AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA ISSN Print: 2151-7517, ISSN Online: 2151-7525, doi:10.5251/abjna.2010.1.6.1253.1259 © 2010, ScienceHuβ, http://www.scihub.org/ABJNA Development of species specific primer for the early detection of Cylindrocladium quinqueseptatum causing leaf and seedling blight in Eucalyptus Amit Pandey, Partha Sarathi Mohanty and Pooja Arya Forest Pathology Division, Forest Research Institute, Dehradun, INDIA ABSTRACT 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. Keywords: Cylindrocladium quinqueseptatum, internal transcribed spacer, nested PCR INTRODUCTION: Large scale seedling mortality in Eucalyptus nurseries of North Indian states caused by Cylindrocladium quinqueseptatum has posed serious threat to paper and pulp industries of this region fearing decline in production, as majority of industry- sponsored nurseries raised by local farmers for achieving the plantation targets have witnessed this disease in epipytotic proportions in the recent years. Major losses in planting stock material is expected in future also if new advances in control and detection technologies are not made. Worst affected North Indian states are Uttarakhand, Punjab and Haryana. Losses from Cylindrocladium quinqueseptatum seedling blight is due to out right killing of the entire seedling within a couple of weeks time during monsoon season and blightening of leaves in plantations thus adversely affecting the tree growth. Apparently healthy seedlings develop blight symptoms and die quickly in short span of 8 to 15 days leaving little time for the application of remedial measures. The three sources of inoculum are the perennating C. quinqueseptatum in plant debris of nursery soil, infected propagules and the adjoining Eucalyptus trees harboring pathogen on their twigs and leaves. The seedlings raised in the subsequent years eventually suffer heavy losses with the every passing year due to increase in inoculum build up. The severity of recent losses and the fact that the infected planting materials are transported across the three states of northern India viz. Punjab, Haryana and Uttarakhand in different plantation programms may have served as the primary source of inoculum, has necessitated the development of rapid and sensitive method for detection of leaf and twig blight of Eucalyptus in early stages, preferably prior to nursery raising and before the onset of monsoon. Sites selection for raising nurseries also needs to be checked for residual presence of pathogen over wintering in the soil and on surrounding Eucalyptus plantations. Such a test would allow the determination of levels of infection in host propagules, nursery soil and adjoining Eucalyptus plantations which are being utilized for the production or as source material for raising planting stock. Among the nursery managers and farmers, currently the disease detection is based solely on visual inspection of seedlings, a procedure in which infected but symptomless Eucalyptus saplings escape