Linda E. Hanson

Virulence, distribution and diversity of Rhizoctonia solani from sugar beet in Idaho and Oregon

Abstract Rhizoctonia root rot causes serious losses on sugar beet worldwide. In order to help explain why Rhizoctonia root rot management practices have not performed well in some areas of the Intermountain West (IMW), a survey was conducted. In the IMW from 2004 to 2006, 94 Rhizoctonia solani field isolates were collected from sugar beet roots. These field isolates were compared with 19 reference strains and 46 accessions from GenBank for genetic diversity based on sequencing of the ITS-5.8S rDNA region. Greenhouse pathogenicity tests on sugar beet and silage corn were conducted and plant damage was assessed using a randomized complete block design with at least four replications. The majority of the isolates had sequence identity with the AG-2-2 IIIB (47%) or AG-4 subgroups (44%). Most of the AG-2-2 isolates (87%) were associated with fields in the western portion of the production area, while 71% of the AG-4 isolates came from the eastern portion of the production area. Isolates from AG-2-2 IIIB were frequently more virulent on sugar beet and sequence of the ITS-5.8s region required cloning because of genetic diversity within isolates. Seven (all AG-2-2 IIIB) of 18 isolates tested could attack both sugar beet and corn, while two of the seven virulent isolates caused less root rot on corn. To reduce Rhizoctonia root rot on sugar beet and corn, crop rotations and the isolates utilized for selecting host resistance could be given further consideration.

Characterizing a novel strain of Bacillus amyloliquefaciens BAC03 for potential biological control application.

To identify and characterize a bacterial strain BAC03, evaluate its biological control activity against potato common scab (Streptomyces spp.) and characterize an antimicrobial substance produced by BAC03.

First report of in vitro fludioxonil-resistant isolates of Fusarium spp. causing potato dry rot in Michigan

Fusarium dry rot of potato (Solanum tuberosum) is a postharvest disease caused by several Fusarium spp. Dry rot is managed primarily by reducing tuber bruising and promoting rapid wound healing. Dry rot symptomatic tubers were collected from Michigan seed lots in 2009 and 2010. The isolates may not have been exposed to fludioxonil because currently applications are restricted to seed not intended for seed production (3). Small sections were cut from the margins of necrotic regions with a scalpel, surface sterile in 10% sodium hypochlorite for 10 s, rinsed twice in sterile distilled water, and blotted with sterile filter paper. The tissue pieces were plated on half-strength potato dextrose agar (PDA) amended with 0.5 g/liter of streptomycin sulfate. The dishes were incubated at 23°C for 5 to 7 days. Cultures resembling Fusarium spp. were transferred onto water agar and hyphal tips from the margin of actively growing isolates were removed with a sterile probe and plated either on carnation leaf agar (CLA) or on half-strength PDA to generate pure cultures. Fusarium isolates were obtained and used for further studies. Among them, 54 were identified as Fusarium oxysporum and 23 as F. sambucinum. Identification was based on colony and conidial morphology on PDA and CLA, respectively. The identity was confirmed through DNA extraction followed by amplification and sequencing of the translation elongation factor (EF-1α) gene region. The Fusarium-ID v. (2) and the NCBI database were used to obtain the closest match to previously sequenced materials. Pathogenicity testing was done on disease-free potato tubers, cv. FL 1879. Tubers were surface sterilized for 10 min in 10% sodium hypochlorite and rinsed twice in distilled water. Three tubers per isolate were injected with 20 μl of a conidial suspension (106 conidia/ml) made from cultures grown on PDA for 7 days. Control tubers were injected with 20 μl of sterile distilled water. All tubers inoculated with F. sambucinum and F. oxysporum developed typical potato dry rot symptoms consisting of dry brown decay lesions. F. sambucinum and F. oxysporum were reisolated from all symptomatic tubers. An effective concentration for 50% reduction in growth (EC50) was determined for each F. sambucinum and F. oxysporum isolate for thiabendazole (TBZ), fludioxonil, and difenoconazole using the spiral gradient endpoint method (1). Sensitive and resistant F. sambucinum and F. oxysporum isolates were reported. Fifteen isolates of F. sambucinum and thirty-four of F. oxysporum were resistant to fludioxonil with EC50 greater than 130 mg/liter. The remainder was sensitive to fludioxonil with EC50 ranging from 0.8 to 4.9 mg/liter. To our knowledge, this is the first report of resistance to fludioxonil in isolates of F. sambucinum and F. oxysporum in Michigan. Fusarium insensitivity in laboratory studies may not translate directly to commercial production. This disparity may result from interactions not experienced in mixed populations or within a living host. There has been no compelling evidence to suggest that fludioxonil has failed to perform because of insensitivity to Fusarium. The occurrence of such isolated strains necessitates the development and registration of partner chemistries that can preempt any future concerns on lack of performance of products in use. References: (1) H. Förster et al. Phytopathology 94:163, 2004. (2) D. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (3) R. D. Peters et al. Plant Dis. 92:172, 2008.

Influence of Glyphosate on Rhizoctonia Crown and Root Rot (Rhizoctonia solani) in Glyphosate-Resistant Sugarbeet

Abstract Previous greenhouse studies with a noncommercial glyphosate-resistant sugarbeet variety indicated that susceptibility to Rhizoctonia crown and root rot could increase after glyphosate was applied. Greenhouse and field experiments were conducted in 2008 and 2009 to determine if glyphosate influenced disease severity in potential commercially available varieties of glyphosate-resistant sugarbeet. In the first greenhouse experiment in 2008, Hilleshög 9027RR, the most tolerant variety to Rhizoctonia crown and root rot, exhibited an increase in disease severity when glyphosate was applied. There were no significant differences between herbicide treatments in Hilleshög 9028RR, and glyphosate decreased disease severity in Hilleshög 9032RR when compared with the no-herbicide treatment. Experiments conducted to determine if glyphosate influenced Rhizoctonia solani growth in vitro indicated that glyphosate did not increase the radial growth of R. solani, except at 10× (190 µg ae ml−1) the normal rate of glyphosate plus ammonium sulfate (AMS). Field and additional greenhouse experiments were conducted using four commercial varieties. Differences in disease severity were observed when comparing varieties, but glyphosate did not significantly influence the severity of Rhizoctonia crown and root rot when compared with the no-herbicide control. Choosing a glyphosate-resistant sugarbeet variety with the best demonstrated tolerance to Rhizoctonia crown and root rot is an important factor in reducing disease severity and maintaining sugarbeet yield. Nomenclature: Glyphosate; sugarbeet, Beta vulgaris L.; Rhizoctonia crown and root rot, Rhizoctonia solani Kühn.

First report of Fusarium torulosum causing dry rot of seed potato tubers in the United States.

Fusarium dry rot of potato (Solanum tuberosum L.) is a postharvest disease caused by several Fusarium species and is of worldwide importance. Thirteen species of Fusarium have been implicated in fungal dry rots of potatoes worldwide. Among them, eight species have been reported in the northern United States (2). In Michigan potato production, F. sambucinum was the predominant species reported to be affecting seed potato in storage and causing seed piece decay after planting (3). Some previous identifications of F. sambucinum as dry rot may have been F. torulosum since F. torulosum was previously classified within F. sambucinum (4). To further investigate this, dry rot symptomatic tubers were collected from Michigan seed lots in the summers of 2009 and 2010. Small sections from the margins of necrotic regions were cut with a scalpel, surface sterilized in 0.5% sodium hypochlorite for 10 s, rinsed twice in sterile distilled water, and blotted with sterile filter paper. The tissue pieces were plated on half-strength potato dextrose agar (PDA) amended with 0.5 g/liter of streptomycin sulfate and incubated at 23°C for 5 to 7 days. Cultures resembling Fusarium species were transferred onto water agar, and single hyphal tips from actively growing isolates were removed and plated either on carnation leaf agar (CLA) or on half-strength PDA to generate pure cultures. Among the Fusarium isolates obtained, five isolates were identified as F. torulosum (GenBank Accessions Nos. JF803658-JF803660). Identification was based on colony and conidial morphology on PDA and CLA, respectively. These features included slow growth (2.8 ± 0.2 cm in 5 days), white mycelium that became pigmented with age, narrow concentric rings, red or white pigmentation on agar, macroconidia (32.4 ± 0.4 μm average length) with five septa, a pointed apical cell, and a foot-shaped basal cell (4). The identity was confirmed through DNA extraction followed by amplification and sequencing of the translation elongation factor (EF-1α) gene region (1). The Fusarium-ID.v (1) and the NCBI database were used to obtain the closest match (99%) to previously sequenced materials (GenBank Accession No. AJ543611). Pathogenicity testing was done on disease-free potato tubers cv. Red Norland. Tubers were surface sterilized for 10 min in 0.5% sodium hypochlorite and rinsed twice in distilled water. Three tubers per isolate were injected with 20 μl of a conidial suspension (106 conidia/ml) made from F. torulosum cultures grown on PDA for 7 to 10 days. Control tubers were injected with 20 μl of sterile distilled water. All tubers inoculated with F. torulosum developed typical potato dry rot symptoms consisting of a brown and dry decay. There was no disease incidence on the control tubers. F. torulosum was reisolated from the symptomatic tubers. To our knowledge, this is the first report of F. torulosum causing potato dry rot in the United States. References: (1) D. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) L. E. Hanson et al. Phytopathology 86:378, 1996. (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.

First Report of Streptomyces stelliscabiei Causing Potato Common Scab in Michigan

Potato (Solanum tuberosum L.) common scab can be caused by multiple Streptomyces spp., with S. scabies as a predominant species (2,3). However, according to our survey in August 2007, many symptomatic potato tubers did not have S. scabies in Michigan. To identify the pathogen, potato tubers with scab symptoms were collected from two fields in Michigan, and Streptomyces spp. were isolated using Streptomyces selective medium (STR) (2). Pure cultures of the isolates were obtained by transferring single colonies and incubation at 28°C on STR. Three isolates, designated HER21, HER24, and HER26, were identified as Streptomyces stelliscabiei based on morphological and physiological characterization (1). Bacterial cultures were prepared in liquid yeast malt extract at 28°C on an incubator shaker at 150 rpm. Genomic DNA was extracted from the cultures and used as a template for PCR with species-specific primers for Streptomyces spp. (4). The isolates gave a positive PCR reaction with primers Stel3 and T2st2 for S. stelliscabiei, but negative for any other Streptomyces spp. reported as pathogenic to potato. The 16S rRNA genes were amplified using primers previously reported (4) and amplicons were sequenced and submitted to GenBank (Accession Nos. HM018115, HM018116, and HM018117 for the three isolates, respectively). BLAST analysis of these sequences against the NCBI GenBank database determined these sequences to have 99 to 100% sequence identity with S. stelliscabiei sequences such as Accession No. FJ546728 (4). These isolates were all confirmed by PCR, using the same conditions described above, to have txtAB, nec1, and tomA genes (4), which are associated with pathogenicity of scab-causing Streptomyces spp. To complete Kochs postulates, cell suspensions of the isolates were mixed in vermiculate media (3) at a final concentration of 106 colony-forming units/ml, which were used as inocula. Potato (cv Snowden) tubers were incubated in sterilized potting mix in a growth chamber at 25°C until the seed germinated. Each potato seedling was transferred to a new pot in the greenhouse. Two weeks later, the potting mix was infested with the bacterial spore suspensions of either HER21, HER24, or HER26, with five pots per isolate. Potting mix with only media or media with S. scabies isolate 49173 were used as negative and positive controls, respectively. Three months later, potato tubers were harvested and evaluated for scab symptoms (3). The experiment was done twice. Potato tubers inoculated with either S. stelliscabiei or S. scabies exhibited superficial, raised, or pitted scabby symptoms, and no symptoms were observed on tubers grown in noninfested potting mix. Disease index values from the combined trials averaged 0, 37.8, 26.5, 11.1, and 30.5% for negative control and isolates HER21, HER24, HER26, and 49173, respectively. The pathogen was reisolated from the lesions and confirmed identical to the original isolate by DNA sequences. To our knowledge, this is the first report of S. stelliscabiei causing potato common scab in Michigan (4). References: (1) K. Bouchek-Mechiche et al. Int. J. Syst. Evol. Microbiol. 50:91, 2000. (2) Conn et al. Plant Dis. 82:631, 1998. (3) Hao et al. Plant Dis. 93:1329, 2009. (4) L. A. Wanner. Am. J. Potato Res. 86:247, 2009.

Global genotype flow in Cercospora beticola populations confirmed through genotyping-by-sequencing

Genotyping-by-sequencing (GBS) was conducted on 333 Cercospora isolates collected from Beta vulgaris (sugar beet, table beet and swiss chard) in the USA and Europe. Cercospora beticola was confirmed as the species predominantly isolated from leaves with Cercospora leaf spot (CLS) symptoms. However, C. cf. flagellaris also was detected at a frequency of 3% in two table beet fields in New York. Resolution of the spatial structure and identification of clonal lineages in C. beticola populations using genome-wide single nucleotide polymorphisms (SNPs) obtained from GBS was compared to genotyping using microsatellites. Varying distance thresholds (bitwise distance = 0, 1.854599 × 10−4, and 1.298 × 10−3) were used for delineation of clonal lineages in C. beticola populations. Results supported previous reports of long distance dispersal of C. beticola through genotype flow. The GBS-SNP data set provided higher resolution in discriminating clonal lineages; however, genotype identification was impacted by filtering parameters and the distance threshold at which the multi-locus genotypes (MLGs) were contracted to multi-locus lineages. The type of marker or different filtering strategies did not impact estimates of population differentiation and structure. Results emphasize the importance of robust filtering strategies and designation of distance thresholds for delineating clonal lineages in population genomics analyses that depend on individual assignment and identification of clonal lineages. Detection of recurrent clonal lineages shared between the USA and Europe, even in the relaxed-filtered SNP data set and with a conservative distance threshold for contraction of MLGs, provided strong evidence for global genotype flow in C. beticola populations. The implications of intercontinental migration in C. beticola populations for CLS management are discussed.

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. Kochs 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 Kochs 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.

CERCOSPORA BETICOLA INSENSITIVITY IN MICHIGAN AND MICHIGAN SUGAR COMPANY’S RESISTANCE MANAGEMENT STRATEGIES

Introduction Cercospora leafspot, caused by the fungus Cercospora beticola, is the most serious foliar disease of sugarbeets in Michigan. Research trials conducted in Michigan show that poorly controlled Cercospora leafspot (25 to 50% canopy desiccation) resulted in losses of 2 to 4 tons per acre and from 0.25 to 1.0 point of sucrose. The disease is managed with an integrated program which includes proper rotations, varietal tolerance and fungicide sprays. Most of the varieties in Michigan have relatively good tolerance to Cercospora; however, our highest producing varieties are quite susceptible to the disease. Michigan growers benefit from the predictive model BEETcast, which monitors leaf wetness and temperatures at over 50 locations and predicts optimum fungicide application timings. Cercospora infection levels were high in 2005 and 2006 then trended lower until 2010, 2011 and late season of 2012, when disease levels increased significantly. For the most part, Cercospora leafspot is adequately controlled in Michigan. However, with the introduction of high producing varieties that have less disease tolerance some control problems have occurred. More aggressive Cercospora control programs have been implemented by the Cooperative which has been successful in protecting varieties with low levels of Cercospora resistance from yield and quality losses. The use of strobilurin and triazole fungicides along with the BEETcast spray model improved the level of Cercospora leafspot control in Michigan significantly. By 2008, Michigan Sugar Company growers were planting Roundup Ready and nematode tolerant varieties without adequate tolerance to Cercospora. In 2010, control of Cercospora became more difficult despite more aggressive BEETcast spray recommendations. The following year, QoI resistant isolates were identified to harbor the G143A mutation. This mutation was discovered in Michigan Sugar Company’s efficacy trial near Elkton, MI, and was confirmed by Michigan State University and USDA-ARS (Figure 1). To effectively manage Cercospora in Michigan, tank-mixed fungicides were recommended prior to the first sign of the leafspot. Application timing was also modified by Cercospora tolerance level of the variety being grown. Fungicide insensitivity in sugarbeets can be managed; however, growers needed to follow a resistance management program set by Michigan Sugar Company.

ASSESSMENT OF STROBILURIN RESISTANCE IN CERCOSPORA BETICOLA ON SUGAR BEET IN MICHIGAN AND NEBRASKA, USA

Cercospora leaf spot (CLS) caused by Cercospora beticola Sacc. is the most important foliar disease of sugar beet (Beta vulgaris) worldwide (Jacobsen & Franc, 2009). CLS is controlled mainly with fungicides, including strobilurins (FRAC group 11). Resistance to strobilurins in C. beticola was first confirmed in 2011 from several fields in Michigan and in one field in Nebraska, USA (Kirk et al. 2012) following anecdotal reports of reduced fungicide efficacy. In these fields, sugar beet treated with strobilurins had severe CLS and diminished control was also noted in small plot trials in Michigan. Individual leaf spot lesions were sampled from leaves and grown on sugar beet leaf extract agar (SBLEA). A conidial germination bioassay was done on SBLEA amended with pyraclostrobin, azoxystrobin or trifloxystrobin at 0, 0.001, 0.01, 0.1, 1, 10, or 100 μg/ml supplemented with salicylhydroxamic acid (SHAM) to block the alternate oxidation pathway (Olaya et al., 1998). After 24 h incubation at 22°C, under ambient light, the percentage of germinated conidia (n = 50) was calculated from three replicates per treatment. Germination was recorded as positive when the germ tube was at least half the length of the conidium. A representative wild type isolate was unable to germinate over the 0.01 μg/ml concentration. Effective concentration for 50% reduction in germination (EC50) values for each isolate were calculated by regression analysis of percentage spore germination vs. the log fungicide concentration using Sigmaplot Version 9.01 (Systat Software, Chicago). The EC50 for the sensitive isolate was <0.01 μg/ml. Isolates from several counties in Michigan had uninhibited germination and EC50 values exceeded the highest concentration tested. Isolates also grew on spiral gradient dilution plates (Forster et al., 2004) amended with the three strobilurins. Two isolates were obtained from Nebraska and each showed a similar response to strobilurin fungicides in amended plate assays. In 2012, widespread strobilurin resistance was recorded in isolates of C. beticola collected in Michigan although a few isolates submitted to the program were sensitive. Pure cultures of a subset of resistant isolates were grown in potato dextrose broth at 125 rpm, and DNA extracted. A fragment of the cytochrome b (CYTB) gene was amplified by PCR using the C. beticola primers of Malandrakis et al. (2011) to amplify the region of the CYTB gene likely to contain resistance mutations (Malandrakis et al., 2011). This fragment was sequenced at the Genomics Technology Support Facility (MSU, East Lansing, MI) and showed 99% identity with both the C. beticola cytochrome b mRNA, partial sequence (GenBank Accession No. EF176921.1) and the C. kikuchii mitochondrial gene for cytochrome b partial sequence (AB231863.1). Sequence results revealed that each resistant isolate contained a change in codon 143 that is predicted to lead to a substitution of G143A, which was demonstrated to confer QoI resistance in several other fungi (Ma & Michailides, 2005). All Michigan isolates with the G143A mutation germinated at 100 μg/ml pyraclostrobin (50% of conidia), while sensitive isolates that lacked the mutation failed to grow. Additional isolates that contained the G143A mutation included representatives from Michigan and Nebraska. A high proportion of isolates (~90%) from the commercial growing region that were screened in 2012 have been found to contain the G143A mutation by PCR-RFLP screening using digestion of the above PCR products. These findings reveal that reduced CLS control in some commercial sugar beet fields may be due to the development of resistance to strobilurins. In 2012, three consecutive applications of pyraclostrobin treatments failed to adequately control C. beticola at the Michigan State University (MSU) Saginaw Valley Research and Extension Center. In addition to the strobilurin sensitivity monitoring, the program at MSU also tests for development of insensitivity in the CLS population to Difenoconazole (Inspire); Tetraconazole (Eminent); Prothioconazole (Proline), Flutriafol (Topguard), Thiophanate-methyl (Topsin) and TPTH (Tin). Some triazole-insensitive isolates have been recovered in MI beet fields over the past 10 years but the proportion is low for all these fungicides, however in 2012, nearly 100% of isolates tested were insensitive to Thiophanate-methyl. The challenges for the sugarbeet industry with respect to CLS control include a lack of products from diverse FRAC groups; a general increase in inoculum due to the prevalence of CLS susceptible cultivars grown in the Michigan and Ontario sugar beet production regions; an increase in proportion of isolates insensitive to strobilurins (e.g. pyraclostrobin); the industry is inexperienced with tank mixes and has a perceived distrust of TPTH (label restrictions). In addition, to the lack of available chemistries from diverse FRAC groups there is confusion on what to start and end a program with in order to delay the onset of insensitivity to other fungicides. A strobilurin is the mainstay for Rhizoctonia control in MI therefore there is a need for new products with alternative modes of action so that a CLS disease management program starts with a product not used earlier in the season for other disease management. Acknowledgements The authors would like to thank Tom Goodwill for his excellent technical assistance.