Cross-Resistance of Succinate Dehydrogenase Inhibitors (SDHI) in Botrytis cinerea and Development of Molecular Diagnostic Tools for SDHI Resistance Detection.

Succinate dehydrogenase inhibitors (SDHIs) are keystone synthetic fungicides used to manage Botrytis cinerea in several hosts. In this study, we investigated the cross-resistance between five new SDHIs (pyraziflumid, isofetamid, benzovindiflupyr, fluxapyroxad, and pydiflumetofen) with commonly used SDHIs boscalid and fluopyram. Different mutations were detected in the sdhB gene in B. cinerea collected from Michigan grapes, and their frequency and EC50 value were determined. Among 216 B. cinerea boscalid-resistant isolates, five different mutations were detected, including H272R/Y, P225F/H, and N230I, at frequencies of 82.6, 4.3, 11.5, 0.4, and 5.3%, respectively. Five isolates of each genotype were used to screen the cross-resistance of the SDHIs. We classified the resistance profile of our mutants into five patterns. We report that all tested mutants were sensitive to benzovindiflupyr, indicating that it can be used as an effective fungicide against all B. cinerea mutants identified in this study. In addition, fluopyram, pydiflumetofen, and isofetamid can provide effective control according to which type of mutation is present in the field. We also developed and compared two molecular diagnostic tools, rhAMP and TaqMan assays, for rapid detection of SDHI resistance-associated mutants in B. cinerea. We report that the TaqMan assay was more successful than the rhAMP assay in detecting the B. cinerea mutant DNA at ≤10 pg and in a single assay was capable of monitoring two amino acid positions. Our results provide essential information about new SDHIs and provide molecular tools for monitoring SDHI resistance mutations, which will assist in gray mold disease control.

Carboxylic Acid Amide but Not Quinone Outside Inhibitor Fungicide Resistance Mutations Show Clade-Specific Occurrence in Pseudoperonospora cubensis Causing Downy Mildew in Commercial and Wild Cucurbits.

Since its reemergence in 2004, Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew (CDM), has experienced significant changes in fungicide sensitivity. Presently, frequent fungicide applications are required to control the disease in cucumber due to the loss of host resistance. Carboxylic acid amides (CAA) and quinone outside inhibitors (QoI) are two fungicide groups used to control foliar diseases in cucurbits, including CDM. Resistance to these fungicides is associated with single nucleotide polymorphism (SNP) mutations. In this study, we used population analyses to determine the occurrence of fungicide resistance mutations to CAA and QoI fungicides in host-adapted clade 1 and clade 2 P. cubensis isolates. Our results revealed that CAA-resistant genotypes occurred more prominently in clade 2 isolates, with more sensitive genotypes observed in clade 1 isolates, while QoI resistance was widespread across isolates from both clades. We also determined that wild cucurbits can serve as reservoirs for P. cubensis isolates containing fungicide resistance alleles. Finally, we report that the G1105W substitution associated with CAA resistance was more prominent within clade 2 P. cubensis isolates while the G1105V resistance substitution and sensitivity genotypes were more prominent in clade 1 isolates. Our findings of clade-specific occurrence of fungicide resistance mutations highlight the importance of understanding the population dynamics of P. cubensis clades by crop and region to design effective fungicide programs and establish accurate baseline sensitivity to active ingredients in P. cubensis populations.

Fungicide Resistance Profiles of Botrytis cinerea Isolates From Michigan Vineyards and Development of a TaqMan Assay for Detection of Fenhexamid Resistance.

Botrytis cinerea on grapes causes bunch rot at both pre- and postharvest stages, in which losses can reach up to 100%. Chemical control primarily relies on the prophylactic use of site-specific fungicides. Repeated applications of these products raise the risk of fungicide resistance development in B. cinerea populations, which can result in disease control failures. To determine the extent of resistance, B. cinerea isolates were collected from grape clusters in the northwest and southwest grape growing regions of Michigan in 2014 and 2018 (n = 115 and 125, respectively). These isolates were phenotyped using discriminatory doses of eight fungicides to determine the levels of resistance. Fungicide resistance increased from 2014 to 2018, mostly affecting the active ingredients fenhexamid, fluopyram, and iprodione. B. cinerea isolates resistant to multiple fungicides were detected in 2014 and 2018, with a higher frequency of resistance in 2018. TaqMan real-time PCR has been developed to detect B. cinerea fungicide resistance to fenhexamid and to differentiate the erg27 F412S/I/V alleles. The TaqMan assay was tested for sensitivity, specificity, and reproducibility on purified DNA and infected grape tissue samples. Our data provide essential information to growers about the efficacy for B. cinerea control using the available botryticides. Furthermore, the developed fenhexamid markers will be transferred to diagnostic clinics to assist growers in the management of bunch rot before resistance-related control failures occur.

First Report of a Binucleate Rhizoctonia (AG-A) from Potato Stems Infecting Potatoes and Sugarbeet in the Pacific Northwest.

Rhizoctonia solani causes economically important diseases on potatoes and sugarbeet throughout the world (2). R. solani is a species complex of 13 anastomosis groups (AGs) of which R. solani AG3-PT is most commonly associated with potato and AG2-2 and AG4 with sugarbeet. However, several AGs, including AG2-2 and AG4, have been recorded causing potato diseases (2,3). In summer 2012, plants in potato fields in Idaho were sampled for R. solani. Isolations were attempted from symptomatic plants. DNA extracted from the resulting pure Rhizoctonia cultures was screened using a real-time PCR assay for AG3-PT (3). For the isolates that tested negative for AG3-PT, AG was determined by amplifying and sequencing the rDNA internal transcribed spacer (ITS) region using the primers ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The resulting sequences of two isolates (isolates 204 and 206, GenBank Accession No. KC782951) shared 99% identity with other AG-A isolates (AY927358 and AY927356). Koch's postulates were confirmed for isolate 206 by placing five 10-mm plugs, from 10-day-old potato dextrose agar (PDA) cultures, onto the surface of a soil-less potting mix (composed of peat moss, perlite, and sand) of 1-liter pots, where non-inoculated PDA plugs served as a control. Each pot contained a 'Rosara' seed tuber or three ungerminated (BETASEED - BTS 27RR10) sugarbeet seeds (n = 5). Pots were incubated in a glasshouse between 18 and 22°C for 1 month and then assessed for disease. For potatoes, a pigmented necrosis was observed at the soil interface in 88% of the stems and plants were stunted relative to the non-inoculated controls. A significant reduction in root growth was observed in 60% of the germinated sugarbeet plants. Control plants of both potatoes and beets were asymptomatic. For reisolation, 1-cm sections were taken from each potato stem and germinated beet plant, surface sterilized, and placed on alkaline water agar. The reisolated fungi were identified using morphology and a subset was confirmed by sequencing. Isolate 206 was successfully recovered from 84% of the potato stems and from 20% of the sugarbeet seedlings. In a similar experiment, 2-month-old potato and sugarbeet plants were inoculated using 50 g of autoclaved barley grains (inoculated with isolate 206) per 1-liter pot. Between 40 and 60% of inoculated plants appeared stunted in both cases. Pigmented necrosis was observed at the soil interface on 45% of the potato stems and reduced root growth was observed in the 50% of the sugarbeet plants. Control plants were asymptomatic. To our knowledge, this is the first report of the binucleate AG-A causing disease in Idaho on potato stems. BNR species have previously been isolated from potato (4) and sugarbeet plants (1). The binucleate Rhizoctonia AG-A caused disease on potato stems and sugarbeet roots and was readily reisolated. Since sugarbeet is commonly grown in rotation with potato in Idaho, such a rotation could increase the risk of soilborne infection to either crop by AG-A. It is known that AGs can differ in fungicide sensitivity (2), and thus a knowledge of which AGs may be present is important when considering disease management strategies. References: (1) C. A. Strausbaugh et al. Can. J. Plant Pathol. 33:210, 2011. (2) L. Tsror. Biology, Epidemiology and Management of Rhizoctonia solani on Potato 158:649, 2010. (3) J. Woodhall et al. Eur. J. Plant Pathol. 136:273, 2013. (4) Y. G. Yang and X. H. Wu. Plant Dis. 97:1246, 2013.

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.

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.

First Report of Blueberry Leaf Rust Caused by Thekopsora minima on Vaccinium corymbosum in Michigan.

Leaf rust symptoms have been noticed sporadically on northern highbush blueberry plants (Vaccinium corymbosum L.) in Michigan for the past 8 years. In 2009, leaf rust was seen in several cultivated blueberry fields and on greenhouse-grown blueberry plants in southwest Michigan. In 2010, leaf rust was widespread throughout western Michigan and particularly evident in the fall, sometimes resulting in premature defoliation. Cultivars Rubel, Jersey, Elliott, Liberty, and Brigitta were most commonly affected. Both the 2009 and 2010 growing seasons were characterized by above-average precipitation in early to mid-summer. Early symptoms on the adaxial leaf surface consisted of roughly circular yellow spots that later developed brown, necrotic centers. Older lesions were more angular and sometimes surrounded by a purplish border. In the fall, a "green island" effect was sometimes apparent around the lesions. On the abaxial side, numerous yellow-to-orange rust pustules (uredinia) were visible. Uredinia were dome shaped, erumpent, 100 to 400 µm in diameter, clustered, and sometimes coalescing. Urediniospores were broadly obovate with dark yellowish content and measured 19 to 25 × 16 to 20 µm (average 22 × 18 µm, n = 30). Spore walls were hyaline, echinulate, and 1.0 to 1.5 µm thick with obscure germ pores. Uredinia were examined with light and scanning electron microscopy for the presence of conspicuous ostiolar cells characteristic of Naohidemyces vaccinii (Wint.) Sato, Katsuya et Y. Hiratsuka, but none were observed. No telia or teliospores were observed. On the basis of morphology, the pathogen was identified as Thekopsora minima P. Syd. & Syd. (3,4) and a sample was deposited in the U.S. National Fungus Collection (BPI 881107). Genomic DNA was extracted from urediniospores of rust isolates from six different locations, and a 267-bp fragment of the ITS2 region was amplified and sequenced using the primers ITS3 and ITS4 (GenBank Accession No. HQ661383). All sequences were identical to each other and shared 99% identity (232 of 234 bp) with a T. minima isolate from South Africa (GenBank Accession No. GU355675). The alternate host, hemlock (mostly Tsuga canadensis L.) is a common and valuable conifer in the Michigan landscape. Hemlock trees were not examined for the presence of aecia but are assumed to play a role in the epidemiology of the disease in Michigan because leaf rust tends to be more severe near hemlock trees. Pucciniastrum vaccinii (G. Wint.) Jorst. was considered the causal agent of blueberry leaf rust until Sato et al. (1,4) identified three unique species. While T. minima has been reported on black huckleberry (Gaylussacia baccata [Wangenh.] K. Koch) in Michigan (4), to our knowledge, this is the first report of T. minima on highbush blueberry in the state. T. minima has been reported on highbush blueberry in Delaware and New York (4), Japan (2), and South Africa (3). The severity of the outbreak in 2010 warrants further research into economic losses, epidemiology, and management of the disease. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Botany and Mycology Laboratory, ARS, UDSA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 2010. (2) T. Kobayashi. Page 1227 in: Index of Fungi Inhabiting Woody Plants in Japan. Host, Distribution and Literature. Zenkoku-Noson-Kyoiku Kyokai Publishing Co., Tokyo, 2007. (3) L. Mostert et al. Plant Dis. 94:478, 2010. (4) S. Sato et al. Trans. Mycol. Soc. Jpn. 34:47, 1993.

First Report of Pythium sterilum Causing Root Rot of Blueberry in the United States.

In September 2009, ~40 declining blueberry plants (Vaccinium corymbosum L. 'Jersey') were observed in a poorly drained area of a 30-year-old field near Fennville, MI. The stunted bushes had yellow leaves and defoliation; others were completely dead. The grower reported that the bushes had been declining over several years. Root samples tested positive in a Phytophthora ELISA test (Agdia Inc., Elkhart IN). Twenty root pieces (2 cm long and 2 to 3 mm in diameter) were surface disinfested and plated on Rye A agar; five yielded fungal-like colonies that were subcultured on potato dextrose agar (PDA). One isolate was white and grew slowly (3 to 4 mm/day at 22 to 24°C). Three isolates were white and grew faster (10 to 12 mm/day at 22 to 24°C) in a chrysanthemal pattern. The fifth was a Fusarium sp. DNA of the white colonies was extracted and the internal transcribed spacer (ITS) region was sequenced using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') primers. The slow-growing morphotype had 99% identity to Phytophthora sp. isolate 92-209C (Accession No. EU106591) in GenBank but failed to induce symptoms in multiple inoculation tests. The fast-growing morphotype (Accession No. HQ398249) had 98% identity to Pythium sterilum UASWS0265 from declining alder stands in Poland (Accession No. DQ525089). Sequencing of the COXII gene using the FM66/FM58 primer set (3) yielded a product (Accession No. HQ721468) with 100% identity to P. sterilum GD32a from forest soil in Poland (Accession No. EF421185). Hyphae were hyaline, coenocytic, and 4 to 7 µm wide with some swellings at the tips (7 to 9 µm wide). No sporangia, oogonia, or antheridia were observed. Mycelium tested positive in the ELISA test described above. According to Agdia Inc., 10 of 19 tested Pythium spp. have shown similar cross reactivity. Pythium spp. are known to cause root rot of blueberries in Oregon (2). In British Columbia, P. sterilum was commonly isolated from roots of declining blueberry bushes (4). P. sterilum Belbahri & Lefort only reproduces asexually (1). Our isolate was similar but did not produce sporangia in water or on PDA, V8 juice agar, Rye A agar, or water agar. Roots of 10 2-month-old 'Bluecrop' cuttings were placed in an aqueous suspension of rinsed mycelium (0.1 g/ml) from 21-day-old cultures grown in V8 broth or in sterile deionized water (control). After 1 h, plants were potted in peat moss/perlite (2:1) or autoclaved sand (five each) and placed in a glasshouse at 25°C. After 7 days, inoculated plants in both soil types had wilted or collapsed with significant necrosis on the roots and primary shoot. Control plants showed no symptoms. In a similar experiment with 6-month-old plants in sand, symptoms appeared after 10 to 12 days. The pathogen was recovered from surface-disinfested root and stem sections of all inoculated plants but not control plants and its identity was confirmed by sequencing of the ITS region. To our knowledge, this is the first report of P. sterilum on blueberries in the United States. While this disease appears to be uncommon in Michigan, it is a potential cause of plant decline, the diagnosis of which may be complicated by cross reactivity in ELISA testing. References: (1) L. Belbahri et al. FEMS Microbiol. Lett. 255:209, 2006. (2) D. R. Bryla and R. G. Linderman. HortScience 43:260, 2008. (3) F. N. Martin. Mycologia 92:711, 2000. (4) S. Sabaratnam. BC Plant Health Fund Final Report. B.C. Retrieved from http://www.agf.gov.bc.ca/cropprot/phf_final_report.pdf , 2008.

First Report of Stem Blight Caused by Calonectria colhounii (Anamorph Cylindrocladium colhounii) on Greenhouse-Grown Blueberries in the United States.

Necrotic stems and leaves were observed on 2- to 4-month-old, rooted microshoot plants (Vaccinium corymbosum L. 'Liberty' and 'Bluecrop', V. angustifolium Aiton 'Putte', and V. corymbosum × V. angustifolium 'Polaris') in a Michigan greenhouse in 2008 and 2009. As the disease progressed, leaves fell off and 80 to 100% of the plants died in some cases. Root rot symptoms were also observed. A fungus was isolated from stem lesions. On potato dextrose agar (PDA), cultures first appeared light tan to orange, then rusty brown and zonate with irregular margins. Chains of orange-brown chlamydospores were abundant in the medium. Macroconidiophores were penicillately branched and had a stipe extension of 220 to 275 × 2.5 µm with a narrowly clavate vesicle, 3 to 4 µm wide at the tip. Conidia were hyaline and cylindrical with rounded ends, (1-)3-septate, 48 to 73 × 5 to 7 (average 60 × 5.5) µm and were held together in parallel clusters. Perithecia were globose to subglobose, yellow, 290 to 320 µm high, and 255 to 295 µm in diameter. Ascospores were hyaline, 2- to 3-septate, guttulate, fusoid with rounded ends, slightly curved, and 30 to 88 × 5 to 7.5 (average 57 × 5.3) µm. On the basis of morphology, the fungus was identified as Calonectria colhounii Peerally (anamorph Cylindrocladium colhounii Peerally) (1,2). The internal transcribed spacer region (ITS1 and ITS2) of the ribosomal DNA and the ß-tubulin gene were sequenced (GenBank Accession Nos. HQ909028 and JF826867, respectively) and compared with existing sequences using BLASTn. The ITS sequence shared 99% maximum identity with that of Ca. colhounii CBS 293.79 (GQ280565) from Java, Indonesia, and the ß-tubulin sequence shared 97% maximum identity with that of Ca. colhounii CBS 114036 (DQ190560) isolated from leaf spots on Rhododendron sp. in North Carolina. The isolate was submitted to the Centraalbureau voor Schimmelcultures in the Netherlands (CBS 129628). To confirm pathogenicity, 5 ml of a conidial suspension (1 × 105/ml) were applied as a foliar spray or soil drench to four healthy 'Bluecrop' plants each in 10-cm plastic pots. Two water-sprayed and two water-drenched plants served as controls. Plants were misted intermittently for 2 days after inoculation. After 7 days at 25 ± 3°C, drench-inoculated plants developed necrotic, sporulating stem lesions at the soil line, while spray-inoculated plants showed reddish brown leaf and stem lesions. At 28 days, three drench-inoculated and one spray-inoculated plant had died, while others showed stem necrosis and wilting. No symptoms were observed on control plants. Fungal colonies reisolated from surface-disinfested symptomatic stem, leaf, and root segments appeared identical to the original isolate. Cy. colhounii was reported to cause a leaf spot on blueberry plants in nurseries in China (3), while Ca. crotalariae (Loos) D.K. Bell & Sobers (= Ca. ilicicola Boedijn & Reitsma) causes stem and root rot of blueberries in North Carolina (4). To our knowledge, this is the first report of Ca. colhounii causing a disease of blueberry in Michigan or the United States. Because of its destructive potential, this pathogen may pose a significant threat in blueberry nurseries. References: (1) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. The American Phytopathological Society, St. Paul, MN, 2002. (2) L. Lombard et al. Stud. Mycol. 66:31, 2010. (3) Y. S. Luan et al. Plant Dis. 90:1553, 2006. (4) R. D. Milholland. Phytopathology 64:831, 1974.

First Report of Grape Root Rot Caused by Roesleria subterranea in Michigan.

In September of 2008, declining grapevines were observed in two vineyards (Vitis interspecific hybrids 'Canada Muscat' and 'Chardonel') in Fennville, MI. Affected vines were stunted with shortened internodes and yellow leaves; others had dead cordons or were entirely dead. The grower reported that vines were losing vigor and collapsing during a period of 2 years. Renewal trunks would collapse during the second season of growth. Several symptomatic vines showed signs of root decay. On one vine, distinctive fruiting bodies (mazaedia) were apparent on the roots below the soil line and resembled those of Roesleria subterranea (Weinm.) Redhead (2,3,4). The mazaedia were 4 to 5 mm tall and 1 mm in diameter with white-to-tan stipes and powdery, gray-to-greenish hemispherical heads. Ascospores were hyaline to light grayish green, disk shaped, and 4 to 6 µm in diameter. This fungus, also known as R. hypogaea Thüm & Pass., has been previously reported to cause grape root rot, vine decline, and replant problems in North America and Europe (2,3,4). The fungus was cultured from ascospores on potato dextrose agar (PDA). Colonies grew slowly (approximately 2 mm per day at 22 to 24°C) and were green in the center. No spores were produced. DNA was extracted, and internal transcribed spacer (ITS) sequences obtained by PCR were compared with known sequences using BLASTn (1). Our isolate had 100% ITS sequence homology to an isolate from Germany, Roesleria subterranea strain IB (Accession No. EF060304.1). To test for pathogenicity, the fungus was grown in potato dextrose broth for 14 days at 22 to 24°C. An aqueous suspension (0.1 g of fungus per ml) was prepared by blending mycelia with sterile deionized water (SDW) in a food processor. Five two-node, rooted 'Chardonnay' cuttings (45 days old) were placed in the suspension. Five other cuttings were placed in SDW (control). After 3 h, plants were removed and repotted in fresh BACTO soil (Michigan Peat Company, Houston, TX) and kept in a growth chamber at 23°C with a 16/8-h light/dark cycle. After 25 days, inoculated plants were, on average, 63% smaller with 77% lower fresh-root weight and fewer fine roots than the control plants. The pathogen was recovered from surface-disinfested roots of all five inoculated plants but not from the control plants. Cultures appeared similar to the original isolate and their identity was confirmed by ITS sequencing. To our knowledge, this is the first report of R. subterranea on grapes in Michigan and the Midwest. This fungus needs to be recognized as a potential cause of vine decline and replant problems in Midwestern vineyards. References: (1) S. F. Altschul et al. J. Mol. Biol. 215:403, 1990. (2) W. Gärtel. Page 40 in: Compendium of Grape Diseases. R. C. Pearson and A. C. Goheen, eds. The American Phytopathological Society, St. Paul, MN, 1988. (3) M. Kirchmair et al. Mycol. Res. 112:1210, 2009. (4) J. R. Liberato et al. Pest and Diseases Image Library. Online publication, 2009.