R.C. Shattock

Variation in DNA content of nuclei of Phytophthora infestans as measured by a microfluorimetric method using the fluorochrome DAPI

Staining with the fluorochrome DAPI was investigated as an alternative to Feulgen staining for measuring DNA contents of zoospore nuclei. Five isolates of P. infestans with variable DNA content gave the same rank order for both methods. DNA contents of hyphal nuclei had similar mean values to those of zoospore nuclei indicating that zoospore nuclei are unreplicated and not in synthesis phase. Both A1 and A2 isolates from selected countries showed a range of DNA contents from a basic 2C level, presumed diploid, through intermediate quantities to 4C, presumed tetraploid. A similar variation was seen in freshly acquired British field isolates. Evidence was produced that one isolate having a bimodal distribution of DNA contents was a heterokaryon with unreplicated nuclei of 2C and 4C values. Chromosome counts on DAPI stained meiotic metaphases of gametangia from 2C × 2C and 4C × 4C matings indicated that 2C isolates had n = 8–12 and 4C isolates had n = 14–20. Thus isolates with approximately 4C values were shown to be tetraploid.

AFLP and RFLP (RG57) fingerprints can give conflicting evidence about the relatedness of isolates of Phytophthora infestans

Selected isolates of Phytophthora infestans from around England and Wales were fingerprinted using both RG57, a multi-locus RFLP probe, and Amplified Fragment Length Polymorphisms (AFLPs). The larger number of polymorphisms detectable with the AFLP method allowed resolution of several similar AFLP genotypes among isolates with identical RG57 fingerprints. However, some isolates with the same RG57 genotype had remarkably dissimilar AFLP genotypes, suggesting that there has been convergent evolution of some RG57 fingerprints. Also, some isolates with dissimilar RG57 fingerprints had similar or identical AFLP fingerprints. Both techniques distinguished isolates of mitochondrial DNA haplotype la from those of haplotype IIa. However, with AFLPs only, most of the isolates of A2 mating type were very similar and were distinguished from those of A1 mating type, suggesting that gene flow between A1 and A2 genotypes is limited and that sexual recombination is rare.

Inheritance of DNA contents in sexual progenies of Phytophthora infestans

Sexual progeny were raised from matings between 2C (diploid) × 4C (tetraploid), 2C × 2C and 4C × 4C isolates of Phytophthora infestans. The percentage germination of oospores and establishment of colonies were generally lower in the 2C × 4C and 4C × 4C crosses than in the 2C × 2C cross. Isoenzyme analysis of progeny from some matings revealed that most were hybrids but that the proportion of selfs was generally higher in 2C × 4C crosses. The fluorochrome DAPI was used to determine DNA contents in samples of all progenies. Hybrids from the 2C × 2C cross were all 2C whereas those from 2C × 4C crosses ranged from 2C to > 3C. Progenies from the 4C × 4C crosses ranged from 2C to 4C.

A histological study of downy mildew (Peronospora rubi) infection of leaves, flowers and developing fruits of Tummelberry and other Rubus spp.

Genotypes of Rubus spp. and some of their hybrids, grown in vitro, in a plastic tunnel and in the field were inoculated with Peronospora rubi or exposed to inoculum of this pathogen to study histologically the infection of leaves, flowers, developing fruits and stems. Samples were fixed, stained and mounted in aniline blue and examined by fluorescence or differential interference contrast microscopy. Conidia germinated on adaxial and abaxial leaf surfaces and penetrated the epidermal cell walls to enter the mesophyll. Points of penetration were stained intensely, probably due to callose formation by the host. Some germ-tubes infected leaves by entering the stoma, but this was less common than direct penetration. Intercellular hyphae colonized the mesophyll extensively and formed simple dichotomously branched haustoria. Spread of the pathogen into the vascular tissues of veins was restricted and accounted for the interveinal distribution of lesions. Hyphae which entered the veins and petioles and spread to the cortical tissues of the stem tended to produce more complex haustoria, but less frequently than in other tissues. In petioles and veins of leaves, fanshaped laterally fused hyphal branches (fasciated hyphae) formed in intercellular spaces and evidence was obtained that anastomosis may have occurred between hyphae growing parallel within these tissues. Oogonia with paragynous antheridia and thick-walled aplerotic oospores developed abundantly in leaves grown in vitro. Oogonia also developed in petals and stamens from flowers of Tummelberry grown in the field and spray-inoculated in the laboratory. An extensive network of mycelium developed in the mesocarp and the receptacle of developing fruits.

The duplication cycle and DAPI-DNA contents in nuclei of germinating zoospore cysts of Phytophthora infestans

The duplication cycle of nuclei in germinating zoospores of Phytophthora infestans was determined. One isolate, a presumed diploid, had a duplication cycle of 7–8 h, whereas a presumed tetraploid isolate had a cycle of 10–12 h. Nuclei from uninucleate germinating zoospore cysts, just prior to the first nuclear division, had twice the DAPI-DNA contents of nuclei from postmitotic binucleate germlings and of nuclei from zoospores. Isolated nuclei from zoospores, therefore, are at G1 in the duplication cycle.