Spatial and Temporal Sensitivity of Alternaria Species Associated With Potato Foliar Diseases to Demethylation Inhibiting and Anilino-Pyrimidine Fungicides.

Early blight and brown spot, caused by Alternaria solani and Alternaria alternata, respectively, are important foliar diseases of potato, affecting both tuber yield and quality. Most of the commercial cultivars lack resistance; therefore, the application of foliar fungicides remains a primary disease management strategy. Baseline sensitivities of A. solani to difenoconazole and metconazole (demethylation inhibitors) using mycelial growth assay exhibited similar intrinsic activity against the pathogen with mean EC50 (the effective concentration at which the fungal growth is inhibited by 50%) values of 0.09 µg/ml. However, the sensitivity of individual baseline A. solani isolates to each fungicide varied substantially, resulting in very low and nonsignificant correlation coefficients among fungicides. Mean EC50 values for baseline A. alternata isolates in response to difenoconazole and metconazole were 0.14 and 0.26 µg/ml, respectively. The sensitivity of the majority of A. solani and A. alternata isolates collected from 2010 to 2014 from various potato production states was consistent with baseline isolates, therefore, these potato pathogens remain sensitive to the two demethylation inhibitor chemistries used to manage it. Baseline sensitivity assays of pyrimethanil (anilino-pyrimidine) also indicated great intrinsic activity against both foliar pathogens with mean EC50 values of 0.44 and 0.35 µg/ml for A. solani and A. alternata, respectively. Although A. alternata remains largely sensitive to pyrimethanil, 6 out of 245 A. solani isolates collected from 2010 to 2014 exhibited reduced-sensitivity to the fungicide in in vitro assays. Reduced-sensitive isolates were not controlled at most pyrimethanil doses except at 100 µg/ml in greenhouse in vivo efficacy tests. These chemistries remain valuable options for fungicide rotation programs in areas of high disease pressure.

Effect of Soil Moisture Management on the Development of Potato mop-top virus-Induced Tuber Necrosis.

Potato mop-top virus (PMTV), transmitted by Spongospora subterranea f. sp. subterranea, the causal agent of powdery scab in potato, has become one of the most important tuber necrosis viruses in the United States. The virus has been confirmed in six major potato-producing states in the United States since its identification in 2003. Currently, no control methods are available for PMTV or its vector. A growth-chamber experiment was conducted to investigate the potential of using moisture regime adjustments to manage tuber necrosis caused by PMTV. Two commercial potato cultivars with varying levels of sensitivity to PMTV, 'Dakota Crisp' and 'Ivory Crisp', were grown in soil obtained from a PMTV-infested field. Over the course of the plant growth cycle, plants of each cultivar were subjected to moisture regimes of wet throughout (WT), wet early/dry late (WEDL), dry early/wet late (DEWL), and dry throughout (DT). Soil moisture levels of 90 and 60% field capacity were considered wet and dry, respectively, while early and late refer to first and last 50 days after planting, respectively. Results of visual assessment conducted 3 months after storage showed significant differences in root gall formation, powdery scab on tubers, and PMTV tuber necrosis among moisture regimes. Powdery scab incidence was significantly higher in the WT and DEWL regimes than WEDL and DT regimes. PMTV tuber necrosis incidence did not differ between the WT and DEWL or between DEWL and WEDL moisture regimes. However, PMTV tuber necrosis incidence was significantly higher in WT than under WEDL and DT regimes. The incidence of PMTV tuber necrosis was significantly lower in the DT regime than in the other three moisture regimes. Severity of PMTV tuber necrosis was significantly higher in the WT than the other regimes, which did not differ statistically among themselves. A significant interaction was found between cultivar and moisture regime on root gall formation, with the highest number of galls found on Ivory Crisp grown in the WT moisture regime. A significant correlation was found between powdery scab incidence on tubers and PMTV-induced tuber necrosis incidence. The results of this study provide useful basic information upon which further work can be built for formulating irrigation management schemes that have the potential of reducing the likelihood of powdery scab infection and PMTV-induced tuber necrosis development in potato.

Sensitivity of Potato Cultivars to Potato mop-top virus-Induced Tuber Necrosis.

Potato (Solanum tuberosum) cultivars representing four market classes were assessed for sensitivity to Potato mop-top virus (PMTV)-induced tuber necrosis in three separate trials in a field in North Dakota known to be infested with PMTV. Reverse-transcription polymerase chain reaction confirmed the presence of PMTV in randomly selected samples. Results of tuber necrosis assessments conducted during storage demonstrated that sensitivity to PMTV-induced tuber necrosis among cultivars follows a continuum of tolerant to sensitive. As a group, the russet-skinned cultivars had a lower incidence of tuber necrosis than red-, yellow-, and white-skinned cultivars. The incidence and severity of PMTV-induced tuber necrosis in trial 1 were significantly correlated with those parameters in trial 2 across years. Significant correlations also existed between the incidence of powdery scab on tubers and the incidence of PMTV-induced tuber necrosis in trial 1 across years. A significant correlation was also found between root gall numbers and powdery scab incidence and severity on tubers as well as PMTV-induced tuber necrosis incidence in trial 2. The results of this study provide growers with disease management options by avoiding cultivars highly sensitive to PMTV-induced tuber necrosis development and potentially replacing them with tolerant cultivars in the same market class. It is apparent from these studies that field assessments can be used for the development of PMTV-resistant germplasm for use in future breeding strategies.

First Report of Potato mop top virus Causing Potato Tuber Necrosis in Colorado and New Mexico.

Potato mop top virus (PMTV) is considered the type member of the genus Pomovirus. PMTV is an important pathogen of potato vectored by the plasmodiophorid Spongospora subterranea f. sp. subterranea (Sss), which causes powdery scab of potato (1). Sss and PMTV are usually associated with cool and humid environments. PMTV-infected potato tubers generally exhibit internal hollow necrotic spots or concentric rings, and the virus is known to cause significant economic losses in Northern Europe, North and South America, and Asia (4). PMTV in the United States was first reported in Maine (2). Potato (Solanum tuberosum L.) tubers cv. FL2048 and cv. Atlantic were sent to our laboratory from fields in Saguache County in Colorado and in San Juan County in New Mexico, respectively, during the spring of 2013. The tubers from both locations had multiple, internal, concentric, necrotic arcs and circles. Internal tissue with necrotic lesion from six symptomatic tubers from each location were crushed in liquid nitrogen followed by ribonucleic acid extraction using a Total RNA Isolation kit (Promega Corp., Madison, WI). These extracts were tested by reverse transcription (RT)-PCR using three different sets of previously published primers for molecular detection of PMTV. The primer set H360/C819 targeting the coat protein (CP) on RNA 3 of PMTV yielded an amplicon (H360-CO and H360-NM) of 460 bp (4). The second set of primers, pmtF4/pmtR4 (5), amplified a 417-bp product (PMTF-CO and PMTF-NM) in RNA 2, and the third set, PMTV-P9/PMTV-M9 (3), designed to amplify the region encoding an 8-KD cysteine-rich protein in RNA 3 of PMTV, yielded a 507-bp amplicon (PMTV9-CO and PMTV9-NM). The amplicons generated from RT-PCR using all three sets were cloned (PGEMT-easy) and sequenced. Since the sequences from symptomatic tuber extracts from each location were identical to their respective primer sets, a consensus sequence from each primer set was submitted to National Center for Biotechnology Information (NCBI) GenBank. Sequences obtained from the H360/C819 primer set (GenBank Accession Nos. KM207013 and KM207014 for H360-CO and H360-NM, respectively) were 100% identical to the corresponding CP regions of PMTV isolates from North Dakota (HM776172). Sequences from the pmtF4/pmtR4 primer set (KM207015 and KM207016 for PMTF-CO and PMTF-NM, respectively) were 100% identical to the corresponding protein in RNA2 of PMTV isolates from North Dakota (GenBank HM776171), and sequences from the PMTV-P9/PMTV-M9 primer set (KM207017 and KM207018 for PMTV9-CO and PMTV9-NM respectively) were 99% identical to the corresponding protein in RNA3 of PMTV isolates (AY187010). The 100-99% homology of the sequences from this study to the corresponding PMTV sequences published in NCBI confirmed the occurrence of symptoms in the tubers from both Colorado and New Mexico due to PMTV. None of the symptomatic tubers tested positive for Tobacco rattle virus, Tomato spotted wilt virus, Alfalfa mosaic virus, Potato leafroll virus, or the necrotic strains of Potato virus Y by RT-PCR. To our knowledge, this is the first report of PMTV in potato in states of Colorado and New Mexico. References: (1) R. A. C. Jones and B. D. Harrsion. Ann. Appl. Biol. 63:1, 1969. (2) D. H. Lambert et al. Plant Dis. 87:872, 2003. (3) T. Nakayama et al. Am. J. Pot. Res. 87:218, 2010. (4) J. Santala et al. Ann. Appl. Biol. Online publication. DOI: 10.1111/j.1744-7348.2010.00423.x (5) H. Xu et al. Plant Dis. 88:363, 2004.

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.

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.

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.

First Report of Potato mop-top virus in North Dakota.

Potato mop-top virus (PMTV) is the type member of the genus Pomovirus. PMTV is an important pathogen of potato, causing significant economic losses in Northern Europe, North and South America, and Asia (3). PMTV in the United States was first reported in Maine (2). PMTV is vectored by the plasmodiophoromycete Spongospora subterranea cv. subterranea, which causes powdery scab of potato (1). S. subterranea and PMTV are usually associated with cool and humid environments. In the spring of 2010, six potato tubers of cv. Russet Burbank were received from a commercial potato farm in Grand Forks County in North Dakota. The tubers had multiple, internal, concentric, necrotic arcs and circles. The presence of PMTV in the necrotic lesions was verified by a positive double-antibody sandwich-ELISA (Agden Ltd., Ayr, Scotland). The tuber lesions had an absorbance value (405 nm) at least two times greater than that of the negative control sample, which consisted of a healthy tuber. Total RNA was extracted from lesions of six different tubers that tested positive by ELISA using a Total RNA Isolation kit (Promega Corp. Madison, WI). These extracts were tested for PMTV by reverse transcription (RT)-PCR using two different sets of primers. The primer set H360/C819 targeted the coat protein (CP) of PMTV and yielded an amplicon of 460 bp (4). The amplicons generated from the necrotic lesions were cloned (TOPO Cloning; Invitrogen, Carlsbad, CA) and sequenced. Another set of primers, pmtF4/pmtR4, designed to bind to a region in RNA 2 of PMTV, yielded a 417-bp amplicon that also was cloned and sequenced (3). The sequences from all six tuber lesions were identical for the respective primer sets. A consensus sequence for each primer pair was submitted to GenBank (Accession No. HM776171 for primers pmtF4/pmtR4 and No. HM776172 for primers H360/C819). The sequences obtained from the H360/C819 and pmtF4/pmtR4 amplicons were 99% identical to the corresponding regions of PMTV isolates from Northern Europe (GenBank Accession Nos. AM503629 and AJ277556, respectively). Freeze-dried, necrotic tuber tissue from all six tubers was also tested at a USDA Laboratory in Prosser, WA by RT-PCR with the H360/C819 primer pair (4), confirming the results above. Cloning and sequencing of one of the amplicons revealed 100% similarity to the sequence described above for these primers (GenBank Accession No. HM776172), confirming the presence of PMTV in the symptomatic tubers. None of the symptomatic tubers tested positive for Tobacco rattle virus, Tomato spotted wilt virus, Alfalfa mosaic virus, Potato leafroll virus, or the necrotic strains of Potato virus Y by RT-PCR. To our knowledge this is the first report of PMTV in North Dakota. References: (1) R. A. C. Jones and B. D. Harrsion. Ann. Appl. Biol. 63:1, 1969. (2) D. H. Lambert et al. Plant Dis. 87:872, 2003. (3) J. Santala et al. Ann. Appl. Biol. Online publication. DOI: 10.1111/j.1744-7348.2010.00423.x (4) H. Xu et al. Plant Dis. 88:363, 2004.

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.

First Report of Tobacco rattle virus Associated with Corky Ringspot in Potatoes Grown in North Dakota.

Tobacco rattle virus (TRV) belongs to the genus Tobravirus and causes a stem mottle of potato (Solanum tuberosum) foliage and necrotic arcs and rings in tubers referred to as corky ringspot. This virus is generally transmitted by a number of species of stubby-root nematode. The virus is widespread and has been reported in California, Colorado, Florida, Idaho, Michigan, Oregon, Washington, Minnesota, and Wisconsin (2). In the spring of 2009, we received potato tubers of cv. Russet Burbank with internal necrotic arcs very similar to those caused by TRV from potato storages located in Grand Forks and Dickey counties of North Dakota. Total RNA was extracted from the necrotic lesions of two tubers from each location using the Total RNA Isolation kit (Promega Corp., Madison WI). These extracts were tested for TRV by reverse transcription (RT)-PCR using primers complementary to nucleotides 6555 to 6575 (Primer A) and identical to nucleotides 6113 to 6132 (Primer B) within the 3' terminus of TRV-SYM RNA-1 (GenBank Accession No. X06172) (3). The expected 463-bp amplicons from two separate tuber samples from each county were cloned (TOPO Cloning; Invitrogen, Carlsbad, CA) and sequenced. The sequences obtained from the four clones at both locations were found to be identical to each other and were 99% identical to the corresponding regions of TRV isolates from Michigan and Florida (GenBank Accession Nos. EU315226.1 and AF055912.1, respectively). Since sequences from all four clones were identical, only one of the sequences was submitted to Genbank (Accession No. GQ223114) and thus represents a consensus sequence. The extracts also tested positive in RT-PCR with a second set of primers corresponding to sequences in TRV RNA-2 yielding a 3.8-kbp amplicon (1). No evidence was found by RT-PCR for several other viruses that cause tuber necrosis in potato (Potato mop top virus, Tomato spotted wilt virus, Alfalfa mosaic virus, and tuber necrosis strains of Potato virus Y). The virus was mechanically transmitted by inoculating sap from symptomatic tubers from both counties to tobacco cv. Samsun NN, which showed typical bright yellow patches and spots on leaves 2 weeks postinoculation. TRV was confirmed in tobacco by RT-PCR from total RNA extracted from tobacco leaves with both sets of the aforementioned primers. To our knowledge, this is the first report of TRV in North Dakota and the first report of corky ringspot disease of potato in this state. References: (1) J. M. Crosslin et al. Virus Res. 96:99, 2003. (2) N. C. Gudmestad et al. Plant Dis. 92:1254, 2008. (3) D. J. Robinson. J. Virol. Methods 40:57, 1992.

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.

First Report of Tomato spotted wilt virus Causing Potato Tuber Necrosis in Texas.

In the summer of 2008, potato (Solanum tuberosum L.) tubers (cvs. FL1867, FL2053, and FL1922) from commercial fields near Dalhart, TX were observed with distinct external erumpent rings and severe internal discolorations including blotches, spots, and dry, cork-like tissue. The presence of rings suggested the possible involvement of one or more viruses. Nucleic acid from seven of eight symptomatic tubers received in Washington (cvs. FL1867 and FL1922) tested positive for Tomato spotted wilt virus (TSWV) by reverse transcription (RT)-PCR with primers TSWV 1 and 2 (3). Similarly, tubers (cvs. FL1867 and FL2053) received in North Dakota tested positive for TSWV with forward (S1983) and reverse (S2767) primers of Tsompana et al. (4). The 777-bp amplicon obtained with primers TSWV 1 and 2 and the 803-bp amplicon obtained with primers S1983 and S2767 were cloned and three clones of each were sequenced. Analysis of the consensus sequences and BLAST comparisons confirmed the Washington and North Dakota sequences were indeed TSWV in origin and were each 98 to 99% identical to the corresponding nucleocapsid region of a number of TSWV isolates and most closely related to an isolate detected in eastern black nightshade from Colorado (GenBank No. AY777475). The deduced amino acid sequences of the 777-bp nucleocapsid open reading frame differed from AY777475 at only two residues in each of the Washington and North Dakota sequences. The Washington and North Dakota derived sequences were deposited with GenBank (Nos. FJ882069 and FJ882070, respectively). None of the eight symptomatic tubers tested positive for Tobacco rattle virus (TRV), Alfalfa mosaic virus (AMV), or the necrotic strains of Potato virus Y (PVY) by RT-PCR. Mechanical transmission tests were conducted by grinding symptomatic tissue of a TSWV-positive FL1867 tuber in 10 volumes of 30 mM potassium phosphate buffer, pH 8.0, containing 10 mM of sodium diethyldithiocarbamate and 10 mM of sodium thioglycollate and rub inoculated onto Carborundum-dusted leaves of Samsun NN tobacco. Approximately 10 days after inoculation, chlorotic-necrotic rings were present on the inoculated leaves and circular necrotic lesions developed on the upper leaves. Dark stem lesions were also present on inoculated tobacco, and after 3 weeks, the upper leaves developed severe, spreading lesions. Tissue from the symptomatic tobacco tested positive for TSWV by RT-PCR (primers TSWV 1 and 2) and also with a TSWV-specific ImmunoStrip (Agdia, Inc., Elkhart, IN), but tested negative for TRV, AMV, and PVY by RT-PCR. TSWV has been reported on field-grown potatoes in North Carolina (1) and has been reported on potatoes in Australia (2) and in other parts of the world (referenced in 1). To our knowledge, this is the first report associating TSWV with tuber necrosis on potatoes in Texas. References: (1) J. A. Abad et al. Am. J. Potato Res. 82:255, 2005. (2) L. J. Latham and R. A. C. Jones. Aust. J. Agric. Res. 48:359, 1997. (3) R. Navarre et al. Am. J. Potato Res. 86:88, 2009. (4) M. Tsompana et al. Mol. Ecol. 14:53, 2005.

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.

First Report of a Defect of Processing Potatoes in Texas and Nebraska Associated with a New Phytoplasma.

An outbreak of a new potato disease occurred in Texas and Nebraska causing a serious defect in potato chips produced from commercial processing potatoes. The defect consists of patchy brown discoloration of chips and can be a cause for rejection of contracted potatoes by the processor. Infected potato plants exhibit symptoms of the purple top wilt syndrome similar to those of the purple top disease in processing potatoes caused by clover proliferation phytoplasma recently found in Washington and Oregon (3). Foliar symptoms include stunting, chlorosis, slight purple coloration of new growth, swollen nodes, proliferated axillary buds, and aerial tubers. Tuber symptoms include mild vascular discoloration and brown flecking of medullary rays. Seed potatoes from affected plants produce hair sprouts. Total nucleic acid was extracted from leaf and stolon tissue of symptomatic plants in the field and from tuber samples exhibiting the defect from commercial storages. Nested polymerase chain reactions (PCR) were performed using phytoplasma-universal 16SrDNA-based primers (P1/P7 and R16F2n/R16R2) (2) to detect the presence of phytoplasmas in these samples. A negative control, devoid of DNA templates in the reaction mix, was included in all PCR assays. In 2004, 13 foliar samples tested positive for phytoplasmas using PCR. None of the apparently symptomless plants or tubers tested positive. Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA using enzymes AluI, MseI, HhaI, BfaI, and Tsp509I indicated that four samples are associated with a phytoplasma belonging to subgroup A (16SrI-A) of the "Candidatus Phytoplasma asteris" (aster yellows phytoplasma) group (16SrI), and nine plant samples were associated with a new phytoplasma related to, but distinct from, the stolbur phytoplasma group (16SrXII). Nucleotide sequence analysis of cloned 16S rDNAs (GenBank Accession Nos. DQ174114-DQ174123) confirmed the results on the basis of RFLP analyses. Sequences of cloned 16S rDNAs were analyzed with previously described phytoplasma strains available in GenBank using DNAStar's (Madison, WI) Lasergene software MegAlign program. The new phytoplasma is only distantly related to the stolbur phytoplasma, sharing 96.6% sequence homology. In 2005, 14 defective tuber samples from storage and 16 symptomatic plants from the field tested positive for the new phytoplasma. In Texas and Nebraska, it appears that at least two distinct phytoplasmas seem to be involved in the disease complex contributing to the defects of processed products produced from infected potatoes. Previous reports have suggested a similar defect of chipping potatoes, but the phytoplasma associated with the disease was not identified (1). To our knowledgek, this the first report of this new phytoplasma associated with disease and defects of potato and the first report of this phytoplasma in the United States. References: (1) E. E. Bantarri et al. Trans. ASAE 33:221, 1990. (2) I.-M. Lee et al. Int. J. Sys. Bacteriol. 48:1153, 1998. (3) I.-M. Lee et al. Plant Dis. 88:429, 2004.

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.