Phytophthora infestans isolates (n = 26) collected in the Columbia Basin of Oregon and Washington in 1993, which had been characterized previously for mating type, metalaxyl sensitivity, and alleles at the glucose-6-phosphate isomerase locus, were analyzed for nuclear restriction fragment length polymorphism (RFLP) bands detected by probe RG57 and mitochondrial haplotype. Analyses involving the larger set of markers indicated that this group of isolates satisfied expectations of a sexual progeny: they contained much greater genetic diversity than has been reported for most other epidemic populations of P. infestans in the United States and Canada (16 unique multilocus genotypes); both mating types were present in proximity; all possible combinations of alleles occurred at many pairs of polymorphic loci; and two distinct mitochondrial haplotypes were distributed among the isolates. An in vitro laboratory cross involving the putative parents (US-6 and US-7) as parental strains produced progeny with the same general characteristics as the field isolates. Among the field progeny were two genotypes, US-11 and US-16, that had been described previously but from subsequent and largely clonal collections. Isolates obtained from tomatoes (n = 40) and potatoes (n = 7) in 24 counties in California in 1998 were analyzed as described above, and all except one US-8 isolate from potatoes were of the US-11 clonal lineage, consistent with the hypothesis that the US-11 lineage is an especially fit clonal lineage that has survived over time and can dominate pathogen populations over a large area. We conclude that the 1993 Columbia Basin collection represents a sexual progeny that generated the US-11 lineage, and that this lineage is particularly fit when tomatoes are part of the agroecosystem.

Potato virus Y (PVY) is a serious potato pathogen that affects potato seed and commercial production crops. In recent decades, novel PVY strains have been described that cause necrotic symptoms on tobacco foliage and/or potato tubers. The major PVY strains that affect potato include PVY(O) and PVY(N), which have distinct serotypes that can be differentiated by immunoassay. Other economically important strain variants are derived from recombination events, including variants that cause tuber necrotic symptoms (PVY(NTN)) and PVY(O) serotypes that cause tobacco veinal necrosis (PVY(N)-W, PVY(N:O)). Although the PVY(NTN) and PVY(N)-W variants were first reported in Europe, apparently similar strains have been appearing in North America. Confirmation of the existence of these recombinant strains in North America is important, as is whether they spread from a common source or were derived by independent recombination. Whole genome sequencing can be used to positively identify strain variants and begin to address the issue of origins. Symptomology, serology, RT-PCR, and partial sequencing of the coat protein region were used to identify isolates of the PVY(NTN), PVY(N), PVY(NA-N), and PVY(N:O) for whole-genome sequencing. Sequencing confirmed the presence of PVY(NTN) and PVY(N) isolates that were >99% identical to European sequences deposited in GenBank in the 1990's. Sequences of the PVY(NA-N) and PVY(N:O) types were 99.0% and 99.5% identical to known sequences, respectively. There was no indication that recombinant strains PVY(NTN) or PVY(N:O) had different parental origins than recombinant strains previously sequenced. This is the first confirmation by whole-genome sequencing that "European"-type strain variants of PVY(N) and PVY(NTN) are present in North America, and the first reported full-length sequence of a tuber necrotic isolate of PVY(N:O).

The aggressiveness of 22 isolates of Phytophthora infestans collected from naturally infected potato plants in the Columbia Basin of Washington and Oregon was determined on detached potato leaflets at 18 degrees C in an incubator. Selected isolates were evaluated on whole plants in a greenhouse. Aggressiveness was measured by using the area under the lesion expansion curve (AULEC), incubation period, latent period, sporulation capacity, and lesion size on detached leaflets and the area under the disease progress curve and sporulation capacity on whole plants. The detached-leaflet assay was useful in that a large number of isolates were tested, several components of aggressiveness were studied, and significant differences were found among isolates. Significant variation for components of aggressiveness was found within and among isolates classified according to genotype. Significant interactions among isolates and cultivars were found for some components of aggressiveness, so results were pooled according to cultivar. On average, US-8 and US-11 isolates had higher AULEC scores, indicating aggressiveness higher than that of US-7, US-6, and US-1 genotypes. One US-8 genotype isolate had a higher standardized sporulation capacity than isolates of the other genotypes. US-6 genotype isolates were the least aggressive group, as indicated by low AULEC, sporulation capacity, and lesion size values. The replacement of the US-1 genotype by the US-8 genotype in the Columbia Basin may be partially explained by the increased aggressiveness of US-8 isolates. Additionally, potato growers may need to shorten intervals between fungicide applications and begin applications earlier.

The environmental release of genetically engineered (transgenic) plants may be accompanied by ecological effects including changes in the plant-associated microflora. A field release of transgenic potato plants that produce the insecticidal endotoxin of Bacillus thuringiensis var. tenebrionis (Btt) was monitored for changes in total bacterial and fungal populations, fungal species diversity and abundance, and plant pathogen levels. The microflora on three phenological stages of leaves (green, yellow and brown) were compared over the growing season (sample days 0, 21, 42, 63 and 98) for transgenic potato plants, commercial Russet Burbank potato plants treated with systemic insecticide (Di-Syston)and commercial Russet Burbank potato plants treated with microbial Btt (M-Trak). In addition, plant and soil assays were performed to assess disease incidence of Fusarium spp., Pythium spp., Verticillium dahliae, potato leaf roll virus (PLRV) and potato virus Y (PVY). Few significant differences in phylloplane microflora among the plant types were observed and none of the differences were persistent. Total bacterial populations on brown leaves on sample day 21 and on green leaves on sample day 42 were significantly higher on the transgenic potato plants. Total fungal populations on green leaves on sample day 63 were significantly different among the three plant types; lowest levels were on the commercial potato plants treated with systemic insecticide and highest levels were on the commercial potato plants treated with microbial Btt.Differences in fungal species assemblages and diversity were correlated with sampling dates, but relatively consistent among treatments. Alternaria alternata, a common saprophyte on leaves and in soil and leaf litter, was the most commonly isolated fungus species for all the plant treatments. Rhizosphere populations of the soilborne pathogens Pythium spp., Fusarium spp. and V. dahliae did not differ between the transgenic potato plants and the commercial potato plants treated with systemic insecticide. The incidence of tuber infection at the end of the growing season by the plant pathogen g dahliae was highest for the transgenic potato plants but this difference was related to longer viability of the transgenic potato plants. This difference in longevity between the transgenic potato plants and the commercial + systemic insecticide potato plants also made comparison of the incidence of PVY and PLRV problematic. Our results indicate that under field conditions the microflora of transgenic Btt-producing potato plants differed minimally from that of chemically and microbially treated commercial potato plants.

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Isolates of Phytophthora infestans collected from 1992 to 1995 from potato fields in the Columbia Basin of Oregon and Washington were analyzed for compatibility type, metalaxyl sensitivity, and glucose-6-phosphate isomerase (Gpi) genotype. In 1992, 30 of 31 isolates were of the US-1 multilocus genotype. A single metalaxyl-resistant isolate of the US-6 (A1 Gpi 86/100) genotype was found near the end of the growing season. In 1993, only 2 of the 59 isolates collected were A1 isolates with Gpi 86/100. Ten isolates were of the A2 compatibility type, seven with Gpi 100/111, two with Gpi 100/100, and one was undetermined. The remaining isolates were metalaxyl-resistant A1 compatibility types with either Gpi 100/100 or 100/111. The first A2 isolates in the Columbia Basin were found in 1993. In 1994, 10 of 18 isolates were of the US-1 genotype. The remaining isolates were US-6 and US-8 genotypes. In 1995, 97% of 268 isolates tested were of the US-8 genotype. Five isolates were A2 compatibility type with Gpi 100/122. One A2, metalaxyl-resistant isolate was Gpi 100/100/111, and two A1 isolates were Gpi 100/111/122. The population of P. infestans quickly changed between 1992 and 1995, from a population comprised almost exclusively of the US-1 genotype to a population represented by new or recombinant genotypes.