Meta-Analysis of the Effects of QoI and DMI Fungicide Combinations on Fusarium Head Blight and Deoxynivalenol in Wheat.

Field trials were conducted in 17 U.S. states to evaluate the effects of quinone outside inhibitor (QoI) and demethylation inhibitor (DMI) fungicide programs on Fusarium head blight index (IND) and deoxynivalenol (DON) toxin in wheat. Four DMI-only treatments applied at Feekes 10.5.1, five QoI-only treatments applied between Feekes 9 or Feekes 10.5, three QoI+DMI mixtures applied at Feekes 10.5, and three treatments consisting of a QoI at Feekes 9 followed by a DMI at Feekes 10.5.1 were evaluated. Network meta-analytical models were fitted to log-transformed mean IND and DON data and estimated contrasts of log means were used to obtain estimates of mean percent controls relative to the nontreated check as measures of efficacy. Results from the meta-analyses were also used to assess the risk of DON increase in future trials. DMI at Feekes 10.5.1 were the most effective programs against IND and DON and the least likely to increase DON in future trials. QoI-only programs increased mean DON over the nontreated checks and were the most likely to do so in future trials, particularly when applied at Feekes 10.5. The effects of QoI+DMI combinations depended on the active ingredients and whether the two were applied as a mixture at heading or sequentially. Following a Feekes 9 QoI application with a Feekes 10.5.1 application of a DMI reduced the negative effect of the QoI on DON but was not sufficient to achieve the efficacy of the Feekes 10.5.1 DMI-only treatments. Our results suggest that one must be prudent when using QoI treatments under moderate to high risk of FHB, particularly where the QoI is used without an effective DMI applied in combination or in sequence.

Use of PCR-RFLP Analysis to Monitor Fungicide Resistance in Cercospora beticola Populations from Sugarbeet (Beta vulgaris) in Michigan, United States.

Genetic resistance to Quinone outside inhibitor (QoI) and benzimidazole fungicides may be responsible for a recent decline in efficacy of chemical control management strategies for Cercospora leaf spot (CLS) caused by Cercospora beticola in Michigan sugarbeet (Beta vulgaris) fields. The target genes and fungicide resistance mutations are known for these two fungicides. Based on this, two standard polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays were developed to detect the G143A and E198A point mutations in the fungal mitochondrial cytochrome b and the ß-tubulin genes, respectively. These mutations confer a high level of resistance to either QoI or benzimidazole fungicides. The presence of the G143A and E198A mutations was monitored within C. beticola populations recovered from Michigan sugarbeet production fields collected in 2012. Both the QoI-resistant cytochrome b allele and the benzimidazole-resistant ß-tubulin allele were detected directly from leaf tissue following a PCR-RFLP assay. Using either detection assay, the G143A and E198A mutations were detected in over 90% of the 118 field samples originating from Michigan sugarbeet production under fungicide management programs for CLS control. Monitoring of the G143A and E198A mutations in fields located in 9 counties and 58 townships indicated that the mutations were widespread in Michigan sugarbeet production areas. The PCR-based assays used and developed in this study were effective in detecting the presence of the G143A and E198A mutations in C. beticola field populations from Michigan.

First Report of Late Blight Caused by Phytophthora infestans Clonal Lineage US-23 on Potato in Idaho.

Late blight, caused by Phytophthora infestans (Mont.) de Bary, is a destructive disease of potato (Solanum tuberosum) and tomato (S. lycopersicum) in the United States. Prior to 2007, the US-8 clonal lineage was the predominant genotype in the United States (4). Since 2007, a significant genetic change in the population of P. infestans occurred in the eastern United States with the appearance of new isolates with unique genotypes and epidemiological characteristics (3). These new genotypes US-22, US-23, and US-24 are sensitive to metalaxyl and represent mating types A2, A1, and A1, respectively (1,2). Prior to 2012, only US-8 had been documented in Idaho (5). In 2013, late blight was discovered in late August on potato crops (cv. Russet Norkotah) in Bingham and Madison counties, ID. Infected foliage (four samples from Bingham County and five from Madison) was sent to Michigan State University and the University of Wisconsin for confirmation of P. infestans and characterization of the isolates. Five sections from the leading edge of lesions were excised with a sterilized scalpel and placed on potato tuber slices ('Dark Red Norkotah'). Pathogen sporulation on the excised lesions was enhanced by incubation in plastic boxes lined with moistened paper towels for 5 days at 18°C in the dark. The sporulating lesions were transferred onto pea agar medium (160 g peas, 5 g sucrose, 15 g agar, 700 ml distilled water) amended with 50 mg/ml vancomycin. Ten pure cultures were obtained for each of 4 isolates per county by hyphal tipping. Cellulose acetate electrophoresis was conducted to determine Gpi allozyme genotype of the 4 isolates (4). The allozyme banding patterns were 100/100 at the Gpi locus, consistent with previously reported analyses of the US-23 genotype (1,2). Genomic DNA was extracted from 10 pure cultures using the DNeasy Plant Mini Kit (Qiagen, Germantown, MD), and SSR analyses were performed. Microsatellite markers Pi02, Pi4B, Pi63, PiG11, and D13 were used in SSR analyses. Pi02, Pi4B, and Pi63 had alleles of 162/164, 213/217, and 270/279 bp in size, respectively which is consistent with the reference US-23 genotype (1). However, heterozygosity was detected at locus D13 in the Idaho genotype with allele size of 134/210 bp and an additional allele of 140/155/176 bp at locus PiG11. This is different from the standard US-23 genotype (homozygous alleles 134/134 at locus D13 and two alleles 140/155 at locus PiG11). These allele changes indicate the isolates may be variants of US-23 isolates as all other phenotypic characteristics were similar to those of reference US-23 isolates. The Idaho genotypes were sensitive to metalaxyl both in vitro on rye A agar medium amended with metalaxyl at <0.1 ppm, and in vivo on Ridomil treated foliage tests at <0.1 ppm (1,2). Mating type assays confirmed the pathogen to be the A1 mating type. In the 2009 and 2010 late blight epidemics in the eastern United States, US-23 was the predominant genotype, but to our knowledge this genotype has never been reported previously in Idaho. Thus, this is the first known report of P. infestans genotype US-23 causing late blight on potato in Idaho, indicating a change in the population of P. infestans. In Idaho, the source of this genotype remains unknown, although infected tomatoes have been implicated in the widespread dissemination of this genotype of P. infestans in the eastern United States. References: (1) G. Danies et al. Plant Dis. 97:873, 2013. (2) C. Hu et al. Plant Dis. 96:1323, 2012. (3) K. Deahl. (Abstr.) Phytopathology 100:S161, 2010. (4) S. B. Goodwin et al. Plant Dis. 79:1181, 1995. (5) USAblight. Recent US Genotypes. Online: www.usablight.org/node/52 , retrieved 3 January 2014.

First Report of Fusarium proliferatum Causing Dry Rot in Michigan Commercial Potato (Solanum tuberosum) Production.

Fusarium dry rot of potato (Solanum tuberosum L.) is a postharvest disease caused by several Fusarium spp. Thirteen Fusarium spp. have been implicated in dry rot of potatoes worldwide. Among them, 11 species have been reported causing potato dry rot of seed tubers in the northern United States (1). Historically, Fusarium sambucinum was the predominant species in Michigan potato production (3). Dry rot symptomatic tubers (n = 972) were collected from Michigan commercial potato storage facilities in 2011 and 2012 to determine the composition of Fusarium spp. Sections were cut from the margins of necrotic tissue with a sterile scalpel and surface disinfested in 0.6% sodium hypochlorite for 10 s, rinsed twice in sterile distilled water, and dried on sterile filter paper. The tissue sections were plated on half-strength potato dextrose agar (PDA) amended with 0.5 g/liter of streptomycin sulfate. Dishes were incubated at 23°C in the dark for 7 days. Putative Fusarium isolates were transferred onto water agar and hyphal tips from the margin of actively growing cultures were removed with a sterile scalpel and plated to carnation leaf agar (CLA) and half-strength PDA to generate pure cultures. Seven hundred and thirty Fusarium isolates were collected using these techniques. Preliminary identification of the 730 isolates was based on colony and conidial morphology on PDA and CLA, respectively. While F. oxysporum and F. sambucinum were isolated as expected from prior reports (3), three isolates of F. proliferatum were also identified. On CLA, macroconidia of F. proliferatum were sparse, slender, and mostly straight, with three to five septae (4). Microconidia were abundant, usually single celled, oval or club-shaped in short chains or false heads on monophialides and polyphialides (4), and chlamydospores were absent. On PDA, abundant white mycelium was produced and turned violet with age. Koch's postulates were confirmed through pathogenicity testing on disease-free potato tubers cvs. Atlantic and Russet Norkotah. Tubers were surface disinfested for 10 min in 0.6% sodium hypochlorite and rinsed twice in distilled water. Three tubers of each cultivar per isolate were wounded at the apical end of the tuber to a depth of 4 to 10 mm with a 4 mm diameter cork-borer. Tubers were inoculated by inserting a mycelial plug from a 7-day-old culture grown on PDA into the wound and incubating the tubers at 20°C for 21 days. All Fusarium isolates were tested. Control tubers were inoculated by inserting a water agar plug. Pathogenicity and virulence testing were replicated three times and repeated. Tubers inoculated with F. proliferatum developed typical potato dry rot symptoms but no dry rot symptoms were observed on control tubers. Fusarium proliferatum was re-isolated from symptomatic tubers, confirming Koch's postulates. To our knowledge, this is the first report of F. proliferatum causing potato dry rot in Michigan. References: (1) E. Gachango et al. Plant Dis. 96:1767. (2) D. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (3) M. L. Lacy and R. Hammerschmidt. Fusarium dry rot. Extension Bulletin. Retrieved from http://web1.msue.msu.edu/msue/iac/onlinepubs/pubs/E/E2448POT, 23 May 2010. (4) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Wiley-Blackwell, Hoboken, NJ, 2006.

Field efficacy of nonpathogenic Streptomyces species against potato common scab.

AIMS: The primary objective of these experiments was to reduce pathogenicity and virulence of endemic soil pathogenic Streptomyces strains that cause potato common scab (CS) using nonpathogenic Streptomyces strains to suppress CS in a field situation. METHODS AND RESULTS: Nonpathogenic Streptomyces strains that had shown potential for mitigating CS in greenhouse assays were used in Michigan and Pennsylvania fields known to have high CS disease pressure. Five biocontrol (BC) strains and three potato cultivars were used in 2009, and three BC strains and three cultivars were used in 2010 in each location. The effects of BC strains on CS disease incidence and severity differed between locations, years and potato cultivars. When overall means of individual BC treatments were compared with nontreated controls, CS incidence and severity were decreased by all BC strains in PA2009, PA2010 and MI2010, particularly in cultivar 'Yukon Gold' in MI. Biocontrol treatments also significantly shifted the proportions of superficial, raised and pitted lesion types in some cultivar/biocontrol treatment combinations. CONCLUSIONS: All BC strains significantly reduced CS incidence and severity on 'Yukon Gold' in three of four trials, and one BC strain significantly improved the lesion severity profile in cultivar 'Atlantic'. No BC strain significantly reduced CS incidence and severity on all potato cultivars in the different years and locations. SIGNIFICANCE AND IMPACT OF THE STUDY: Several nonpathogenic Streptomyces strains showed potential to reduce CS incidence and severity on two important potato-chipping cultivars in the field. These results can be further applied to reduce CS disease severity in potatoes.

First Report of QoI Insensitive Cercospora beticola on Sugar Beet in Ontario, Canada.

Cercospora beticola Sacc. causes Cercospora leaf spot (CLS) of sugar beet (Beta vulgaris L.) and is the most destructive foliar disease of sugar beet worldwide (1). The QoI fungicide pyraclostrobin has been an important management tool for CLS in Canada since 2003. Beginning in 2010, some growers reported poor disease control after applying pyraclostrobin. Leaf disk samples with CLS lesions were collected in September 2012 from 16 commercial fields located in Kent and Lambton Counties, Ontario, Canada. These counties (ca. 300,000 ha) encompass the major commercial sugar beet production area in Ontario (ca. 3,925 ha). CLS severity ranged from low to severe among the sampling sites. Leaf discs with a single leaf spot were cut from leaves using a hole punch. Spots were up to 5 mm in diameter with tan, light brown, or sometimes gray centers. DNA was extracted from leaf discs using a Qiagen DNeasy Plant Mini Kit (Germantown, MD) according to the manufacturer's instructions. PCR was used to amplify a fragment of the C. beticola cytochrome b (CYTB) gene (4). Pure cultures were obtained by placing plant tissue in a moist chamber and transferring single spores to V8 juice agar. PCR products were sequenced for 32 samples at the Genomics Technology Support Facility (Michigan State University, East Lansing, MI) and 25 were confirmed to have 100% identity with the sequence of QoI-resistant C. beticola from Michigan (2) and to QoI-resistant isolates from GenBank (Accession Nos. JQ619933 and JQ360628). The remaining seven had 100% identity with a sensitive isolate (EF176921.1). Each resistant isolate contained a change in codon 143 that is predicted to lead to a substitution of G143A in the cytochrome b gene. This G143A mutation has been associated with QoI resistance in a number of fungi (3). To confirm the result, a conidium germination bioassay was carried out using nine isolates with the G143A mutation on sugar beet leaf agar covered with water agar amended with pyraclostrobin at concentrations ranging from 0 to 54.3 µg/ml and distributed on a spiral gradient using an Eddyjet II spiral plater. The medium was supplemented with salicylhydroxamic acid (SHAM) to block the alternate oxidation pathway. Following incubation at 25°C for 2 days, the distance between the center of the plate at which conidial germination was 50% of the maximum observed growth (EC) and the point at which conidial germination terminated were measured (TEC). The EC50 values were determined from the SGE software for each isolate by entering the EC and TEC values, respectively. The estimated EC50 for a representative wild type (sensitive) isolate was 0.03 µg/ml, while the value for the resistant isolate could not be calculated because it was greater than the highest concentration tested (54.3 µg/ml). Additionally, in the controls with no SHAM or fungicide, the resistant isolate showed a consistent reduced germination rate compared to the sensitive isolate (30.0% and 93.5% germination, respectively). Confirmation of fungicide insensitivity will require a re-evaluation of current management practices in Ontario to minimize economic losses due to CLS. References: (1) B. J. Jacobsen and G. D. Franc. Compendium of Beet Diseases and Pests, 2nd ed, APS Press, St. Paul, MN, 2009. (2) W. Kirk et al. New Dis. Rep. 26:3, 2012. (3) Z. Ma and T. J. Michailides. Crop Prot. 24:853, 2005. (4) A. Malandrakis et al. Pestic. Biochem. Physiol. 100:87092, 2011.

First Report of Boscalid and Penthiopyrad-Resistant Isolates of Alternaria solani Causing Early Blight of Potato in Michigan.

Early blight of potato (Solanum tuberosum) is caused by Alternaria solani and occurs annually in Michigan. If left uncontrolled, it can result in yield losses exceeding 20% and impact stored potatoes. The disease is commonly managed using succinate dehydrogenase inhibitor (SDHI) fungicides (1). Unfortunately, recent studies have shown that SDHI resistance has increased dramatically over the past 2 years in A. solani populations (1,2). To investigate the occurrence of SDHI resistance in Michigan, potato leaves with early blight symptoms were collected from fields in Montcalm and Ionia counties, MI, in 2012. To obtain A. solani isolates from leaves, small pieces of leaf tissue (5 × 5 mm) were excised from the center of lesions and transferred on to water agar. Plates were incubated at 25°C overnight to allow conidia to germinate. Single germinated A. solani conidia were transferred to potato dextrose agar (PDA) and incubated at 25°C for 7 days. The identity of cultures was confirmed by colony and conidial morphology (3). Nineteen A. solani isolates were obtained and each was screened for sensitivity to the SDHI fungicides boscalid, penthiopyrad, and fluopyram, using a 50 ppm discriminatory dose based on EC50 values previously determined (2). Mycelial plugs (~5.5 mm) were transferred to amended and non-amended PDA plates that were incubated at 25°C for 7 days. An isolate was considered highly resistant if fungal growth relative to control plates exceeded 50%, moderately resistant if it was between 35 and 50%, and sensitive if it was less than 35% (2). A sensitive A. solani isolate (AS11) from Bonners Ferry, ID, was used as a control in these experiments. Of all isolates tested, 11% were highly resistant to both boscalid and penthiopyrad and 5% were moderately resistant to both fungicides, 21% were moderately resistant to penthiopyrad alone, and the remaining isolates (84 and 68% respectively) were sensitive to the two fungicides. None of the isolates tested were resistant to fluopyram. Recently, two major mutations, H227R in SdhB and H133R in SdhD, have been identified in highly resistant A. solani isolates in Idaho (2). Because the majority of the identified mutations occur near the 3' end of each subunit, this region was amplified and sequenced using the following primer sets: SdhB (5'-TACTGGTGGAACCAGGAGGAGTA-3' and 5'-CATACCACTCTAGGTGAAG-3'), SdhC (5'-CCAAATCACCTGGTACGCCTCG-3' and 5'-TCATCCGAGGAAGGTGTAGTAAAGGCTG-3'), and SdhD (5'-CCGACTCTATTCTCTGCGCCCT-3' and 5'-CTCGAAAGAGTAGAGGGCAAGACCCA-3'). In this study, all of the isolates that were highly resistant to both boscalid and penthiopyrad were found to contain the H133R mutation in SdhD, which correlated with the strongest resistance phenotype. To our knowledge, this is the first report of resistance to SDHI fungicides in populations of A. solani on potato in Michigan. These data are concerning as they indicate that the highly resistant isolates have already developed cross-resistance between boscalid and penthiopyrad, despite penthiopyrad not yet having regular use in Michigan. Although all of the isolates tested were sensitive to fluopyram, the discovery of isolates resistant to boscalid and penthiopyrad suggests that all SDHI fungicides should be considered at high risk of resistance development in Michigan. References: (1) K. Fairchild et al. Crop Prot. 49:31, 2013. (2) T. Miles et al. Plant Pathol. doi: 10.1111/ppa.12077, 2013. (3) P. Wharton et al. Plant Dis. 96:454, 2012.

First Report of Rhizoctonia solani AG2-2IIIB Infecting Potato Stems and Stolons in the United States.

Rhizoctonia solani is an important pathogen of potato (Solanum tuberosum) causing qualitative and quantitative losses. It has been associated with black scurf and stem canker. Isolates of the fungus are assigned to one of 13 known anastomosis groups (AGs), of which AG3 is most commonly associated with potato disease (2,4). In August 2011, diseased potato plants originating from Rupert, ID (cv. Western Russet) and Three Rivers, MI (cv. Russet Norkotah) were received for diagnosis. Both samples displayed stem and stolon lesions typically associated with Rhizoctonia stem canker. The presence of R. solani was confirmed through isolation as previously described (4) and the Idaho and Michigan isolates were designated J11 and J8, respectively. AG was determined by sequencing the rDNA internal transcribed spacer (ITS) region using primers ITS5 and ITS4 (3). The resulting sequences of the rDNA ITS region of isolates J8 and J11 (GenBank Accession Nos. HE608839 and HE608840, respectively) were between 97 and 100% identical to that of other AG2-2IIIB isolates present in sequence databases (GenBank Accession Nos. FJ492075 and FJ492170, respectively). Koch's postulates were confirmed for each isolate by carrying out the following protocol. Each isolate was cultured on potato dextrose agar for 14 days. Five 10-mm agar plugs were then placed on top of seed tubers (cv. Maris Piper) in 1-liter pots containing John Innes Number 3 compost (John Innes Manufacturers Association, Reading, UK). Pots were held in a controlled environment room at 18°C with 50% relative humidity and watered as required. After 21 days, plants were removed and assessed for disease. Typical Rhizoctonia stem lesions were observed and R. solani was successfully reisolated from symptomatic material. To our knowledge, this is the first report of AG2-2IIIB causing disease on potatoes in the United States. In the United States, AGs 2-1, 3, 4, 5, and 9 have all been previously implicated in Rhizoctonia potato disease (2). AG2-2IIIB should now also be considered a potato pathogen in the United States. Knowledge of which AG is present is invaluable when considering a disease management strategy. AG2-2IIIB is a causal agent of sugar beet (Beta vulgaris) root rot in Idaho (1). Sugar beet is commonly grown in crop rotation with potato and such a rotation could increase the risk of soilborne infection to either crop by AG2-2IIIB. References: (1) C. A. Strausbaugh et al. Can. J. Plant Pathol. 33:210, 2011. (2) L. Tsror. J. Phytopatol. 158:649, 2010. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, Inc., New York, 1990. (4) J. W.Woodhall et al. Plant Pathol. 56:286, 2007.

Fusarium spp. Causing Dry Rot of Seed Potato Tubers in Michigan and Their Sensitivity to Fungicides.

A survey of seed potato tubers in Michigan seed production storage facilities was carried out during 2009 and 2010. Fusarium spp. associated with tuber dry rot symptoms were identified to species and tested for sensitivity to difenoconazole, fludioxonil, and thiabendazole. Symptomatic tubers (n = 370) were collected from a total of 51 seed lots, from which 228 isolates of Fusarium were recovered and identified to 11 species. Fusarium oxysporum was the most commonly isolated species (30.3%), followed by F. equiseti (19.3%). F. sambucinum and F. avenaceum were third most prevalent (each at 13.6%). Less prevalent species (each at 4 to 10%) included F. cerealis, F. solani, and F. acuminatum; and species present at ≤3% included F. sporotrichioides, F. torulosum, F. tricinctum, and F. graminearum. Representative isolates of all species were pathogenic when inoculated onto seed tubers ('Dark Red Norland'). Isolates of F. sambucinum were the most virulent. All 228 isolates of Fusarium were sensitive to difenoconazole (effective fungicide concentration that caused 50% inhibition of mycelial growth [EC50] < 5 mg/liter). Insensitivity to fludioxonil (EC50 > 100 mg/liter) was detected only for F. sambucinum and F. oxysporum isolates at 8.9 and 20.4%, respectively. All isolates were sensitive to thiabendazole (EC50 < 5 mg/liter), except for those of F. sambucinum (EC50 > 100 mg/liter). Therefore, knowledge of what Fusarium spp. are present in seed potato storage facilities in Michigan may be important if using fludioxonil or thiabendazole for seed piece treatment but not when using difenoconazole.

Strobilurin (QoI) Resistance in Populations of Erysiphe necator on Grapes in Michigan.

Powdery mildew, caused by Erysiphe necator, is the most common and destructive disease of grapes (Vitis spp.) worldwide. In Michigan, it is primarily controlled with fungicides, including strobilurins (quinone outside inhibitors [QoIs]). Within the United States, resistance to this class of fungicides has been reported in E. necator populations in some east coast states. Among 12 E. necator isolates collected from five Michigan vineyards in 2008, one carried the G143A single-nucleotide mutation responsible for QoI resistance. This isolate was confirmed to be resistant in a conidium germination assay on water agar amended with trifloxystrobin at 0.001, 0.01, 0.1, 1, 10, or 100 µg/ml and salicylhydroxamic acid (100 mg/liter). The mutant isolate was able to germinate on media amended with 100 µg/ml trifloxystrobin, whereas a representative wild-type isolate did not germinate at concentrations higher than 0.1 µg/ml. In 2009, 172 isolates were collected from a total of 21 vineyards (juice and wine grapes): three vineyards with no fungicide application history (baseline sites), six research vineyards, and 12 commercial vineyards. QoI resistance was defined as the effective concentration that inhibited 50% of conidial germination (EC50) > 1 µg/ml. Isolates from baseline sites had EC50 values mostly below 0.01 µg/ml, while isolates that were highly resistant to trifloxystrobin (EC50 > 100 µg/ml) occurred in five research and three commercial wine grape vineyards at frequencies of 40 to 100% and 25 to 75% of the isolates, respectively. The G143A mutation was detected in every isolate with an EC50 > 1 µg/ml. These results suggest that fungicide resistance may play a role in suboptimal control of powdery mildew observed in some Michigan vineyards and emphasizes the need for continued fungicide resistance management.

Characterization of a New Streptomyces Strain, DS3024, That Causes Potato Common Scab.

A novel strain of Streptomyces (named DS3024) was isolated from a potato field in Michigan in 2006. The taxonomy of the organism was determined by morphology, biochemistry, and genetic analysis. Analysis of the 16S ribosomal RNA gene sequence indicated that the organism was most similar to an isolate of Streptomyces sp., ME02-6979.3a, which is not pathogenic to potato tubers but is distinct from other known pathogenic Streptomyces spp. Strain DS3024 has genes that encode thaxtomin synthetase (txtAB), which is required for pathogenicity and virulence, and tomatinase (tomA), which is a common marker for many pathogenic Streptomyces spp. However, the nec1 gene (associated with virulence in most pathogenic Streptomyces spp.) was not detected. The new strain was capable of growth at pH 4.5, caused necrosis on potato tuber slices, and produced thaxtomin A. In greenhouse experiments, DS3024 caused scab symptoms on potato tubers similar to those caused by Streptomyces scabies on tubers of potato cv. Atlantic, which is scab susceptible. We propose that DS3024 is a new strain of Streptomyces capable of causing common scab on potato tubers. The prevalence of this strain of Streptomyces in potato-producing areas in the north-central United States has not been determined.

Optimizing Fungicide Timing for the Control of Rhizoctonia Crown and Root Rot of Sugar Beet Using Soil Temperature and Plant Growth Stages.

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

First Report of Corky Ringspot Caused by Tobacco rattle virus on Potatoes (Solanum tuberosum) in Michigan.

Potato is the fourth most important agricultural commodity in Michigan and is grown for table stock, chip processing, and seed. Tubers are either processed or fresh packed immediately following harvest or sent to storage. Tubers of potato cv. FL1879 representing two separate fields in Huron County were removed from separate storage facilities in February and March of 2007, and substantial internal necrosis was observed in 1 to 2% of the tubers. Symptoms included arcs similar to those caused by Tobacco rattle virus (TRV). This virus is a member of the genus Tobravirus and is transmitted by a number of species of stubby-root nematodes (Paratrichodorus or Trichodorus spp.). Stubby-root nematodes have been reported previously from Michigan (1). To confirm the presence of TRV, nucleic acid extractions were made from these symptomatic tubers (3). Samples were initially tested for TRV by reverse transcription (RT)-PCR using primers specific to the 16 kDa open reading frame on genomic RNA-1 (2). Samples from both storage facilities were positive. The 463-bp RT-PCR product, amplified with these primers, was sequenced (GenBank Accession No. EU315227) and was 99.6% identical to the corresponding region of two TRV isolates from Florida and Washington (GenBank Accession Nos. AF055912 and EU315226, respectively). In addition, sap from cv. FL1879 tuber samples was used to transmit the virus mechanically to tobacco cv. Samsun NN, which produced typical TRV symptoms following inoculation, and sap extracts from the tobacco leaves also tested positive with antiserum specific to TRV upon subsequent ELISA testing. Corky ringspot can result in substantial losses, with entire potato fields being rejected because of internal tuber damage. Once found, fields must be considered permanently at risk to this disease due to the large host range of both the virus and the nematode vector. This disease has been previously found in the United States in California, Colorado, Florida, Idaho, Washington, Oregon, and it is likely present in Indiana. To our knowledge, this is the first report of corky ringspot and TRV on potato in Michigan. References: (1) G. W. Bird and N. Knobloch. Plant Dis. Rep. 60:76, 1976. (2) J. M. Crosslin and P. E. Thomas. Am. Potato J. 72:605, 1995. (3) G. G. Presting et al. Phytopathology 85:436, 1995.

First Report of Potato Tuber Sprout Rot Caused by Fusarium sambucinum in Michigan.

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

The Generation and Quantification of Resistance to Dimethomorph in Phytophthora infestans.

The generation of dimethomorph resistance in Phytophthora infestans was attempted using ethidium bromide/UV light mutagenesis and repeated culturing on dimethomorph-amended medium. Ethidium bromide/UV mutagenesis created two isolates of P. infestans with resistance factors for dimethomorph >20, i.e., the ratio of the 50% effective concentration (EC50) of the mutant to that of the wild-type. With repeated culturing on dimethomorph-amended medium, the rate of growth (mm diameter/day) increased until the tenth subculture for most P. infestans isolates. Resistance factors generated from repeated culturing were <8 for all isolates. For most isolates, the generation of dimethomorph resistance resulted in reduced growth rates on nonamended medium, regardless of the level of resistance or induction treatment. Additionally, the frequency of infection of leaf disks and whole tubers was significantly reduced in >20% of the isolates repeatedly subcultured on dimethomorph-amended medium. Regardless of the induction treatment, reduced fitness was common for all P. infestans isolates, indicating a potential biological cost associated with dimethomorph resistance. Based on these results, the development of field resistance to dimethomorph in P. infestans is unlikely with the currently employed resistance management strategies.

Variations in the Sensitivity of Phytophthora infestans Isolates from Different Genetic Backgrounds to Dimethomorph.

The sensitivities of 11 isolates of Phytophthora infestans to dimethomorph were examined at all stages of the asexual life cycle and when inoculated onto potato leaf discs. In vitro zoospore encystment and cyst germination were highly sensitive to dimethomorph with 50% reduction of mycelial growth and cyst germination (EC50) values for most isolates <0.20 µg/ml, whereas direct sporangia germination and in vitro hyphal growth and sporulation were less sensitive (means of 0.45 and 0.22 µg/ml, respectively). Zoosporogenesis was not significantly inhibited at the maximum dimethomorph concentration examined, 10 µg/ml. Significant differences (Fisher's least significant difference, P = 0.05) in the EC50 values were present between isolates for all stages of the asexual life cycle, except direct sporangia germination and zoosporogenesis. Sensitivity ratios between the least- and most-sensitive isolates were 6.11, 12.14, 12.36, and 10.56 for hyphal growth, in vitro sporulation, zoospore encystment, and cyst germination, respectively. Application of dimethomorph at 1,000 µg/ml to potato leaf discs at 24 or 48 h before inoculation completely inhibited symptom incidence for most isolates, whereas application after inoculation generally was not significantly different from the untreated control, regardless of concentration. Sporulation from leaf discs treated with dimethomorph at 24 or 48 h after inoculation was completely inhibited for all isolates with dimethomorph at 1,000 µg/ml, even when symptom incidence was not significantly reduced.

Effect of Host Plant Resistance and Reduced Rates and Frequencies of Fungicide Application to Control Potato Late Blight.

Field experiments were conducted during 1998 to 2000 to determine the response of commercial potato cultivars and advanced breeding lines (ABL) differing in susceptibility to foliar late blight (caused by Phytophthora infestans) to reduced rates and frequencies of residual, contact fungicide applications. When environmental conditions were most favorable for the development of late blight, the lowest application rate of the fungicides chlorothalonil or fluazinam (33% of the manufacturers' recommended application rate [MRAR]) gave unsatisfactory control of potato late blight. Under conditions moderately conducive for late blight development, effective control was achieved with 33 to 66% MRAR with either fungicide. The Michigan State University advanced selection, MSG274-3, was the least susceptible ABL tested and, during 1998 to 2000, late blight was effectively managed using reduced rates of fungicides. Application rates of chlorothalonil (33 to 100% MRAR) significantly reduced late blight in the cultivar Snowden (5-day application interval) compared with the nontreated control; whereas, late blight was not effectively controlled in Snowden even at 100% MRAR of chlorothalonil at either 10- or 15-day application intervals in 1999 or 2000. The ABL MSG274-3 was the least susceptible of all cultivars and ABL used in this study, and required minimal chemical protection against late blight. The study demonstrates that ABL with reduced susceptibility to late blight can be managed with reduced fungicide rates and longer application intervals, thus offering more economical control of this disease.