3.3.58
PCR-BASED DETECTION OF PHYTOPHTHORA FRAGARIAE VAR. RUBI IN RASPBERRY ROOTS IN RELATION TO DISEASE DEVELOPMENT
PCR-BASED DETECTION OF PHYTOPHTHORA FRAGARIAE VAR. RUBI IN RASPBERRY ROOTS IN RELATION TO DISEASE DEVELOPMENT

A J INMAN1, KJD HUGHES1, PA BEALES1, RTCOOK,CFULTON2,ADKMcREYNOLDS3


1 Central Science Laboratory, MAFF, York Y04 I LZ; 2 The Queens University of Belfast, Agriculture & Food Science Centre, BT9 5PX 3 Department of Agriculture for Northern Ireland, Belfast, BT9 5PX

Background and objectives

Raspberry root rot (Phytophthora fragariae var. rubi) is the most important disease of raspberries in Europe. Recently, PCR protocols have been developed for screening planting material for low levels of the pathogen in roots of plants showing no aerial symptoms [1]. Such PCR methods have proved more rapid and reliable than traditional bait tests. This study investigated the ability of PCR to detect the pathogen during the course of disease development and in relation to the development of fungal structures within roots.

Materials and methods

Individual raspberry plants, cv. Glen Moy, were inoculated in a I 50C growth room by eluting the soil of each pot with c. 100 ml spore suspension on each of three consecutive days (spore concentrations in zoospores lml: first inoculation, 1900; second inoculation 4200; third inoculation 15700). Zoospores were obtained by floating 5 mm agar plugs, taken from the margins of French bean agar cultures, in sedge peat water (II water eluted through 1 kg sedge peat). Uninoculated plants acted as healthy controls. Five plants were destructively sampled on each of the following days after the last inoculation: 1, 2, 3, 5, 8, 12, 16, 20, 25, 30, 35, 40 and 50. The roots were washed free of soil and frozen prior to PCR. At the same time, some roots were examined microscopically for the presence of coenocytic mycelium and reproductive structures. DNA was extracted for PCR using a modified CTAB extraction method. The purified DNA sample, together with a tenfold and a hundredfold dilution, were tested by PCR using a primer designed from the rDNA 1T51 region (5' TCGATGTCAAACTTGA 3') [2] and the primer 1T54 (5' TCCTCCGCTTAUGATATGC 3'). GeneReleaser (Bioventures, Inc.) was added to the reaction tubes to prevent PCR inhibition. The product of PCR was approximately an 800 base pair fragment. Quantification of the amount of DNA product was estimated by comparison with a low DNA mass ladder (Gibco).

Results and conclusions

lnfection in roots was detected by PCR within one day of the inoculations. This correlated with the observed presence of coenocytic mycelium in the roots. Infection could still be detected at 40 days, but not at 50. The greatest levels of detection occurred between 1-8 days, with the amount of PCR product generally declining after 12 days. Levels of detection corresponded well with observed changes in fungal structures in roots. Coenocytic mycelium was found abundantly from 1-12 days but appeared to degenerate after sexual reproduction occurred, being seen only occasionally from 16-20 days, and rarely thereafter. In comparison, oogonia were present from 3-8 days and only occasionally at 12 days. Oospores began to form within 5 days and almost all developed thick walls within 8-12 days. The decrease in detection with time reflected the degeneration of coenocytic mycelium occurring after the switch from active pathogenesis to sexual reproduction, rather than any increase in PCR inhibition with time. Clearly, once the coenocytic mycelium degenerates, most of the fungal DNA in the root is locked-up inside thick walled oospores. Since oospores are not easily cracked open by conventional methods, the target DNA remains unavailable for PCR. Novel methods for cracking open oospores are currently under evaluation.

References

1. Lacourt I, Cooke DEL, Duncan JM. 1996. BCPC Symposium Proceedings: Diagnostics in Crop Production, No.65, 145-149. 2. Fulton C. 1992. BSc (Hons) Project: Queens University of Belfast.