3.3.32
DETECTION OF PHYTOPHTHORA INFESTANS OOSPORES FROM SOILS IN MEXICO

A FLORES-OLIVAS, E GOMEZ-DELGADO, N GRUNWALD and V OLALDE-PORTUGAL

1Unidad de Biotecnolog’a e Ingenier’a Genetica de Plantas, CINVESTAV-IPN U. Irapuato, PO Box 629, Irapuato, Guanajuato, Mexico; 2PICTIPAPA, PO Box 12-2, Conjunto SEDAGRO, Metepec, CP52142, Edo. de Mexico, Mexico

Background and objectives
The potato late blight caused by the fungus Phytophthora infestans, has its origin in central Mexico where both A1 and A2 mating types were found since the 1950s. From the two, only the A2 was found exclusively confined to Mexico until its rapid widespread around the world during the 1980s. This fact has drawn a great attention to study the oospores impact in the late blight epidemics under natural conditions (1). Current information of the P. infestans oospores role as inoculum source, generation of pathogenic strains and fungicides resistant strain in Mexico is scarce. For this reason our research objective is the P. infestans oospores detection, in order to determine epidemiological studies, using microbiological, serological and molecular techniques.

Materials and methods
Fifty one isolates of P. infestans from diverse geographical regions, mainly from the Toluca Valley in Mexico were used in this study. The isolates were cultured using rye B agar or clarified V-8 juice agar at 18 *C in the dark. Mating types were identified by pairing isolates from A1 and A2 reference strains. In vitro oospores were induced by pairing isolates of opposite compatibility type. Oospores were extracted from isolates by mixing and homogenizing mycelia and oospores into destilled water. The mixture was sieved using a 38 micron diameter mesh, and centrifugated at 3000 rpm in 60, 50, and 25% of sucrose gradient concentration during ten minutes. The oospores were extracted of the gradient. Fifty milliliters with four thousand oospores per milliliter were added into sterilized soil and soil from a field where potato was cultured and with high late blight incidence. Both type of soils were processed by glycerol flotation and sucrose gradient to extract oospores. PCR amplification of oospores DNA was performed using primers already published (2).

Results and conclusions
Isolates from Central Mexico were 41% and 59 % from A1 and A2 respectively. All 100% isolates from northern Mexico were A2 and 100% isolates eastearn Mexico were A1. All fifty paired isolates produced oospores but when the A2 mating type from Toluca Valley was used, more oospores were significantly produced. Sieving, glycerol flotation and centrifugation in 60% sucrose gradient proved to be efficient to obtain oospores from soil. PCR using of Phytophthora infestans oospores isolated directly from soil has been a difficult but not an impossible task. Optimization of PCR and serological techniques to overcome these difficulties are being carried out in our laboratory.

References
1. Andrivon, D. 1995. Phytopathology 85:1053-1056.
2. Niepold F, Schober-Butin B, 1995. Microbiol. Res. 150:379-385.