Molecular characterization and detection of mutations associated with resistance to succinate dehydrogenase-inhibiting fungicides in Alternaria solani.

Early blight, caused by Alternaria solani, is an economically important foliar disease of potato in several production areas of the United States. Few potato cultivars possess resistance to early blight; therefore, the application of fungicides is the primary means of achieving disease control. Previous work in our laboratory reported resistance to the succinate dehydrogenase-inhibiting (SDHI) fungicide boscalid in this plant pathogen with a concomitant loss of disease control. Two phenotypes were detected, one in which A. solani isolates were moderately resistant to boscalid, the other in which isolates were highly resistant to the fungicide. Resistance in other fungal plant pathogens to SDHI fungicides is known to occur due to amino acid exchanges in the soluble subunit succinate dehydrogenase B (SdhB), C (SdhC), and D (SdhD) proteins. In this study, the AsSdhB, AsSdhC, and AsSdhD genes were analyzed and compared in sensitive (50% effective concentration [EC50] < 5 µg ml(-1)), moderately resistant (EC50 = 5.1 to 20 µg ml(-1)), highly resistant (EC50 = 20.1 to 100 µg ml(-1)), and very highly resistant (EC50 > 100 µg ml(-1)) A. solani isolates. In total, five mutations were detected, two in each of the AsSdhB and AsSdhD genes and one in the AsSdhC gene. The sequencing of AsSdhB elucidated point mutations cytosine (C) to thymine (T) at nucleotide 990 and adenine (A) to guanine (G) at nucleotide 991, leading to an exchange from histidine to tyrosine (H278Y) or arginine (H278R), respectively, at codon 278. The H278R exchange was detected in 4 of 10 A. solani isolates moderately resistant to boscalid, exhibiting EC50 values of 6 to 8 µg ml(-1). Further genetic analysis also confirmed this mutation in isolates with high and very high EC50 values for boscalid of 28 to 500 µg ml(-1). Subsequent sequencing of AsSdhC and AsSdhD genes confirmed the presence of additional mutations from A to G at nucleotide position 490 in AsSdhC and at nucleotide position 398 in the AsSdhD, conferring H134R and H133R exchanges in AsSdhC and AsSdhD, respectively. The H134R exchange in AsSdhC was observed in A. solani isolates with sensitive, moderate, highly resistant, and very highly resistant boscalid phenotypes, and the AsSdhD H133R exchange was observed in isolates with both moderate and very high EC50 value boscalid phenotypes. Detection and differentiation of point mutations in AsSdhB resulting in H278R and H278Y exchanges in the AsSdhB subunit were facilitated by the development of a mismatch amplification mutation assay. Detection of these two mutations in boscalid-resistant isolates, in addition to mutations in AsSdhC and AsSdhD resulting in an H134R and H133R exchange, respectively, was achieved by the development of a multiplex polymerase chain reaction to detect and differentiate the sensitive and resistant isolates based on the single-nucleotide polymorphisms present in all three genes. A single A. solani isolate with resistance to boscalid did not contain any of the above-mentioned exchanges but did contain a substitution of aspartate to glutamic acid at amino acid position 123 (D123E) in the AsSdhD subunit. Among A. solani isolates possessing resistance to boscalid, point mutations in AsSdhB were more frequently detected than mutations in genes coding for any other subunit.

Development and Validation of a Real-Time PCR Assay for the Quantification of Verticillium dahliae in Potato.

An increase in the stringency for higher quality potato tubers and restrictions on the use of soil fumigants, among other factors, has garnered renewed interest in Verticillium wilt, particularly in russet-skinned cultivars grown for processing. In response to the needs of producers, breeders have increased efforts in the development of potato cultivars with resistance to Verticillium dahliae Kleb., the primary cause of Verticillium wilt. These efforts have resulted in the release of numerous russet-skinned cultivars with purported resistance to the pathogen. However, because efficient and effective methods to screen germplasm for true resistance do not exist, breeders typically have reported resistance based on the development of wilt symptoms alone. The studies reported here demonstrate the efficiency and practicality of a QPCR method for quantification of V. dahliae in potato stem tissue. This method, developed to detect the target trypsin protease gene of the pathogen, was compared with traditional methods for V. dahliae quantification which involve plating stem tissue or sap onto semi-selective media, as well as to a recently developed QPCR assay which amplifies a region of the ß-tubulin gene of V. dahliae. The QPCR assay developed in the studies reported here was demonstrated to be sensitive to 0.25 pg of DNA. Use of the duplex real-time PCR assay, utilizing the potato actin gene to normalize quantification, resulted in clearer differentiation of levels of resistance among eight russet-skinned potato cultivars inoculated in greenhouse trials when compared with traditional plating assays. However, relative levels of resistance among cultivars were similar between traditional plating and QPCR methods, resulting in correlation coefficients greater than 0.93. The assay described here also detected the pathogen in inoculated stem tissue at higher frequencies than both traditional plating assays and a previously developed QPCR assay. The QPCR assay developed here demonstrates rapid, efficient, and accurate quantification of V. dahliae, providing a tool amenable for use by breeding programs on large numbers of clones and selections, and will aid researchers evaluating other control strategies for Verticillium wilt.

Prevalence and Impact of SDHI Fungicide Resistance in Alternaria solani.

Early blight, caused by Alternaria solani, is an important chronic foliar disease of potato (Solanum tuberosum) present every growing season in the Midwestern United States. Most currently grown potato cultivars lack resistance to early blight; therefore, foliar fungicides are relied upon for disease management. Foliar fungicides with high efficacy against the pathogen, such as boscalid, frequently are used under high disease pressure situations, such as potatoes grown under overhead irrigation. Boscalid is a member of the succinate dehydrogenase inhibiting (SDHI) fungicide group and was registered for use on potato in 2005. Baseline sensitivity of A. solani to the SDHI fungicides boscalid, penthiopyrad, and fluopyram using a spore germination assay demonstrated similar intrinsic activity against A. solani with mean EC50 values of 0.33, 0.38, and 0.31 µg/ml, respectively. However, isolates varied in their sensitivity to each of these fungicides, resulting in very low correlations (r) among isolate sensitivity to each fungicide. Resistance to boscalid in A. solani was detected in the states of North Dakota, Minnesota, Nebraska, Texas, Idaho, Wisconsin, and Florida from early blight samples collected in 2010 and 2011. Two phenotypes of boscalid resistance were detected. Approximately 80% of all A. solani assayed were found to have some level of resistance to boscalid with about 5 and 75% of the population moderately resistant (5 to 20 µg/ml) and highly resistant (>20 µg/ml), respectively, to the fungicide. Nearly 99% of all boscalid resistant isolates possessed the F129L mutation in the cytrochrome b gene, indicating that an A. solani population with dual fungicide resistance predominates in the states surveyed. However, A. solani isolates resistant to boscalid remained sensitive to fluopyram, and a large proportion of moderately resistant and resistant isolates were sensitive to penthiopyrad. Disease control data from in vivo trials demonstrated a significant loss of fungicide efficacy when boscalid and fluxapyroxad were used to control moderately and highly resistant isolates of A. solani relative to the control these fungicides provided wild-type isolates. Fluopyram, however, controlled boscalid resistant isolates as well as it controlled wild-type isolates of A. solani. These data will assist potato growers in regions where boscalid resistance is prevalent by assisting them in avoiding fungicides that do not effectively control early blight and in selecting SDHI fungicide molecules that remain efficacious.

Colonization of Potato by Colletotrichum coccodes: Effect of Soil Infestation and Seed Tuber and Foliar Inoculation.

Colonization of potato (Solanum tuberosum) tissue, including roots, stolons, and above and below ground stems, by Colletotrichum coccodes, the causal agent of black dot, was evaluated following soil infestation, inoculation of seed tubers and foliage, and every combination thereof, in field trials over two growing seasons in North Dakota and Minnesota. A total of 107,520 isolations for C. coccodes performed across four site-years allowed for an extensive comparison of fungal colonization of the host plant and disease severity. The black dot pathogen was detected in potato stems at the first sampling date in all four site-years, as early as 14 days prior to emergence. Colonization of above and below ground stems occurred at a higher frequency than in roots and stolons in all four site-years, resulting in significantly higher relative area under the colonization progress curves (RAUCPCs) (α = 0.05). Although fungal colonization and disease incidence were higher in inoculated and/or infested treatments, sufficient natural inoculum was present to result in substantial levels of disease in noninoculated and noninfested plots. However, noninoculated and noninfested plots displayed the lowest RAUCPC values across three of four site-years and those treatments with multiple inoculation events tended to have higher RAUCPC values. Isolates belonging to vegetative compatibility group (VCG)2 and -5 were recovered from plants sampled in 2004 more frequently than isolates belonging to VCG1 and -3. A significant difference in disease incidence on stems was observed only in North Dakota in 2004 and Minnesota in 2003 (α = 0.05). Noninoculated and noninfested plots displayed the lowest disease incidence, whereas those treatments with more than one inoculation and/or infestation event tended to have higher disease incidence. Results of this study, including the disease severity and yield data, provide a better understanding of colonization of potato plants by C. coccodes and its impact.

Characterization and Epidemiological Significance of Potato Plants Grown from Seed Tubers Affected by Zebra Chip Disease.

An emerging disease of potato in the United States, known as "Zebra Chip" or "Zebra Complex" (ZC), is increasing in scope and threatens to spread further. Here, we report on studies performed to understand the role of tuberborne ZC in the epidemiology of this disease. Depending on variety, up to 44% of ZC-affected seed tubers (ZCST) were viable, producing hair sprouts and weak plants. Chip discoloration in progeny tubers of ZCST was more severe than those from ZC-asymptomatic seed tubers but varied depending on whether progeny tubers or foliage were positive or negative for 'Candidatus Liberibacter solanacearum'. A low percentage of greenhouse-grown plants produced by ZCST tested positive for 'Ca. Liberibacter'. No adult potato psyllids became infective after feeding upon these plants but they did acquire 'Ca. Liberibacter' from field-grown plants produced by ZCST. Plants with new ZC infections near plants produced by ZCST were not significantly different from healthy plants, whereas plants affected with ZC from infectious potato psyllids had significantly more ZC infections near either plants produced by ZCST or healthy plants. We conclude that, in areas where ZC is currently established, plants produced by ZCST do not significantly contribute to ZC incidence and spread within potato fields.

Detection, Distribution, and Genetic Variability of 'Candidatus Liberibacter' Species Associated with Zebra Complex Disease of Potato in North America.

The specificity and sensitivity of polymerase chain reaction (PCR) primers developed for 'Candidatus Liberibacter solanacearum' and 'Candidatus Liberibacter psyllaurous' were evaluated in conventional and real-time PCR assays. All PCR primers were specific for 'Ca. L. psyllaurous' and 'Ca. L. solanacearum' insomuch as they did not detect other prokaryotic plant pathogens that affect potato except for the putative pathogens associated with psyllid-yellows and haywire. Conventional PCR assays were capable of detecting 0.19 to 1.56 ng of total DNA per reaction, and real-time PCR was found capable of detecting 1.56 to 6.25 ng of total DNA per reaction, depending on the specific PCR primer set used. 'Ca. Liberibacter' species associated with zebra complex disease (ZC) was confirmed in plants affected by this disease throughout Texas from 2005 to 2008, in seed tubers produced in Wyoming in 2007, and in Colorado, Kansas, Nebraska, and Mexico in 2008. A multiplex PCR assay using 'Ca. L. solanacearum'-specific primers and primers specific for the ß-tubulin DNA regions from potato was developed, providing possible utility of the multiplex assay for 'Ca. Liberibacter' detection in different solanaceous plant species. Preliminary studies suggest silverleaf nightshade (Solanum elaeagnifolium), wolfberry (Lycium barbarum), black nightshade (S. ptychanthum), and jalapeno pepper (Capsicum annuum) as additional solanaceous hosts for the ZC-associated bacterium. The 'Ca. Liberibacter' species detected in all samples divided into two clusters sharing similarity of 99.8% in their partial 16S rRNA gene sequences and 99.3% in their partial intergenic spacer region (ISR)-23S rRNA gene sequences. Genetic variation in the 16S rDNA region consistently matched that of the ISR-23S rDNA region. In this partial 16S-ISR-23S rDNA region, there was a total of eight single nucleotide polymorphisms among 'Ca. L. psyllaurous' and 'Ca. L. solanacearum' "strains" investigated in this study. 'Ca. L. solanacearum' and 'Ca. L. psyllaurous' were shown to be very closely related bacteria, if not the same, by successful amplification using a combination of forward primer of 'Ca. L. solanacearum' and reverse primer of 'Ca. L. psyllaurous' in ZC-affected potato samples. This finding clarifies the current taxonomic status of 'Ca. L. solanacearum' and 'Ca. L. psyllaurous'. The detection of 'Ca. L. solanacearum' from haywire-symptomatic potato samples demonstrates that this bacterium might also be associated with this disease.

Resistance to QoI Fungicides in Ascochyta rabiei from Chickpea in the Northern Great Plains.

Ascochyta blight, caused by Ascochyta rabiei (teleomorph: Didymella rabiei), is an important fungal disease of chickpea (Cicer arietinum). A monitoring program was established in 2005 to determine the sensitivity of A. rabiei isolates to the QoI (strobilurin) fungicides azoxystrobin and pyraclostrobin. A total of 403 isolates of A. rabiei from the Northern Great Plains and the Pacific Northwest were tested. Ninety-eight isolates collected between 2005 and 2007 were tested using an in vitro spore germination assay to determine the effective fungicide concentration at which 50% of conidial germination was inhibited (EC50) for each isolate-fungicide combination. A discriminatory dose of 1 µg/ml azoxystrobin was established and used to test 305 isolates from 2006 and 2007 for in vitro QoI fungicide sensitivity. Sixty-five percent of isolates collected from North Dakota in 2005, 2006, and 2007 and from Montana in 2007 were found to exhibit a mean 100-fold decrease in sensitivity to both azoxystrobin and pyraclostrobin when compared to sensitive isolates, and were considered to be resistant to azoxystrobin and pyraclostrobin. Under greenhouse conditions, QoI-resistant isolates of A. rabiei caused significantly higher amounts of disease than sensitive isolates on azoxystrobin- or pyraclostrobin-amended plants. These results suggest that disease control may be inadequate at locations where resistant isolates are present.

Variability in morphology and aggressiveness among North American vegetative compatibility groups of Colletotrichum coccodes.

North American isolates of Colletotrichum coccodes, representing six vegetative compatibility groups (NA-VCG), were used to study morphological and pathogenic variability. The objective was to determine if variability in conidial and microsclerotial size was related to pathogenicity. Significant differences were detected in length, width, and length/width ratios of conidia as well as in the length and width of microsclerotia among the NA-VCGs. The longest and widest conidia were produced by isolates belonging to NA-VCG1 and the largest microsclerotia were produced by isolates of NA-VCG2. Conidial and microsclerotial lengths and widths also were affected significantly by type of growth medium. There was no relationship between the size of conidia and the size of microsclerotia among the NA-VCGs studied. Conidial and microsclerotial size may affect inoculum potential and survival as isolates of NA-VCG2 have been demonstrated to occur more frequently than other NA-VCGs. Aggressiveness of 17 isolates of C. coccodes representing six NA-VCG's was studied on three potato cultivars using foliar and root inoculation methods. C. coccodes infection reduced tuber weight in all cultivars with both inoculation methods although tuber weight reductions were significantly higher following root inoculations than foliar inoculations. Pathogenic aggressiveness varied among NA-VCGs. Isolates belonging to NA-VCG2 and 3 were the least aggressive on potato foliage and isolates of NA-VCG1, 2, 3, 4, and 5 produced higher microsclerotial density on all three cultivars compared with isolates of NA-VCG6. Across inoculation methods, isolates of C. coccodes belonging to NA-VCG2 and 6 were the most aggressive based on reductions in tuber weight. Umatilla Russet was the most susceptible cultivar to C. coccodes compared to other cultivars regardless of inoculation method. These results demonstrate variability in morphology and pathogenic aggressiveness among the NA-VCGs of C. coccodes but these traits are not related.

First Report of Reduced Sensitivity to a QoI Fungicide in Isolates of Alternaria solani Causing Early Blight of Potato in Canada.

Early blight of potato (Solanum tuberosum L.) caused by Alternaria solani Sorauer is a frequent concern for potato growers in Canada. Management of early blight has relied on foliar fungicides that often include quinone outside inhibitor (QoI) fungicides such as azoxystrobin. In recent years, isolates of A. solani with reduced sensitivity to QoI fungicides, conferred by the presence of the F129L mutation (in the cytochrome b gene causing amino acid substitution of phenylalanine with leucine at position 129), have become widespread in potato-production areas of the United States, leading to a reduced efficacy of these products (3). Observations of reduced fungicide efficacy, following application of QoI fungicides to commercial fields in Manitoba, Canada in 2007, prompted an examination of the fungicide sensitivity of isolates of A. solani collected from fields in this province. Nine isolates of A. solani were obtained from potato foliage with typical early blight symptoms from four fields in Manitoba using standard protocols (2). Isolates were maintained on clarified V8 agar (1) and identified to species level based on conidial morphology (4). The sensitivity of each isolate to azoxystrobin was determined by assessing conidial germination on water agar plates amended with 0, 0.001, 0.01, 0.1, 1.0, or 10.0 mg/liter of azoxystrobin with protocols described previously (1). Two reference isolates of A. solani from North Dakota with known sensitivities to azoxystrobin and one isolate from Prince Edward Island (PEI), Canada, (a province yielding only isolates sensitive to azoxystrobin in previous surveys; R. D. Peters, unpublished data) were included in the assays. Calculated effective concentration (EC50) values (azoxystrobin concentration inhibiting conidial germination by 50%) were determined for each isolate response from two replications of the assays. The reference isolates of A. solani from North Dakota were sensitive or had reduced sensitivity to azoxystrobin with mean EC50 values of 0.02 and 0.2 mg/liter, respectively. The isolate from PEI was sensitive to azoxystrobin with a mean EC50 value of 0.04 mg/liter. By contrast, isolates of A. solani from Manitoba had reduced sensitivity to azoxystrobin with mean EC50 values from 0.2 to 0.8 mg/liter. Real-time PCR analysis of each isolate was performed (2) and confirmed the presence of the F129L mutation in the Manitoba isolates and the isolate with reduced sensitivity to azoxystrobin from North Dakota. The F129L mutation was absent in the azoxystrobin-sensitive wild-type isolates from PEI and North Dakota. To our knowledge, this is the first report of isolates of A. solani with reduced sensitivity to azoxystrobin in Canada. Since cross resistance among QoI fungicides has been demonstrated in A. solani isolates with the F129L mutation (3), adoption of resistance management strategies, including alternating QoI fungicides with fungicides having different modes of action and further monitoring pathogen populations for QoI sensitivity in Canadian production areas, is recommended. References: (1) J. S. Pasche et al. Plant Dis. 88:181, 2004. (2) J. S. Pasche et al. Plant Dis. 89:269, 2005. (3) J. S. Pasche and N. C. Gudmestad. Crop Prot. 27:427, 2008. (4) J. Rotem. The Genus Alternaria: Biology, Epidemiology, and Pathogenicity. The American Phytopathological Society, St. Paul, MN, 1994.

Baseline Sensitivity of Ascochyta rabiei to Azoxystrobin, Pyraclostrobin, and Boscalid.

Ascochyta rabiei, causal agent of Ascochyta blight on chickpea (Cicer arietinum), can cause severe yield loss in the United States. Growers rely on applications of fungicides with site-specific modes of action such as the quinone outside inhibiting (QoI) fungicides azoxystrobin and pyraclostrobin, and the carboximide fungicide boscalid, to manage disease. In all, 51 isolates collected prior to QoI fungicide registration and 71 isolates collected prior to boscalid registration in the United States were tested in an in vitro assay to determine the effective fungicide concentration at which 50% of conidial germination was inhibited (EC50) for each isolate-fungicide combination. The effect of salicylhydroxamic acid (SHAM) on conidia of A. rabiei in the presence and absence of azoxystrobin also was assessed to determine whether the fungus is capable of using alternative respiration. Five of nine A. rabiei isolates tested had significantly higher (P ≤ 0.05) EC50 values when SHAM was not included in media amended with azoxystrobin, indicating that A. rabiei has the potential to use alternative respiration to overcome fungicide toxicity in vitro. EC50 values of azoxystrobin and pyraclostrobin ranged from 0.0182 to 0.0338 µg/ml and from 0.0012 to 0.0033 µg/ml, with mean values of 0.0272 and 0.0023 µg/ml, respectively. EC50 values of boscalid ranged from 0.0177 to 0.4960 µg/ml, with a mean of 0.1903 µg/ml. Establishment of these baselines is the first step in developing a monitoring program to determine whether shifts in sensitivity to these fungicides are occurring in the A. rabiei pathogen population.

First Report of Tobacco rattle virus Causing Corky Ringspot in Potatoes Grown in Minnesota and Wisconsin.

In July 2007, potato tubers cv. Russet Burbank (RB) with necrotic arcs and spots were detected in three fields in Buffalo County, Wisconsin and one field in Benson County, Minnesota. Umatilla Russet (UR) potatoes harvested from the west half of a field in Swift County, MN had similar, but visually distinct necrotic lesions. Portions of one field in Minnesota were abandoned, and the stored potato crop from two fields in Wisconsin was rejected by processors, representing a total crop loss due to tuber necrosis. Tuber symptoms displayed in both cultivars resembled those described for corky ringspot caused by Tobacco rattle virus (TRV) (4). Total RNA was isolated from necrotic tuber tissue crushed in liquid nitrogen and extracted using the Total RNA Isolation Kit (Promega Corp., Madison, WI). These extracts were tested for the presence of TRV by reverse transcription (RT)-PCR using primers complementary to nucleotides 6555 to 6575 and identical to nucleotides 6113 to 6132 within the 3' terminal open reading frame of TRV RNA-1 (3). The expected 463-bp fragments were amplified from RB tubers. Nucleotide sequences from a Wisconsin and Minnesota isolate (GenBank Accession Nos. EU569290 and EU569291, respectively) were 99 to 100% identical to the corresponding region in a published TRV sequence (GenBank Accession No. AF055912). A 396-bp fragment was amplified from UR tubers and sequence data (GenBank Accession No. EU569292) indicated a unique 63 nucleotide sequence was substituted for a 129 nucleotide sequence spanning residues 227 to 357 of the 463-bp amplicon from the RB TRV isolates. Seven fragments were sequenced from different UR tubers and the 396-bp fragment was identical among them. The sequence outside the substituted region had 92% identity to the published TRV sequence. Amplification of the full-length TRV RNA2 using primers 179/180 located in the 5' and 3' untranslated regions (2) was successful for 28 and 0% of the RB and UR samples, respectively, suggesting that the RNA2 is not present in these strains or has undergone significant mutation. TRV-infected sap from both potato cultivars was mechanically transmitted to tobacco cv. Samsun NN and these plants subsequently tested positive for TRV by ELISA using ATCC antiserum PVAS 820. Ninety tubers exhibiting mild to severe symptoms of TRV were planted in the greenhouse. Each tuber was bisected laterally; necrotic tissue was removed from one half of the tuber and tested for the presence of TRV using RT-PCR protocols described above for RNA1. The remaining half was bisected horizontally and both sections were planted. Foliage from each emerged plant was subsequently also tested by RT-PCR for TRV RNA1. All RB tubers from Wisconsin tested positive for TRV, but only 7 of 24 emerged plants tested positive. Only 72% of the UR tubers and 4 of 25 emerged plants tested positive. TRV has been confirmed in California, Colorado, Florida, Idaho, Michigan (1), Oregon, and Washington. To our knowledge, this is the first report of corky ringspot in potato caused by TRV in Minnesota and Wisconsin. References: (1) W. W. Kirk et al. Plant Dis. 92:485, 2008. (2) S. A. MacFarlane. J. Virol. Methods. 56:91, 1996. (3) D. J. Robinson. J. Virol. Methods 40:57, 1992. (4) S. A. Slack. Tobacco rattle virus. Page 71 in: Compendium of Potato Diseases. 2nd ed. W. R. Stevenson et al., eds. The American Phytopathological Society, St. Paul, MN, 2001.

Effect of the F129L Mutation in Alternaria solani on Fungicides Affecting Mitochondrial Respiration.

Isolates of Alternaria solani previously collected from throughout the Midwestern United States and characterized as being azoxystrobin sensitive or reduced sensitive were tested for sensitivity to the Quinone outside inhibitor (QoI) fungicides famoxadone and fenamidone and the carboxamide fungicide boscalid. All three fungicides affect mitochondrial respiration: famoxadone and fenamidone at complex III, and boscalid at complex II. A. solani isolates possessing reducedsensitivity to azoxystrobin also were less sensitive in vitro to famoxadone and fenamidone compared with azoxystrobin-sensitive isolates, but the shift in sensitivity was of lower magnitude, approximately 2- to 3-fold versus approximately 12-fold for azoxystrobin. The in vitro EC50 values, the concentration that effectively reduces germination by 50% relative to the untreated control, for sensitive A. solani isolates were significantly lower for famoxadone and azoxystrobin than for fenamidone and boscalid; whereas, for reduced-sensitive isolates, famoxadone EC50 values were significantly lower than all other fungicides. Isolates of A. solani with reducedsensitivity to azoxystrobin were twofold more sensitive in vitro to boscalid than were azoxystrobin-sensitive wild-type isolates, displaying negative cross-sensitivity. All isolates determined to have reduced-sensitivity to azoxystrobin also were determined to possess the amino acid substitution of phenylalanine with leucine at position 129 (F129L mutation) using real-time polymerase chain reaction. In vivo studies were performed to determine the effects of in vitro sensitivity shifts on early blight disease control provided by each fungicide over a range of concentrations. Reduced-sensitivity to azoxystrobin did not significantly affect disease control provided by famoxadone, regardless of the wide range of in vitro famoxadone EC50 values. Efficacy of fenamidone was affected by some azoxystrobin reduced-sensitive A. solani isolates, but not others. Boscalid controlled azoxystrobin-sensitive and reduced-sensitive isolates with equal effectiveness. These results suggest that the F129L mutation present in A. solani does not convey cross-sensitivity in vivo among all QoI or related fungicides, and that two- to threefold shifts in in vitro sensitivity among A. solani isolates does not appreciably affect disease control.

Shift in Sensitivity of Alternaria solani in Response to QoI Fungicides.

Isolates of Alternaria solani, cause of potato early blight, collected in 1998 through 2001 from various potato growing areas across the midwestern United States, were tested for sensitivity to azoxystrobin. Isolates collected in 1998, prior to the introduction of azoxystrobin, were tested to establish the baseline sensitivity of the fungus to this fungicide. Isolates collected in subsequent years, not necessarily from the same sites as baseline isolates, were tested to determine if populations of A. solani had become less sensitive to azoxystrobin. Azoxystrobin sensitivity was determined utilizing an in vitro spore germination assay. The effective fungicide concentration that inhibited spore germination by 50% (EC50) was determined for each isolate. There was no significant difference in mean EC50 values between baseline isolates and all other isolates collected through 1999. Mean azoxystrobin EC50 values of A. solani isolates collected in 2000 and 2001 were significantly higher compared with means from previous years, and mean azoxystrobin EC50 values from 2001 were significantly higher than means from isolates collected in 2000. A subset of 54 A. solani isolates was evaluated in vitro for cross-sensitivity to pyraclostrobin and trifloxystrobin. A highly significant and strong correlation among the isolates tested for fungicide cross-sensitivity was detected between azoxystrobin and pyraclostrobin; however, the correlation between azoxystrobin and trifloxystrobin, and between trifloxystrobin and pyraclostrobin, was significant but weak. A second subset of five isolates was chosen for in vivo assessment of azoxystrobin, pyraclostrobin, and trifloxystrobin sensitivity. Disease severity on plants treated with azoxystrobin and pyraclostrobin was significantly greater with reduced-sensitive A. solani isolates compared with sensitive isolates. Disease severity was not statistically different between azoxystrobin reduced-sensitive and sensitive A. solani isolates on plants treated with trifloxystrobin. This is the first report of a shift in sensitivity to QoI fungicides in a fungus possessing only an anamorphic stage.