A simple and reliable method for preparation of whole nuclei of a common oomycete, Phytophthora infestans, is described for laser flow cytometry. The ease of preparation, the absence of detectable debris and aggregates, and the precision in determinations of DNA content per nucleus improve interpretation and understanding of the genetics of the organism. Phytophthora infestans is the pathogen that causes potato and tomato late blight. The genetic flexibility of P. infestans and other oomycete pathogens has complicated understanding of the mechanisms of variation contributing to shifts in race structure and virulence profiles on important agricultural crops. Significant phenotypic and genotypic changes are being reported in the apparent absence of sexual recombination in the field. Laser flow cytometry with propidium iodide is useful in investigating the nuclear condition of the somatic colony of field strains of P. infestans. The majority of the studied strains contain a single population of nuclei in nonreplicated diplophase. However, mean DNA content per nucleus varies considerably among isolates confirming the heterogeneity of the nuclear population in regard to C-value, for field isolates. Nuclear DNA content varies from 1.753 to 0.753 that of nuclei in a standard strain from central Mexico. Some strains contain two to three populations of nuclei with differing DNA contents in the mycelium and are heterokaryons. Such a range in DNA content suggests DNA-aneuploidy, but direct confirmation of aneuploidy will require microscopy of chromosomes. Heterokaryosis and populations of nuclei of differing DNA content necessarily confound standardized assays used worldwide in crop breeding programs for determination of race profiles and virulence phenotypes of this important pathogen. '

Competition between genotypes of Phytophthora infestans was studied by inoculating potato cultivars with differing susceptibility to late blight in field experiments over three years in Northern Ireland, UK, and Michigan, USA. Multiple isolates of six genotype groups of P. infestans were chosen from the local populations in both N. Ireland and Michigan for inoculation of separate field trials planted in 2003, 2004 and 2005. Four cultivars were used in each trial; two (susceptible cv. Atlantic and the partially resistant cv. Stirling) were common to both locations, whereas the two additional cultivars (with partial resistance to late blight) were cvs Santé and Milagro in N. Ireland and cvs Pike and Jacqueline Lee in Michigan. Single-lesion isolates of P. infestans were obtained from leaves at 1% level of infection, characterized using pre-assigned markers and re-assigned to their respective genotype groups. Extreme selection occurred within the population of genotypes of P. infestans in N. Ireland in each year, with different genotype groups dominating the infection of different cultivars. Selection was observed on all cultivars tested, but was greatest on the more resistant cultivars. Over the 3 years, all of the 114 isolates obtained from cv. Milagro belonged to a single group, whereas among the 118 isolates from cv. Atlantic all six groups were represented. By contrast, in Michigan, the US-8 genotype dominated infection in all cultivars in each year; only 12 of 374 isolates characterized belonged to other genotypes (11 US-14 and a single US-10 isolate).

Seed pieces of different potato cultivars and advanced breeding lines (ABLs) from north central US breeding programmes were inoculated with different genotypes of Phytophthora infestans (US-1, US-1.7, US-8, . The effect of these genotypes of P. infestans on seed piece rot severity after re-storage was assessed using an image analysis technique. P. infestans genotypes demonstrated variable ability to cause seed piece rot and to reduce plant emergence measured as final plant stand (%) and the relative area under the plant emergence curve (RAUEPC). The US-8 genotype of P. infestans was the most aggressive genotype, as indicated by tuber rot severity across all cultivars/ABLs tested, followed by US-14 in both years. The US-1, US-1.7 and US-11 genotypes were the least aggressive, causing only moderate seed piece rotting across cultivars/ABLs tested. Similar trends were observed in two field experiments, where the US-8 and US-14 genotypes delayed or reduced emergence. Values of final plant stand (%) and RAUEPC demonstrated that the cultivars/ABLs Atlantic, MSJ453-4Y and Torridon were the least susceptible across all P. infestans genotypes. In both experiments cv. Pike was the most susceptible. Other cultivars/ABLs demonstrated variable

Azoxystrobin is applied early in the sugar beet growing season in north-central United States for control of Rhizoctonia damping-off and Rhizoctonia crown and root rot caused by Rhizoctonia solani anastomoses groups (AGs) 4 and 2-2, respectively. Fungicide application timings based on crop growth stage and soil temperature thresholds were evaluated in inoculated small-scale trials and in commercial fields with a history of Rhizoctonia crown and root rot. Soil temperature thresholds of 10, 15, and 20°C were selected for fungicide application timings and used to test whether soil temperature could be used to better time applications of azoxystrobin. In both small- and large-plot trials, timing applications after attainment of specific soil temperature thresholds did not improve efficacy of azoxystrobin in controlling damping-off or Rhizoctonia crown and root rot compared with application timings based on either planting date, seedling development, or leaf stage in a susceptible (E-17) and a resistant (RH-5) cultivar. Application rate and split application timings of azoxystrobin had no significant effect on severity of crown and root rot. Other environmental factors such as soil moisture may interact with soil temperature to influence disease development. Cv. RH-5 had higher sugar yield attributes than the susceptible cultivar (E-17) in seasons conducive and nonconducive to crown and root rot development. All isolates recovered from both small- and large-plot trials in all years were AG 2-2. R. solani AG 4 was not identified in any samples from any year.

Fusarium dry rot is one of the most important diseases of potato (Solanum tuberosum L.), affecting tubers in storage and whole seed or seed pieces after planting (2). Fusarium sambucinum Fuckel (teleomorph Giberella pulicaris) is the most common pathogen causing dry rot of stored tubers in North America. (4). Cut seed potato tubers of cvs. FL1879 and Pike with severe sprout rot were collected in Michigan during May 2006. As well as having rotted sprouts, all diseased tubers had dry rot. When diseased sprouts were cut in half, brown, necrotic lesions could be seen spreading down the center of the sprout in vascular tissue and at the base of the sprout in tuber tissue. Pathogen isolations were made from both infected tuber tissue and diseased sprouts on potato dextrose agar (PDA). In both cases, only F. sambucinum was isolated from diseased sprout and tuber tissue. Identification of the pathogen was based on colony and conidial morphology. This included white, fluffy mycelium on the surface and crimson coloration of the colonies viewed from the underside of PDA plates and large distinctive macroconidia (3). Identification was confirmed by comparison of ITS (internal transcribed spacer) sequence data with reference isolates. The ITS region of rDNA was amplified by polymerase chain reaction (PCR) with primers ITS1/ITS4 and sequenced. BLASTn analysis (1) of the sequence obtained showed a 100% homology with F. sambucinum Fuckel. For inoculum production, isolates were grown on PDA at 8°C for 14 days prior to inoculation. Pathogenicity was tested in potato tubers of cv. FL1879 with a single isolate collected from diseased sprouts. Whole seed tubers with 4 mm long sprouts were cut in half longitudinally with a sterile knife to ensure that seed pieces had viable sprouts. The cut surfaces of seed pieces were spray inoculated with 200 ml of conidial suspension (1 × 104 conidia ml-1) over the entire cut surface to give a final dosage of approximately 1 ml per seed piece. Care was taken to limit inoculum spray to the cut surface so that sprouts were not inoculated. Seed pieces (40 per replicate × 4 replicates) were then placed in plastic boxes (30 × 15 × 10 cm) and incubated in the dark at 18°C and 95% relative humidity for 30 days in a controlled environment chamber. As a control, cut seed pieces were spayed with sterile distilled water and incubated as above. All tubers inoculated with the pathogen developed typical Fusarium dry rot symptoms consisting of a brown, dry decay of tuber tissue with mycelial lined cavities. Sprouts on inoculated tubers developed symptoms that were observed in the initially collected seed pieces, and F. sambucinum was reisolated from all infected sprouts. The noninoculated control tubers did not develop any symptoms of dry rot. The results of the pathogenicity tests indicate that F. sambucinum caused sprout rot on potato seed pieces. Since only the cut surfaces of tubers were inoculated, it is assumed that infection of sprouts is systemic through the tuber. To our knowledge, this is the first report of F. sambucinum causing a sprout rot of developing sprouts on seed tubers in the United States. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) L. E. Hanson et al. Phytopathology 86:378, 1996. (3) P. E. Nelson et al. Pages 118–119 in: Fusarium Species: An Illustrated Manual for Identification. The Pennsylvania State University, University Park and London, 1983. (4) G. A. Secor and B. Salas. Fusarium dry rot and Fusarium wilt. Pages 23–25 in: Compendium of Potato Diseases. 2nd ed. W. R. Stevenson et al., eds. The American Phytopathological Society, St. Paul, MN, 2001.