Black spot caused by Alternaria brassicicola is an important fungal disease affecting cruciferous crops, including Korean cabbage (Brassica rapa subsp. pekinensis). The interaction between Arabidopsis thaliana and Alt. brassicicola 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 Ara. thaliana 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 A. brassicicola SCYTALONE DEHYDRATASE1 (AbSCD1), 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 in planta 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.
BaumgartnerK.,
BhatR.,
FujiyoshiP.(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]
BretagneS.,
CostaJ. M.,
Marmorat-KhuongA.,
PoronF.,
CordonnierC.,
VidaudM.,
Fleury-FeithJ.(1995). Detection of Aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR. J Clin Microbiol 33:1164–1168[PubMed]
BrouwerM.,
LievensB.,
Van HemelrijckW.,
Van den AckervekenG.,
CammueB. P. A.,
ThommaB. 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]
EntzS. C.,
JohnsonD. L.,
KawchukL. 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]
FanC. Y.,
KöllerW.(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]
FärberP.,
GeisenR.,
HolzapfelW. H.(1997). Detection of aflatoxinogenic fungi in figs by a PCR reaction. Int J Food Microbiol 36:215–220 [View Article][PubMed]
FeinbergA. P.,
VogelsteinB.(1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13 [View Article][PubMed]
FiererN.,
JacksonJ. A.,
VilgalysR.,
JacksonR. 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]
GachonC.,
SaindrenanP.(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]
Van BrouwershavenI. R.,
BruilM. L.,
Van LeeuwenG. C. M.,
KoxL. 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]
KulikT.(2011). Development of TaqMan assays for 3ADON, 15ADON and NIV Fusarium genotypes based on Tri12 gene. Cereal Res Commun 39:200–214 [View Article]
MandalP. K.,
BiswasA. K.,
ChoiK.,
PalU. K.(2011). Methods for rapid detection of foodborne pathogens: an overview. Am J Food Technol 6:87–102 [View Article]
MartinF.,
DelaruelleC.,
HilbertJ.-L.(1990). An improved ergosterol assay to estimate fungal biomass in ectomycorrhizas. Mycol Res 94:1059–1064 [View Article]
MatsenkoN. U.,
RijikovaV. S.,
KovalenkoS. 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]
NicholsonP.,
RezanoorH. N.,
SimpsonD. R.,
JoyceD.(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]
NowickiM.,
NowakowskaM.,
NiezgodaA.,
KozikE.(2012).Alternaria black spot of crucifers: symptoms, importance of disease, and perspectives of resistance breeding. Vegetable Crops Res Bull 76:5–19 [View Article]
PavónM. A.,
GonzálezI.,
MartínR.,
García LacarraT.(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]
PengY.-L.,
ShishiyamaJ.,
YamamotoM.(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]
RoduB.,
ChristianC.,
SynderR. C.,
RayR.,
MillerD. M.(1991). Simplified PCR-based detection and typing strategy for human papillomaviruses utilizing a single oligonucleotide primer set. Biotechniques 10:632–637[PubMed]
SalvioliA.,
LuminiE.,
AncaI. A.,
BianciottoV.,
BonfanteP.(2008). Simultaneous detection and quantification of the unculturable microbe Candidatusglomeribactergigasporarum inside its fungal host Gigaspora margarita.
. New Phytol 180:248–257 [View Article][PubMed]
SouthernE. M.(1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517 [View Article][PubMed]
StewartC. N.Jr,
ViaL. E.(1993). A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Biotechniques 14:748–750[PubMed]
Su’udiM.,
ParkJ. M.,
KangW. R.,
ParkS. R.,
HwangD. J.,
AhnI.-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]
ThommaB. P. H. J.,
EggermontK.,
TierensK. F. M.-J.,
BroekaertW. 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]
ThorntonC. R.,
SlaughterD. C.,
DavisR. 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]
WheelerM. H.(1983). Comparisons of fungal melanin biosynthesis in ascomycetous, imperfect and basidiomycetous fungi. Trans Br Mycol Soc 81:29–36 [View Article]
WightW. D.,
KimK. H.,
LawrenceC. B.,
WaltonJ. 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]
YinJ. L.,
ShackelN. A.,
ZekryA.,
McGuinnessP. H.,
RichardsC.,
Van Der PuttenK. ,
McCaughanG. W.,
ErisJ. M.,
BishopG. 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]
ZhaoS.,
ClarkeB. B.,
ShenQ. R.,
ZhangL. S.,
ZhangN.(2012). Development and application of a TaqMan real-time PCR assay for rapid detection of Magnaporthe poae.
. Mycologia 104:1250–1259 [View Article][PubMed]