Aggressiveness and Fungicide Sensitivity of Alternaria dauci from Cultivated Carrot.

Isolates of Alternaria dauci causing Alternaria leaf blight (ALB) were collected from commercial carrot (Daucus carota var. sativus) fields in northeastern North America during 2004. Twenty-two isolates representing a range of genetic diversity were analyzed for their aggressiveness on three commercial carrot varieties (Bolero, Enterprise, and Heritage) varying in disease susceptibility as well as their in vitro response to three fungicides (azoxystrobin, chlorothalonil, and boscalid) commonly used for ALB control. Severity of leaf and petiole blight and leaf chlorosis varied among isolates and carrot varieties in each of three experiments. Visible differences in disease severity, which ranged from 10.9 to 45.1% of the leaf area affected, were apparent 16 days after inoculation. Intensity of chlorosis correlated strongly with blight severity among all isolates. Significant differences were noted among carrot varieties in response to ALB. These varieties may prove useful as differentials capable of distinguishing isolates because variety by isolate interactions were detected. Inhibition of conidial germination ranged from 0.01 to 0.37 µg/ml for azoxystrobin, 0.009 to 0.08 µg/ml for chlorothalonil, and 0.09 to 0.59 µg/ml for boscalid. On average, isolates were more sensitive to chlorothalonil than to azoxystrobin and boscalid. No significant correlation was noted between fungicide sensitivity and aggressiveness. These data provide evidence for phenotypic diversity among A. dauci isolates collected from areas of commercial carrot production.

Evaluation of QoI Fungicide Application Strategies for Managing Fungicide Resistance and Potato Early Blight Epidemics in Wisconsin.

Potato early blight (Alternaria solani) is a yield-limiting disease and control depends primarily on multiple fungicide applications. Azoxystrobin, registered in the United States in 1999, initially provided outstanding early blight control. Within 3 years, approximately 80% of the total potato acreage was being treated with azoxystrobin and other quinone outside inhibitor (QoI), fungicides registered subsequently. Alternaria solani isolates with decreased in vitro sensitivity to azoxystrobin were detected in Wisconsin during 2001. Field experiments were conducted in 2001 to 2003 to evaluate season-long fungicide programs and test fungicide resistance management strategies. The fungicide program recommended to growers at that time, which consisted of three applications of azoxystrobin for weeks 1, 3, and 5 alternated with applications of chlorothalonil at label recommended rates, was effective in controlling early blight when conditions were conducive to disease development. Mean sensitivity in vitro of A. solani isolates from fungicide efficacy field experiments in 2001 to 2003 was numerically highest for isolates from the untreated control plots, chlorothalonil-alone plots, or plots treated with three applications of azoxystrobin alternated with chlorothalonil compared with other treatments tested. Three single-nucleotide polymorphisms (SNPs) can cause the F129L substitution (TTC to TTA, CTC, or TTG) that results in decreased sensitivity to azoxystrobin of A. solani. The TTA mutant was the most frequently recovered mutant type in the field experiments. The frequency of recovery of wild-type isolates in experiments was 22% in 2001, 4% in 2002, and 22% in 2003.

Monitoring and Tracking Changes in Sensitivity to Azoxystrobin Fungicide in Alternaria solani in Wisconsin.

Azoxystrobin is a common fungicide used by farmers of Solanaceous crops against Alternaria solani, but there was growing concern about decreased sensitivity with repeated applications. In 2002 and 2003, monitoring of A. solani from commercial potato fields in Wisconsin indicated increased frequency and a statewide distribution of isolates with decreased in vitro sensitivity to azoxystrobin. Mean effective concentration in inhibiting spore germination by 50% values gathered in 2002 and 2003 were approximately 20-fold higher than baseline isolates of A. solani collected in 1998 from fields that had never been treated with azoxystrobin. This sensitivity decrease was correlated with site-specific mutations in the cytochrome b detected by quantitative real-time polymerase chain reaction. The F129L and the G143A substitution have been shown to cause a reduction in sensitivity or resistance, respectively, to quinone outside inhibitors. All of the recovered A. solani isolates collected in 2002 and 2003 were wild type at position 143. However, all three mutations responsible for the F129L substitution (TTA, CTC, and TTG) were detected in our samples. In addition, the frequency of this amino acid substitution in A. solani isolates was statistically different across sampling sites and years, indicating that sensitivity changes depended on specific disease management practices.

Multiplex Real-Time Quantitative PCR to Detect and Quantify Verticillium dahliae Colonization in Potato Lines that Differ in Response to Verticillium Wilt.

ABSTRACT Potato early dying (PED), also known as Verticillium wilt, caused by Verticillium dahliae, is a seasonal yield-limiting disease of potato worldwide, and PED-resistant cultivars currently represent only a small percentage of potato production. In this study, we developed a real-time quantitative polymerase chain reaction (Q-PCR) approach to detect and quantify V. dahliae. The efficiency of the designed primer pair VertBt-F/VertBt-R, derived from the sequence of the beta-tubulin gene, was greater than 95% in monoplex Q-PCR and duplex (using Plexor technology) procedures with primers PotAct-F/PotAct-R, obtained from the sequence of the actin gene, designed for potato. As few as 148 fg of V. dahliae DNA were detected and quantified, which is equivalent to five nuclei. Q-PCR detected V. dahliae in naturally infected air-dried potato stems and fresh stems of inoculated plants. Spearman correlations indicated a high correlation (upward of 80%) between V. dahliae quantifications using Q-PCR and the currently used plating assays. Moreover, Q-PCR substantially reduced the variability compared with that observed in the plating assay, and allowed for the detection of V. dahliae in 10% of stem samples found to be pathogen free on the culture medium. The described Q-PCR approach should provide breeders with a more sensitive and less variable alternative to time-consuming plating assays to distinguish response of breeding lines to colonization by V. dahliae.

Fungicide Spray Programs for Defender, A New Potato Cultivar with Resistance to Late Blight and Early Blight.

Defender (A90586-11) is a new late blight-resistant potato cultivar which was released from the Tri-State Potato Variety Development Program in 2004. Conventional and reduced fungicide spray programs were compared on Defender and Russet Burbank (3 years) and Ranger Russet (1 year) in Wisconsin experimental field trials. Useful levels of field resistance to both late blight and early blight were observed in Defender in the absence of fungicide sprays and reduced fungicide input programs. Disease progressed slowest on Defender regardless of fungicide program, relative to Russet Burbank and Ranger Russet. Organic, conventional, and reduced fungicide spray programs also were compared on Defender and Russet Burbank in experimental greenhouse and field tests in Washington. Fungicide spray programs performed similarly on both Defender and Russet Burbank; however, area under the disease progress curve values for no-fungicide treatments were either three times (greenhouse) or six times (field) lower on Defender compared with Russet Burbank. Regardless of the fungicide program, total yield was higher for Defender than Russet Burbank. Mean economic returns associated with Defender also were higher than for Russet Burbank ($6,196 versus $4,388/ha). Fungicide and nonfungicide treatment programs generated similar returns on Defender whereas conventional and reduced fungicide programs generated comparable but higher returns than the nonfungicide program on Russet Burbank.

Colonization Dynamics and Spatial Progression of Verticillium dahliae in Individual Stems of Two Potato Cultivars with Differing Responses to Potato Early Dying.

Potato early dying (PED), caused by Verticillium dahliae, is a chronic yield-limiting disease of potato (Solanum tuberosum). In this study, we describe the colonization dynamics of V. dahliae in two potato cultivars with varying responses to PED. We utilized a quantitative real-time polymerase chain reaction (Q-PCR) assay to assess the colonization and spatial progression of V. dahliae in cvs. Ranger Russet (moderately resistant) and Russet Norkotah (highly susceptible). Ninety plants per cultivar were inoculated with a conidial suspension in the greenhouse. Every 2 weeks until week 10, we collected basal samples from 15 plants, and repeatedly sampled the growing apices of another 15 plants. The mean infection coefficient (IC) values in the basal and apical samples were significantly lower in cv. Ranger Russet at all five sampling dates. The pathogen was detected in basal samples of both cultivars by week 2, and in apical samples of cv. Russet Norkotah at week 4 and of cv. Ranger Russet at week 6. Colonization of cv. Russet Norkotah consistently increased in apical and basal samples during the 10 weeks, while it plateaued after week 6 in cv. Ranger Russet. Differences in response to PED appear associated with the speed of colonization and the establishment of a higher population density by V. dahliae in the plant.

A methodology to detect and quantify five pathogens causing potato tuber decay using real-time quantitative polymerase chain reaction.

ABSTRACT Late blight (Phytophthora infestans), pink rot (Phytophthora erythroseptica), leak (Pythium ultimum), dry rot (Fusarium sambucinum), and soft rot (Erwinia carotovora subsp. carotovora and subsp. atroseptica) are particularly damaging diseases of stored potato tubers worldwide. In this study, we present a methodology to detect and quantify the causal agents of the five aforementioned diseases from whole potato tubers, using real-time quantitative-polymerase chain reaction. Six primer pairs were designed to amplify targets smaller than 150-bp DNA in single copy protein-coding gene targets of each of the pathogens and the potato host. Using a large collection of pure culture DNA samples, all primer pairs specifically detected the DNA target in the intended pathogenic species. Amplification efficiencies over a five-log dilution series ranged between 95 and 100% and were unaffected by the presence of large amounts of host DNA. The detection level of the primers reached 0.5 pg of target DNA. Pathogens were detected in 100 pg of total DNA extracted from 170 to 250 g of tubers, 4 days after inoculation, regardless of the presence of symptoms. The presence of P. erythroseptica, Pythium ultimum, or E. carotovora was also detected in 1 ng of DNA extracted from potato tubers collected from a commercial storage facility. This study provides the first step in a methodology to predict the storability of potato tubers following harvest.

Weather-Based Fungicide Spray Programs for Control of Two Foliar Diseases on Carrot Cultivars Differing in Susceptibility.

Foliar diseases of carrot caused by Alternaria dauci and Cercospora carotae occur every year in Wisconsin, requiring repeated foliar fungicide applications to minimize defoliation and yield reduction. Improved timing of fungicide applications combined with the use of disease resistant cultivars offer growers a means to improve disease control with fewer fungicide inputs compared with the current strategy of weekly fungicide applications to a susceptible cultivar. Field experiments in 2002 to 2004 examined fungicide application schedules indicated by a disease forecasting model that calculated the duration of environmental conditions favorable for A. dauci and C. carotae (adaptation of TOM-CAST) on two carrot cultivars differing in susceptibility to these foliar diseases. All fungicide programs were initiated at a 1% disease severity threshold determined by scouting. Intervals for weather-based spray programs were based on in-canopy leaf wetness and temperature data. Fungicide sprays were applied according to 15 and 20 disease severity value (DSV) application thresholds, and were compared with a weekly spray program and an untreated control. Results of this trial demonstrated that fungicide sprays made according to weather data may reduce fungicide inputs in most years compared with current industry-standard, calendar-based spray programs. Host susceptibility affected the efficacy of weather-based spray programs, resulting in longer spray intervals and fewer fungicide applications on the resistant cultivar Bolero when compared with the susceptible cultivar Fontana. Fungicide spray programs based on TOM-CAST diminished fungicide inputs by 30 to 50%, compared with the weekly spray program, by lengthening intervals between applications without compromising disease control or root yield.

Characterization of Phytophthora infestans in Wisconsin from 1993 to 1995.

Late blight of potato (Solanum tuberosum), caused by Phytophthora infestans, recently reappeared in Wisconsin and was a significant production problem in 1994. P. infestans isolates collected in Wisconsin from 1993 to 1995 were characterized for the following traits: mating type, sensitivity to metalaxyl, and allozyme genotype for Glucose-6-phosphate isomerase (Gpi). Characterization of these isolates revealed that a new, more aggressive population (A2 mating type, metalaxyl resistant, and Gpi genotype 100/111/122) is displacing the old population (A1 mating type, metalaxyl sensitive, and Gpi genotype 86/100) in Wisconsin.

Effect of Organic Amendments on Soilborne and Foliar Diseases in Field-Grown Snap Bean and Cucumber.

Several paper mills in Wisconsin have programs for spreading paper mill residuals (PMR) on land. A growing number of vegetable farmers recognize the agronomic benefits of PMR applications, but there have been no investigations on the use of PMR for control of vegetable crop diseases. Our objective was to determine the effect of PMR amendments on soilborne and foliar diseases of cucumber and snap bean grown on a sandy soil. Raw PMR, PMR composted without bulking agent (PMRC), or PMR composted with bark (PMRBC) were applied annually in a 3-year rotation of potato, snap bean, and pickling cucumber. Several naturally occurring diseases were evaluated in the field, along with in situ field bioassays. All amendments suppressed cucumber damping-off and Pythium blight and foliar brown spot of snap bean. Both composts reduced the incidence of angular leaf spot in cucumber. In a separate field experiment planted with snap bean for two consecutive years, all amendments reduced common root rot severity in the second year. In a greenhouse experiment, the high rate of PMRBC suppressed anthracnose of snap bean. These results suggest that the application of raw and composted PMR to sandy soils has the potential to control several soilborne and foliar diseases.

Plant pathogen population dynamics in potato fields.

Modern technologies incorporating Geographic Information Systems (GIS), Global Positioning Systems (GPS), remote sensing, and geostatistics provide unique opportunities to advance ecological understanding of pests across a landscape. Increased knowledge of the population dynamics of plant pathogens will promote management strategies, such as site-specific management, and cultural practices minimizing the introduction and impact of plant pathogens. The population dynamics of Alternaria solani, Verticillium dahliae, and Pratylenchus penetrans were investigated in commercial potato fields. A 0.5-ha diamond grid-sampling scheme was georeferenced, and all disease ratings and nematode samples were taken at these grid points. Percent disease severity was rated weekly, and P. penetrans densities were quantified 4 weeks after potato emergence. Spatial statistics and interpolation methods were used to identify the spatial distribution and population dynamics of each pathogen. Interpolated maps and aerial imagery identified A. solani intra-season progression across the fields as the potato crop matured. Late-season nitrogen application reduced A. solani severity. The spatial distributions of V. dahliae and P. penetrans were spatially correlated.

Malformin in Aspergillus niger-infected onion bulbs (Allium cepa).

Malformin was identified, by its biological activity and chromatography, in acetone extracts of the outer scales of onion bulbs infected with Aspergillus niger. Malformin was not detected in tissue underlying the infected areas or in the central portions of the bulbs, nor was malformein liberated from extracts or extracted tissues after reduction with zinc in acetic acid. This is the first report of naturally occurring malformin.