Development of species specific primer for the early detection of Cylindrocladium quinqueseptatum causing leaf and seedling bl

We developed PCR primers to detect Cylindrocladium quinqueseptatum which infect Eucalyptus causing leaf and seedling blight resulting in heavy seedling mortality in North Indian states. Primers based on sequence analysis of internal transcribed spacer region 1 and 5.8S of ribosomal DNA produced PCR product of 245bp. The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) sub unit repeat was sequenced in 26 isolates of Cylindrocladium quinqueseptatum and sequences were aligned and compared with the ITS sequences of other fungi in GenBank. No amplification resulted from PCR reactions on fungal DNA from 6 common forest fungi, 10 soil contaminates and 6 Eucalyptus pathogens. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was done. First, the entire ITS was amplified with universal fungal primer; a second round of amplification was carried out with species specific primer that amplified a 245 bp PCR product. The method detected leaf and seedling blight in artificially and naturally infected Eucalyptus plants. From the soil also the pathogen was detected using species specific primer. In sampling studies, C. quinqueseptatum was detected by PCR from artificially infected seedlings at 6 days post inoculation, before any visible symptoms were present. The PCR assay and direct tissue extraction methods provide tools which may be used to detect C. quinqueseptatum from soil, plant cuttings and adjoining Eucalyptus plantations serving as recurring source of infection and thus limit the transmission and spread of new aggressive strains of C. quinqueseptatum in Eucalyptus growing regions of India.