2.4.16
USING MOLECULAR METHODS TO STUDY THE EPIDEMIOLOGY OF NECTRIA GALLIGENA IN APPLE ORCHARDS

CV WALLIS1, S LANGRELL3, AM BERRIE1, DJ BARBARA2, AR McCRACKEN4, 1 KOOMEN5, T LOCKE6 and TR SWINBURNE3

1HRI-East Mailing, West Mailing, Kent, ME19 6BJ, UK; 2HRI Wellesbourne, Wellesbourne, Warwick, CV35 9EF, UK; 3Wye College, Wye, Ashford, Kent, TN25 5AH, UK; 4Queens University of Belfast, Newforge Lane, Belfast, BT9 5PX, UK; 5ADAS, Olantigh Road, Wye, Ashford, Kent, TN25 5EL, UK; 6ADAS Rosemaund, Preston Wynne, Hereford, HR1 3PG, UK

Background and objectives
Canker caused by the fungus Nectria galligena is a destructive disease of apple and pear. Total losses of 5-10% of young trees can occur in areas where the disease is prevalent. The fungus enters plants mainly through wounds caused by pruning or natural openings, such as leaf or bud scale scars. N. ;galligena also causes a fruit rot in the orchard and in storage and losses up to 5000 per 100 tons are possible. Recent results [1] suggest that infection by N. ;galligena arising in the nursery may be an important source of canker, particularly in young orchards and this may be a factor in the poor disease control achieved by some growers. However, there is good evidence that disease transmission by spores remains an important component of canker and fruit rot epidemiology [2]. The objective of this collaborative study was to determine the relative importance of infection of trees in the nursery compared with aerial spread in the orchard in terms of the epidemiology of apple canker. Information about the initial sources of infection and subsequent spread is essential for developing more effective control strategies. This work is being funded by MAFF, APRC and DANI.

Materials and methods
Young orchards for the study were selected from farms with a high incidence of canker. Samples were collected on the basis of variety, age of wood and orchard position. The fungus was isolated from cankered wood using selective media. Total DNA was extracted from mycelia grown on cellophane discs over potato dextrose agar. The intergenic spacer (IGS) region of N. ;galligena isolates was amplified using primers developed by Langrell (pers. comm.). The PCR products were digested with four restriction enzymes: HinfI, HhaI, HaeIII, and TaqI. Restriction fragments were separated on an agarose gel and fragment profiles were obtained for individual isolates of N. ;galligena.

Results and conclusions
Cankers were collected from three areas of a young orchard of about 4 ;years old, consisting of the cultivars Cox and Gala of known nursery origin. Samples were also collected from an old cankered pear orchard and an old cankered 'Cox'/'Spartan' orchard, of different nursery origin, adjacent to the young orchard. N. ;galligena was isolated from approximately 50% of the cankers collected and DNA profiles were obtained. Comparison of the populations obtained within and at the interface of these orchards will enable an estimate to be made of the relative frequencies of infections originating in the nursery and those arising from tree-to-tree transmission.

References
1. Brown AE, Muthumeenakshi S, Swinburne TR. Li R, 1994. Plant Pathology 43, 338-343.
2. Swinburne TR, 1975. Review of Plant Pathology 54, 787-799.