A Real-time PCR Assay for Quantitative and Accurate Assessment of Fungicide Effects on Mycosphaerella graminicola Leaf Blotch

A real-time PCR assay was applied to evaluate the effects of two fungicide treatments on Mycosphaerella graminicola leaf blotch in the field and compared with two usual visual methods. The three fungicides Fandango, Input and Prosaro used in the two treatments showed high inhibitions against germ tube growth and conidial germination in vitro, suggesting that they had high curative and protective activities. In the field, the fungicide effect was assessed for two times on 17 May and 14 June, 2004. M. graminicola DNA, disease index and pycnidial number per leaf reduced obviously on most leaf layers after both treatments. According to the real-time PCR data, significant effects were still observed on leaf 3, 4 and 5 even after 25 days of treatment (17 May). And on 14 June, treatment A significantly decreased the DNA amount by 99.42, 87.68, 24.12 and 68.87% on leaf 1, 2, 3 and 4 and treatment B by 98.83, 98.12, 70.79 and 64.15% respectively. In contrast, effects could neither be assessed by disease classification on leaf 5 of treatment B on 17 May and leaf 3 and 4 of treatment A on 14 June and nor by pycnidia-counting method on leaf 3 and 5 of treatment B on 17 May and leaf 4 of both treatments on 14 June. The band intensity of final real-time PCR products could be hardly distinguished on agarose gel amongst those samples with disease index >2.7, although their initial DNA amounts quantified by real-time PCR were different, indicating the superiority of the real-time PCR assay over those PCR assays estimating initial DNA input with endpoint measurement. All this shows that the real-time PCR assay may serve as an alternative method for accurate assessment of the fungicide effects on M. graminicola leaf blotch