2.4.9
IN PLANTA PRODUCTION, SURVIVAL AND GERMINATION OF OOSPORES OF PHYTOPHTHORA INFESTANS

LC BAINES1, RC SHATTOCK1, RA BARDSLEY2

lSchool of Biological Sciences, University of Wales, Bangor, Gwynedd, LL57 2UW, UK; 2AgrEvo UK Ltd, Chesterford Park, Saffron Walden, Essex, CB10 1XL, UK

Background and objectives
Phytophthora infestans readily reproduces sexually in vitro when the two mating types, Al and A2 are paired. The significance of the sexual oospore and its main effect on potato production is still unclear. Oospores are produced in leaves and stems of both potato and tomato plants when inoculated with a mixture of sporangia of the Al and A2 mating types [1]. Oospores are also able to remain viable in soil, when subjected to natural weathering, for at least 10 months, retaining the ability to infect detached potato leaves [2]. This study aims to show that oospores can be formed in planta, with the Al and A2 isolates originating from independent foci. Further, it is the aim of this study to show that, once formed, the oospores can overwinter and remain viable in soil until the following season.

Materials and methods
Sixty-four potato plants were grown in 30-cm pots, in a polythene tunnel, during August and September 1997. The pots were arranged in a 16X4 row arrangement. After 8 ;weeks, two pots, one from each end of the trial were inoculated with an Al or A2 isolate. The crop was misted periodically, with the aid of a leaf wetness indicator to maintain free water within the crop, allowing the spread of the two isolates. The movement of disease through the crop was recorded daily. Samples of leaf material were taken as the disease progressed, until complete necrosis of the haulm occurred. The samples taken were then transferred to rye agar to be assessed for mating type and a DNA fingerprint was determined (using probe RG-57). Leaves from selected plants were also collected to detect the production of oospores by microscopic observation. The viability of the oospores produced was assessed by placing the oospores in a 2 ;M solution of sodium chloride. Viable oospores undergo plasmolysis within 15 ;min. When all the plants were fully necrotized they were removed from the polythene tunnel and placed outside in plastic bags over the winter.

Results and conclusions
From inoculation to 100% necrosis of the crop took 18 ;days. The Al isolate was found to progress much more quickly that the A2, reaching almost all of the plants contained within the experiment. Conversely, the A2 isolate was found in only 22 of the plants, mostly centred around the original focus. Isolates taken from 11 of the 64 plants produced oospores in vitro. (i.e. Al and A2 were present in the same lesion.) In all but one of the 11 plants oospores were found in the leaves themselves. Approximately half of these oospores appeared to be viable when tested by plasmolysis.

It can be seen that the likelihood of Al and A2 isolates coming into contact is high if the conditions for spread are suitable. It has been shown that oospores can be formed in planta when the introduction of Al and A2 isolates is separated temporally and spatially. This is akin to a field situation, where it is unlikely that sporangia of two isolates would be introduced into a crop at the same time. It remains to be shown whether oospores formed in this a way are capable of overwintering and retaining their infection potential.

References
1. Frinking HD, Davidse LC, Limburg H, 1987. Netherlands Journal of Plant Pathology 93, 147-149.
2. Drenth A, Janssen EM, Govers F, 1995. Plant Pathology 44,86-94.