Presymptomatic and accurate diagnosis of Mycosphaerella graminicola leaf blotch is desirable for the disease prediction and the timely application of fungicides. To develop a sensitive PCR assay, four specific primer pairs were designed. They were more specific than three known specific primer pairs. Three of them could detect as little as 0.5 pg M. graminicola DNA in a conventional PCR. A real‐time PCR assay was applied for monitoring the disease progression in both inoculated and naturally infected wheat plants using the primer pair ST‐rRNA F/R. In inoculated plants, M. graminicola DNA could be detected immediately after inoculation and a steady increase was detected before visible symptoms appeared at 8 days. The rapid growth period took place between 6 and 16 days postinoculation. In the field, the disease progression in the top three leaf layers was followed during the epidemic period. The results were significantly correlated to the disease indices (R=0.8986) and also to the number of pycnidia per leaf (R=0.9227). These suggest that the real‐time PCR assay is a reliable approach for the presymptomatic and accurate detection of M. graminicola development in the field.

A modified procedure using the commercial DNAzol reagent was successfully applied to extract genomic DNA from 25 fungal species. The DNA yield varied from 306 to 1,927 microg g(-1) dry mycelia and the A(260)/A(280) ratio from 1.59 to 1.93. Compared with the method of J.L. Cenis (Nucleic Acids Res. 1992, 20: 2380) this procedure generated a higher DNA yield from 17 species and a higher A(260)/A(280) ratio from 23 species. But for four species, Cenis (1992) method was more suitable. No inhibitor of polymerase chain reaction was evident for the DNA extracted by the modified procedure, whereas some inhibitors remained in DNA of eight species extracted by the previous method.

Mycosphaerella graminicola is a prevalent and economically important fungal pathogen in wheat. Presymptomatic and accurate detection of viable pathogen structures in infected host plants is desirable for determining latent periods of epidemics and for timely fungicide application. A reverse transcription polymerase chain reaction (RT-PCR) assay was used to detect and identify M. graminicola in wheat with the specific primer set E1/STSP2R. One single fragment was amplified only from total RNA of M. graminicola and infected leaves, but not from those of healthy leaves or five other common fungal wheat pathogens. The sensitivity of one-step RT-PCR was compared with that of two-step RT-PCR. The detection limit of two-step RT-PCR was markedly lower (100 pg total RNA) than that of one-step RT-PCR (5 ng total RNA). RT-PCR was used to monitor M. graminicola disease development in inoculated wheat plants and in naturally infected F 1 leaves (the second leaf from top) during the epidemic period. RT-PCR could detect M. graminicola at least 4 days before lesions appeared in inoculated plants. After symptoms were visible on the leaves, the band intensities of the amplified RT-PCR products were in general agreement with the visual disease assessment.www.blackwell-synergy.com

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We have modified a quick, inexpensive and less prone to contamination protocol by culturing the cryopreserved or fresh mycelium directly in 96deepwell plate. The method facilitates concom*tant assessment of ssDNA fungal diversity by asymmetric PCR (A-PCR) from a single extraction. The DNA yields from Fusarium spp. isolates was reasonably high, and a clear DNA band was frequently seen when I0 pL. of the PCR product was run in agarose gel. The procedure can be completed in less than 4 h and 96 samples can be processed at the same time.