1887

Abstract

Black spot caused by is an important fungal disease affecting cruciferous crops, including Korean cabbage ( subsp. ). The interaction between and is a representative model system, and objective estimation of disease progression is indispensable for accurate functional analyses. Five strains caused black spot symptom progression on Korean cabbage and ecotype Col-0. In particular, challenge with the strains Ab44877 and Ab44414 induced severe black spot progression on Korean cabbage. Ab44877 was also highly infective on Col-0; however, the virulence of Ab44414 and the remaining strains on Col-0 was lower. To unveil the relationship between mycelial growth in the infected tissues and symptom progression, we have established a reliable quantification method using real-time PCR that employs a primer pair and dual-labelled probe specific to a unigene encoding (), which is involved in fungal melanin biosynthesis. Plotting the crossing point values from the infected tissue DNA on a standard curve revealed active fungal ramification of Ab44877 in both host species. In contrast, the proliferation rate of Ab44414 in Korean cabbage was 3.8 times lower than that of Ab44877. Massive infective mycelial growth of Ab44877 was evident in Col-0; however, inoculation with Ab44414 triggered epiphytic growth rather than actual ramification. Mycelial growth did not always coincide with symptom development. Our quantitative evaluation system is applicable and reliable for the objective estimation of black spot disease severity.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.068205-0
2013-09-01
2021-08-05
Loading full text...

Full text loading...

/deliver/fulltext/micro/159/9/1946.html?itemId=/content/journal/micro/10.1099/mic.0.068205-0&mimeType=html&fmt=ahah

References

  1. Ahn I.-P., Kim S., Kang S., Suh S.-C., Lee Y.-H. ( 2005). Rice defense mechanisms against Cochliobolus miyabeanus and Magnaporthe grisea are distinct. Phytopathology 95:1248–1255 [View Article][PubMed]
    [Google Scholar]
  2. Baumgartner K., Bhat R., Fujiyoshi P. ( 2010). A rapid infection assay for Armillaria and real-time PCR quantitation of the fungal biomass in planta. . Fungal Biol 114:107–119 [View Article][PubMed]
    [Google Scholar]
  3. Bretagne S., Costa J. M., Marmorat-Khuong A., Poron F., Cordonnier C., Vidaud M., Fleury-Feith J. ( 1995). Detection of Aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR. J Clin Microbiol 33:1164–1168[PubMed]
    [Google Scholar]
  4. Brouwer M., Lievens B., Van Hemelrijck W., Van den Ackerveken G., Cammue B. P. A., Thomma B. P. H. J. ( 2003). Quantification of disease progression of several microbial pathogens on Arabidopsis thaliana using real-time fluorescence PCR. FEMS Microbiol Lett 228:241–248 [View Article][PubMed]
    [Google Scholar]
  5. Entz S. C., Johnson D. L., Kawchuk L. M. ( 2005). Development of a PCR-based diagnostic assay for the specific detection of the entomopathogenic fungus Metarhizium anisopliae var. acridum. . Mycol Res 109:1302–1312 [View Article][PubMed]
    [Google Scholar]
  6. Fan C. Y., Köller W. ( 1998). Diversity of cutinases from plant pathogenic fungi: differential and sequential expression of cutinolytic esterases by Alternaria brassicicola. . FEMS Microbiol Lett 158:33–38 [View Article]
    [Google Scholar]
  7. Färber P., Geisen R., Holzapfel W. H. ( 1997). Detection of aflatoxinogenic fungi in figs by a PCR reaction. Int J Food Microbiol 36:215–220 [View Article][PubMed]
    [Google Scholar]
  8. Feinberg A. P., Vogelstein B. ( 1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13 [View Article][PubMed]
    [Google Scholar]
  9. Fierer N., Jackson J. A., Vilgalys R., Jackson R. B. ( 2005). Assessment of soil microbial community structure by use of taxon-specific quantitative PCR assays. Appl Environ Microbiol 71:4117–4120 [View Article][PubMed]
    [Google Scholar]
  10. Francis M., Lin H., Cabrera-La, Rosa J., Doddapaneni H., Civerolo E. L. ( 2006). Genome-based PCR primers for specific and sensitive detection and quantification of Xylella fastidiosa. . Eur J Plant Pathol 115:203–213 [View Article]
    [Google Scholar]
  11. Gachon C., Saindrenan P. ( 2004). Real-time PCR monitoring of fungal development in Arabidopsis thaliana infected by Alternaria brassicicola and Botrytis cinerea. . Plant Physiol Biochem 42:367–371 [View Article][PubMed]
    [Google Scholar]
  12. Gunel T., Kalelioglu I., Surmeli Y., Turken B., Ermis H., Aydinli K. ( 2011). Comparison of real-time polymerase chain reaction assay methods for detection of RHD gene in amniotic fluid. J Nat Sci Biol Med 2:193–197[PubMed] [CrossRef]
    [Google Scholar]
  13. Van Brouwershaven I. R., Bruil M. L., Van Leeuwen G. C. M., Kox L. F. F. ( 2010). A real time (TaqMan) PCR assay to differentiate Monilinia fructicola from other brown rot fungi of fruit crops. Plant Pathol 59:548–555 [View Article]
    [Google Scholar]
  14. Harrison J. G., Barker H., Lowe R., Rees E. A. ( 1990). Estimation of amounts of Phytophthora infestans mycelium in leaf tissue by enzyme-linked immunosorbent assay. Plant Pathol 39:274–277 [View Article]
    [Google Scholar]
  15. Hoffman T., Schmidt J. S., Zheng X., Bent A. F. ( 1999). Isolation of ethylene-insensitive soybean mutants that are altered in pathogen susceptibility and gene-for-gene disease resistance. Plant Physiol 119:935–950 [View Article][PubMed]
    [Google Scholar]
  16. Jarnagin J. L., Harris S. K. ( 1985). A modified plastic culture flask for microscopic observation of fungi. Mycopathologia 90:55–58 [View Article]
    [Google Scholar]
  17. Jurado M., Vázquez C., Marín S., Sanchis V., Teresa González-Jaén M. ( 2006). PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in maize. Syst Appl Microbiol 29:681–689 [View Article][PubMed]
    [Google Scholar]
  18. Kirk P. M., Cannon P. F., Minter D. W., Stalpers J. A. ( 2008). Dictionary of the Fungi, 10th edn. Wallingford: CABI;
    [Google Scholar]
  19. Kolb S., Knief C., Stubner S., Conrad R. ( 2003). Quantitative detection of methanotrophs in soil by novel pmoA-targeted real-time PCR assays. Appl Environ Microbiol 69:2423–2429 [View Article][PubMed]
    [Google Scholar]
  20. Kulik T. ( 2011). Development of TaqMan assays for 3ADON, 15ADON and NIV Fusarium genotypes based on Tri12 gene. Cereal Res Commun 39:200–214 [View Article]
    [Google Scholar]
  21. Mandal P. K., Biswas A. K., Choi K., Pal U. K. ( 2011). Methods for rapid detection of foodborne pathogens: an overview. Am J Food Technol 6:87–102 [View Article]
    [Google Scholar]
  22. Martin F., Delaruelle C., Hilbert J.-L. ( 1990). An improved ergosterol assay to estimate fungal biomass in ectomycorrhizas. Mycol Res 94:1059–1064 [View Article]
    [Google Scholar]
  23. Matsenko N. U., Rijikova V. S., Kovalenko S. P. ( 2008). Comparison of SYBR Green I and TaqMan real-time PCR formats for the analysis of her2 gene dose in human breast tumors. Bull Exp Biol Med 145:240–244 [View Article][PubMed]
    [Google Scholar]
  24. Mühling M., Woolven-Allen J., Murrell J. C., Joint I. ( 2008). Improved group-specific PCR primers for denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities. ISME J 2:379–392 [View Article][PubMed]
    [Google Scholar]
  25. Nicholson P., Rezanoor H. N., Simpson D. R., Joyce D. ( 1997). Differentiation and quantification of the cereal eyespot fungi Tapesia yallundae and Tapesia acuformis using a PCR assay. Plant Pathol 46:842–856 [View Article]
    [Google Scholar]
  26. Nowicki M., Nowakowska M., Niezgoda A., Kozik E. ( 2012). Alternaria black spot of crucifers: symptoms, importance of disease, and perspectives of resistance breeding. Vegetable Crops Res Bull 76:5–19 [View Article]
    [Google Scholar]
  27. Otani H., Kohmoto K., Kodama M. ( 1995). Alternaria toxins and their effects on host plants. Can J Bot 73:Suppl. 1453–458 [View Article]
    [Google Scholar]
  28. Pavón M. A., González I., Martín R., García Lacarra T. ( 2012). ITS-based detection and quantification of Alternaria spp. in raw and processed vegetables by real-time quantitative PCR. Food Microbiol 32:165–171 [View Article][PubMed]
    [Google Scholar]
  29. Peng Y.-L., Shishiyama J., Yamamoto M. ( 1986). A whole-leaf staining and clearing procedure for analyzing cytological aspects of interaction between rice plant and rice blast fungus. Jap J Phytopathol 52:801–808 [View Article]
    [Google Scholar]
  30. Pfaffl M. W. ( 2004). Quantification strategies in realtime PCR. A–Z of Quantitative PCR87–112 Bustin S. A. La Jolla, CA: International University Line;
    [Google Scholar]
  31. Rodu B., Christian C., Synder R. C., Ray R., Miller D. M. ( 1991). Simplified PCR-based detection and typing strategy for human papillomaviruses utilizing a single oligonucleotide primer set. Biotechniques 10:632–637[PubMed]
    [Google Scholar]
  32. Salvioli A., Lumini E., Anca I. A., Bianciotto V., Bonfante P. ( 2008). Simultaneous detection and quantification of the unculturable microbe Candidatus glomeribacter gigasporarum inside its fungal host Gigaspora margarita. . New Phytol 180:248–257 [View Article][PubMed]
    [Google Scholar]
  33. Southern E. M. ( 1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517 [View Article][PubMed]
    [Google Scholar]
  34. Stewart C. N. Jr, Via L. E. ( 1993). A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Biotechniques 14:748–750[PubMed]
    [Google Scholar]
  35. Su’udi M., Park J. M., Kang W. R., Park S. R., Hwang D. J., Ahn I.-P. ( 2012). Quantification of rice brown leaf spot through Taqman real-time PCR specific to the unigene encoding Cochliobolus miyabeanus SCYTALONE DEHYDRATASE1 involved in fungal melanin biosynthesis. J Microbiol 50:947–954 [View Article][PubMed]
    [Google Scholar]
  36. Taylor S. M., Juliano J. J., Trottman P. A., Griffin J. B., Landis S. H., Kitsa P., Tshefu A. K., Meshnick S. R. ( 2010). High-throughput pooling and real-time PCR-based strategy for malaria detection. J Clin Microbiol 48:512–519 [View Article][PubMed]
    [Google Scholar]
  37. Thomma B. P. H. J., Eggermont K., Tierens K. F. M.-J., Broekaert W. F. ( 1999). Requirement of functional ethylene-insensitive 2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea. . Plant Physiol 121:1093–1101 [View Article][PubMed]
    [Google Scholar]
  38. Thornton C. R., Slaughter D. C., Davis R. M. ( 2010). Detection of the sour-rot pathogen Geotrichum candidum in tomato fruit and juice by using a highly specific monoclonal antibody-based ELISA. Int J Food Microbiol 143:166–172 [View Article][PubMed]
    [Google Scholar]
  39. Wheeler M. H. ( 1983). Comparisons of fungal melanin biosynthesis in ascomycetous, imperfect and basidiomycetous fungi. Trans Br Mycol Soc 81:29–36 [View Article]
    [Google Scholar]
  40. Wight W. D., Kim K. H., Lawrence C. B., Walton J. D. ( 2009). Biosynthesis and role in virulence of the histone deacetylase inhibitor depudecin from Alternaria brassicicola. . Mol Plant Microbe Interact 22:1258–1267 [View Article][PubMed]
    [Google Scholar]
  41. Yin J. L., Shackel N. A., Zekry A., McGuinness P. H., Richards C., Van Der Putten K. , McCaughan G. W., Eris J. M., Bishop G. A. ( 2001). Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I. Immunol Cell Biol 79:213–221 [View Article][PubMed]
    [Google Scholar]
  42. Zhao S., Clarke B. B., Shen Q. R., Zhang L. S., Zhang N. ( 2012). Development and application of a TaqMan real-time PCR assay for rapid detection of Magnaporthe poae. . Mycologia 104:1250–1259 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.068205-0
Loading
/content/journal/micro/10.1099/mic.0.068205-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error