Rodrigues TTMS, Berbee ML, Simmons EG, Cardoso CR, Reis A, Maffia LA, Mizubuti ESG (2010) First report of Alternaria tomatophila and A. grandis causing early blight on tomato and potato in Brazil. New Dis Rep 22:28

    Article  Google Scholar 

  • Saharan GS, Mehta N, Meena PD, Dayal P (2016) Alternaria diseases of crucifers: biology, ecology and disease management. Springer, Singapore

    Book  Google Scholar 

  • Shafique S, Bajwa R, Shafique S (2010) Alpha-amylase production by toxigenic fungi. Nat Prod Res 24:1449–1456

    Article  CAS  PubMed  Google Scholar 

  • Shafique S, Shafique S, Ahmed A (2013) Ecofriendly response of citrus peels to Alternaria leaf spots of tomato: exclusive role of peel phenolics. Int J Agric Biol 15

  • Shahbazi H, Aminian H, Sahebani N, Halterman DA (2010) Biochemical evaluation of resistance responses of potato to different isolates of Alternaria solani. Phytopathology 100:454–459

    Article  CAS  PubMed  Google Scholar 

  • Showalter AM (1993) Structure and function of plant cell wall proteins. Plant Cell 5:9–23

    CAS  PubMed  PubMed Central  Google Scholar 

  • Simmons E (2007) Alternaria, an identification manual. CBS Fungal Biodiversity Centre, Utrecht

    Google Scholar 

  • Stammler G, Bohme F, Philippi J, Miessner S, Tegge V (2014) Pathogenicity of Alternaria-species on potatoes and tomatoes. In Fourteenth Euroblight Workshop PPO–Special Report 16:85–96

    Google Scholar 

  • Sunitha VH, Devi DN, Srinivas C (2013) Extracellular enzymatic activity of endophytic fungal strains isolated from medicinal plants. World J Agric Sci 9:1–9

    CAS  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729

  • Ten Have A, Tenberge KB, Benen JA, Tudzynski P, Visser J, van Kan JA (2002) The contribution of cell wall degrading enzymes to pathogenesis of fungal plant pathogens. In: Agricultural Applications. Springer Berlin Heidelberg, Berlin, pp 341–358

    Chapter  Google Scholar 

  • Thomma BP (2003) Alternaria spp.: from general saprophyte to specific parasite. Mol Plant Pathol 4:225–236

    Article  CAS  PubMed  Google Scholar 

  • White TJ, Bruns T, Lee SJWT, Taylor JL (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications 18:315–322

    Google Scholar 

  • Wood TM, Bhat KM (1988) Methods for measuring cellulase activities. In: Methods in enzymology, vol 160. Academic Press, Cambridge, pp 87–112

    Google Scholar 

  • Yang Z, Rogers LM, Song Y, Guo W, Kolattukudy PE (2005) Homoserine and asparagine are host signals that trigger in planta expression of a pathogenesis gene in Nectria haematococca. Proc Natl Acad Sci U S A 102:4197–4202

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yao C, Koller W (1995) Diversity of cutinases from plant pathogenic fungi: different cutinases are expressed during saprophytic and pathogenic stages of Alternaria brassicicola. Mol Plant-Microbe Interact 8:122–130

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Ferdowsi University of Mashhad, Iran, for financial support of this research with project number 3/40740 approved on 27/4/2016.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Parissa Taheri.

Ethics declarations

Conflict of interest

The authors have no conflict of interest to declare.

Ethical approval

This research did not involve human participants and/or animals.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ramezani, Y., Taheri, P. & Mamarabadi, M. Identification of Alternaria spp. associated with tomato early blight in Iran and investigating some of their virulence factors. J Plant Pathol 101, 647–659 (2019). https://doi.org/10.1007/s42161-019-00259-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s42161-019-00259-w

Keywords

Navigation