D.S. Shaw

Inheritance of mitochondrial DNA in Phytophthora infestans

Mature (three-week) and immature (five-day) sexual progeny were raised from two matings between isolates of Phytophthora infestans with 2C-DNA content (and therefore presumed diploids) but which differed for isozyme genotype and restriction fragment length polymorphisms (RFLPs) in their mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Approximately 90% of the progeny which were derived from mature oospores, but approximately 50% of the progeny from immature oospores, were classed as hybrids on the basis of their Gpi 1 genotype. All single-oospore cultures, whether derived from a mature or an immature oospore, contained a single mtDNA type from either the A1 or the A2 parent. All single-zoospore lines derived from a single germ sporangium of a germinating hybrid oospore also had the same mtDNA type, Gpi 1 genotype and mating type. These data suggest that mtDNA is inherited uniparentally in P. infestans with no evidence for segregation, elimination or recombination of types. Mating type segregated differently in hybrid offspring; those with type II mtDNA (from the A1 parent) had a preponderance of A1 mating type. Some nRFLPs also segregated unexpectedly in the population with type II mtDNA, suggesting that any bias in the segregation of mating type is caused by viability disturbance rather than linkage between mating type gene(s) and mtDNA.

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.

An analysis of self-fertility in field isolates of Phytophthora infestans

Some field isolates of Phytophthora infestans from England and Wales had a flat, adpressed, waxy appearance when grown in agar culture. These isolates formed very little aerial mycelium and few sporangia, but abundant oospores were formed on or below the agar surface. When these cultures were propagated following single-sporangium and single-hyphal tip isolation, white, fluffy colonies with aerial mycelium and abundant sporangia were formed. These propagations were usually self-sterile of either A1 or A2 mating type and did not form oospores. Occasionally a propagation from a single protoplasmic unit was self-fertile. When grown together self-sterile propagations of A1 and A2 mating type reformed self-fertile cultures. Self-fertile cultures have also been synthesised from A1 and A2 field isolates following co-cultivation on agar. Self-fertile isolates would therefore appear to be intimate mixtures of A1 and A2 hyphae. However, results also indicate that heterokaryons/heteroplasmons occurred at a low frequency. Other work on self-fertility reports less strict inhibition of sporangiogenesis and more frequent transmission of the character to asexual progeny.

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.

Self-fertility in Phytophthora infestans: heterokaryons segregate several phenotypes

Isolates from potato in an isolated garden in North Wales in 1995 consisted of essentially two distinct phenotypes. Phenotypes P1 and P2 , of A1 and A2 mating type respectively, had distinctive multi-locus (RG57) and telomeric RFLP fingerprints. In addition, about 6% of isolates forming abundant oospores were self-fertile and showed a combination of both fingerprints. Among a total of 40 single-hyphal-tip and single-sporangial propagations from two self-fertile isolates, 16 were P2 whereas only one propagation was P1 ; four propagations were still self-fertile and still carried the molecular markers typical of both P1 and P2. Most of the remainder were A1 but otherwise carried both P1 and P2 molecular markers. Uninucleate zoospore lines from two of these were also of A1 mating type but had segregated molecular markers typical of P2 ; a zoospore line from a third segregated both A2 mating type and markers typical of P2. The original self-fertile isolates appeared to have heterokaryotic hyphae and sporangia with P1 and P2 nuclei and also nuclei of A1 mating type but P2 markers. Tetraploid nuclei within a heterokaryon, resulting from fusion of P1 with P2 nuclei, could have segregated to yield the latter.

The development of sporangia of Phytophthora infestans

The dynamics of sporulation and zoosporogenesis of Phytophthora infestans in a naturally infected crop of potato cv. Maris Bard and on potted plants of the same cultivar infected artificially with the same fungus are described. Development of sporangiophores and sporangia was synchronous after induction. Migration of several nuclei from the sporangiophore into the sporangium initial was followed by nuclear division, then by degeneration of a proportion of the nuclei. Sporangia then became deciduous, developed papillae and became able to release zoospores in rapid succession. When infected leaves were detached and transferred from the field to moist Petri dishes, sporulation was at first synchronous then sequential; sporangiophores were longer and lateral branches more numerous than in vivo. The number of nuclei in mature sporangia was greater in vitro than in vivo.

Inheritance of streptomycin and chloramphenicol resistance in Phytophthora infestans: evidence for co-segregation of mitochondrial DNA and streptomycin resistance

Spontaneous mutant strains of Phytophthora infestans resistant to either streptomycin or chloramphenicol were selected by prolonged exposure of colonies to drug-amended media. Each strain was then crossed with a sensitive strain having different RFLP markers in its mitochondrial (mt) DNA and nuclear (n) DNA. A majority (56 out of 65) of the sexual progeny from the streptomycin mating inherited at least one nRFLP band from each parent. Fifty-two of these hybrids were streptomycin resistant and inherited their mtDNA from the resistant parent, while the four streptomycin sensitive isolates inherited their mtDNA from the sensitive parent. These data are consistent with streptomycin resistance being cytoplasmically inherited, possibly on the mitochondrial genome. The inheritance of chloramphenicol resistance in hybrids did not correlate well with that of mtDNA type but neither was it obviously Mendelian.

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.

Oogonia with multiple oospheres in Phytophthora infestans

Isolates of Phytophthora infestans of A1 mating type from central Mexico and Wales, when mated to a range of A2 isolates from Russian and Mexico, produced a proportion of abnormal oogonia with several oospheres. Similar oogonia were observed in self-fertile lines resulting from treatment of a Russian A1 isolate with a chemical mutagen followed by the fungicide, metalaxyl.