Fungicide Sensitivity Monitoring of Alternaria spp. Causing Leaf Spot of Sugarbeet (Beta vulgaris) in the Upper Great Lakes.

Alternaria leaf spot (ALS), caused by Alternaria spp., can occur wherever sugarbeet is grown. Infection by Alternaria spp. and disease management has historically been considered a minor issue in sugarbeet production in the United States. An increase of both incidence and severity in 2016 of ALS high enough to cause yield loss has been observed in Michigan. With a renewed need to consider potential management of this disease, the sensitivity was determined for populations of Alternaria spp. to three classes of fungicides currently labeled for management of leaf spot on sugarbeet, including demethylase inhibitor (DMI), quinone outside inhibitor (QoI), and organo-tin fungicides. Leaves with symptoms of ALS were sampled from sugarbeet fields in east-central Michigan and southwestern Ontario, Canada. Monoconidial isolates were obtained to determine sensitivity to each fungicide class above. A spiral gradient dilution method was used to estimate the fungicide effective concentration (in milligrams per liter) that caused a 50% inhibition of fungal growth in vitro for all isolates. Significant temporal shifts were detected in the frequencies of sensitivity phenotypes to DMI and QoI but not organo-tin fungicides from 2016 through 2017. Individual isolates of Alternaria spp. were recovered with cross-resistance to DMI and multiple resistance to DMI, QoI, and triphenyltin hydroxide fungicides. To our knowledge, this is the first report of a fungus other than Cercospora beticola with resistance to organo-tin fungicides. Fungicide sensitivity monitoring indicates that an effective integrated disease management approach combining fungicide efficacy trials and monitoring pathogen biology is essential for developing effective resistance management recommendations.

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 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.

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.

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

AIMS: 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. METHODS AND RESULTS: Bacterial strain BAC03, isolated from potato common scab suppressive soil, was identified as Bacillus amyloliquefaciens by analysing sequences of fragments of the recA, recN, cheA and gyrA genes. BAC03 displayed an antagonistic activity against Streptomyces spp. on agar plates using a co-culture method. In glasshouse assays, BAC03 applied in potting mix significantly reduced common scab severity (P < 0·05) and potentially increased the growth of potato plants (P < 0·05). An antimicrobial substance extracted from BAC03 by ammonium sulfate precipitation was identified as an LCI protein using liquid chromatography-mass spectrometry. The antimicrobial activity of either a BAC03 liquid culture or the ammonium sulfate precipitate fraction was stable under a wide range of temperatures, and pH levels, as well as following incubation with several chemicals, but was reduced by all proteinases tested. CONCLUSIONS: Bacillus amyloliquefaciens strain BAC03 displayed a strong antimicrobial activity, that is, the suppression of potato common scab, and may potentially enhance the plant growth. LCI protein is associated with some of the antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial strain BAC03 has the potential to be developed as a commercial biological control agent for potato common scab.

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.

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 Koch's 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.

The Perfect Stage of Powdery Mildew (Erysiphe polygoni) of Beta vulgaris Found in Michigan.

Powdery mildew (Erysiphe polygoni DC [synonym E. betae {Vanha} Weltzien]) affects several different crops of Beta vulgaris, including sugar beet, Swiss chard, and table beet. The disease has been prevalent in many sugar beet-growing areas of the United States since the first major epidemic in beet in 1974 (3). Powdery mildew in the United States was primarily associated with the asexual stage of the pathogen until the perfect stage was found, first in western states such as Idaho and Colorado (2), then in more Midwestern states such as Nebraska, and most recently in North Dakota (1). Similar to North Dakota, powdery mildew has not been a major problem in the Michigan growing area. It does appear sporadically, particularly on sugar beets that have not been sprayed to control other foliar diseases. In 2010, powdery mildew infection on sugar beet was observed in late August in a field in the Saginaw Valley of Michigan. Plants were inspected periodically for the presence of the sexual stage. In early October, sugar beet and Swiss chard plants with heavy powdery mildew infection also were observed at the Michigan State University (MSU) Horticultural Demonstration Gardens in East Lansing and on sugar beet at the MSU Plant Pathology and Botany research farms. On both the Saginaw Valley sugar beet and Swiss chard on the MSU campus, ascomata were observed on a few leaves in mid-October. No ascomata were found on sugar beet at the other two locations. The majority of ascomata were dark brown to black when located, although a few light tan ascomata were observed on the Swiss chard. Ascomata varied from 70 to 100 µm in diameter. Asci contained one to four hyaline or golden yellow ascospores similar to those described in other growing regions on sugar beet (1,2). No ascomata had been detected on powdery mildew-infected sugar beet from either the Saginaw Valley or the MSU research farms the previous two years. These results appear to indicate a spread of the ability to form the perfect stage eastward from the western United States. This may be due to movement of one mating type because E. polygoni has been reported to be heterothallic on some crops (4). The presence of the perfect stage indicates that sexual recombination could occur in E. polygoni on Beta species in Michigan, creating the potential for more rapid development of new strains that might vary in fungicide sensitivity and response to host resistance. References: (1) C. A. Bradley et al. Plant Dis. 91:470, 2007 (2) J. J. Gallian and L. E. Hanson. Plant Dis. 87:200, 2003. (3) E. G. Ruppel. Page 13 in: Compendium of Beet Disease and Insects. E. D. Whitney and J. E. Duffus, eds. The American Phytopathological Society, St. Paul, MN, 1986. (4) C. G. Smith. Trans. Br. Mycol. Soc. 55:355, 1970.

Interaction of Rhizoctonia solani and Rhizopus stolonifer Causing Root Rot of Sugar Beet.

In recent years, growers in Michigan and other sugar beet (Beta vulgaris) production areas of the United States have reported increasing incidence of root rot with little or no crown or foliar symptoms in sugar beet with Rhizoctonia crown and root rot. In addition, Rhizoctonia-resistant beets have been reported with higher levels of disease than expected. In examining beets with Rhizoctonia root rot in Michigan, over 50% of sampled roots had a second potential root rot pathogen, Rhizopus stolonifer. Growing conditions generally were not conducive to disease production by this pathogen alone, so we investigated the potential for interaction between these two pathogens. In greenhouse tests, four of five sugar beet varieties had more severe root rot symptoms when inoculated with both pathogens than when inoculated with either pathogen alone. This synergism occurred under conditions that were not conducive to disease production by R. stolonifer. Host resistance to Rhizoctonia crown and root rot reduced diseases severity, but was insufficient to control the disease when both pathogens were present. This raises concerns about correct disease diagnosis and management practices and indicates that a root rot complex may be important on sugar beet in Michigan.

Stalk Rot of Sugar Beet Caused by Fusarium solani on the Pacific Coast.

In 2006, symptoms of stalk blight (2) were observed on sugar beet (Beta vulgaris L.) plants from roots produced in Oregon that were being grown for seed production in a greenhouse in Salinas, CA using Salinas Valley soil. Symptoms included vascular and cortical browning, necrosis, and death of seed stalks. Isolations were made from the edge of stalk lesions and the crown. In addition to Fusarium oxysporum, the known cause of stalk blight (2), two isolates of F. solani were identified by morphology. For pathogenicity tests, sugar beet plants (FC606 [4]), grown in pasteurized potting mix and induced to flower by exposure at 4 to 7°C for 90 days (1) were used. Bolting plants were maintained in a greenhouse at 24 to 27°C. A 100-µl drop of a spore suspension (104 spores per ml) of each Fusarium isolate was placed on the surface of the seed stalk. The plant was stabbed through the drop with a sterile 18-gauge needle so that the drop was taken into the plant by hygroscopic pressure. Positive and negative control treatments were a stalk blight isolate of F. oxysporum from an Oregon seed production field and sterile water, respectively. Three plants were inoculated per isolate. Each inoculation site was wrapped loosely in Parafilm for 1 week to maintain a high humidity level around the site of inoculation, and seed stalks were covered in cloth bags (1). After 1 week, the Parafilm was removed and plants were examined weekly for symptoms. At 4 weeks, lesion size was measured. After 5 weeks, sections were taken from the seed stalk around the site of inoculation, surface disinfested with 0.5% NaOCl, and plated on potato dextrose agar to confirm the presence of the pathogen. The experiment was done twice. One of the two isolates of F. solani caused dark brown lesions on all inoculated seed stalks. On one plant, at 4 weeks after inoculation when the bag was being removed for observation, the seed stalk broke at the site of inoculation because of a spreading, brown lesion at the site. No lesions were observed on the water control plants. Brown lesions were observed on seed stalks inoculated with the known stalk blight isolate. Lesions were significantly (P = 0.001) larger with F. oxysporum than with F. solani when measured at 4 weeks (mean of 6.3 cm versus 2.2 cm, respectively). Lesions caused by F. solani showed a dark discoloration through the cortical tissue, as opposed to those caused by F. oxysporum, for which most of the initial discoloration was in the vascular bundles and epidermis. Fusarium isolates recovered from inoculated plants were morphologically similar to the isolates used for inoculation. Fusarium spp. were not isolated from the water control plants. While some F. solani isolates cause seedling or mature root disease in sugar beet (3), to our knowledge, this is the first report of a Fusarium species other than F. oxysporum causing a rot of sugar beet stalks. References: (1) E. Biancardi et al. Genetics and Breeding of Sugar Beet Science Publishers, Inc., Enfield, NH, 2005. (2) A. N. Mukhopadhay. Handbook of Diseases of Sugar Beet, Vol. 1. CRC Press, Boca Raton, FL 1987. (3) E. G. Ruppel. Plant Dis. 75:486, 1991. (4) G. A. Smith and E. G. Ruppel. Crop Sci. 19:300, 1980.

First Report of Fusarium Yellows of Sugar Beet Caused by Fusarium oxysporum in Michigan.

Fusarium yellows of sugar beet (Beta vulgaris L.), caused by Fusarium oxysporum Schlechtend.Fr. f. sp. betae (Stewart) Snyd & Hans., has been a long-term problem in the western United States (3) and recently was reported in Minnesota and North Dakota (4). This disease is typified by interveinal yellowing and wilting of the foliage. Roots have no external symptoms but show internal vascular discoloration. In 2005, 12 sugar beet roots from Michigan with yellows-type symptoms were received by the author. Isolations were made from the cortical and vascular tissue of the crown and tap root. Fusarium spp. isolates were obtained from 10 of the beets, and 16 isolates were identified as Fusarium oxysporum on the basis of morphology and pigmentation on potato dextrose agar and spores and phialides on carnation leaf agar (2). F. oxysporum isolates were tested for pathogenicity by dipping roots of 5-week-old susceptible sugar beet plants (FC716) in a suspension of 104 spores per ml for 8 min, 10 plants per isolate. Two known pathogenic isolates of F. oxysporum f. sp. betae, Fob13 and Fob216c (4), were used for comparison. For a negative control, plants were dipped in sterile water. Beets were planted in Cone-tainers (3.8 cm diameter × 21 cm) containing pasteurized potting mix. Plants were placed in a greenhouse at 24 to 27°C and fertilized with 15-30-15 fertilizer every 2 weeks to avoid chlorosis from nutrient deficiency. Plants were rated weekly for foliar symptoms for 6 weeks using a Fusarium yellows rating scale of 0 to 4 in which 0 = no disease and 4 = complete plant death (1). After the final rating, plants were removed from soil and the tap root examined for root symptoms. Root segments were surface disinfested with 0.5% sodium hypochlorite and cultured on potato dextrose agar to confirm presence of the pathogen. The experiment was done twice. Seven F. oxysporum isolates tested caused typical Fusarium yellows symptoms including interveinal yellowing, stunting, and wilting of inoculated plants. Pathogenic isolates were obtained from 7 of the 10 beets that yielded F. oxysporum. Symptoms were indistinguishable from those caused by Fob13 (average ratings ranged from 1.8 to 2.4) and milder than those caused by Fob216c (average rating 3.1). No interveinal chlorosis or wilting was observed on the control plants. Isolations from inoculated plants provided F. oxysporum cultures morphologically similar to those used in inoculation by the methods of Nelson et al. (2). No F. oxysporum was isolated from control plants. To my knowledge, this is the first report of F. oxysporum causing Fusarium yellows on beet in Michigan. References: (1) L. E. Hanson and A. L. Hill. J. Sugar Beet Res. 41:163, 2004. (2) P. E. Nelson et al. Fusarium species: An Illustrated Manual for Identification. The Pennsylvania State University Press, University Park, 1983. (3). C. L. Schneider and E. D. Whitney. Fusarium Yellows. Page 18 in: Compendium of Beet Diseases and Insects. C. L. Schneider and E. D. Whitney, eds. The American Phytopathological Society, St. Paul, MN, 1986. (4) C. E. Windels et al. Plant Dis. 89:341, 2005.

Fusarium Yellowing of Sugar Beet Caused by Fusarium graminearum from Minnesota and Wyoming.

In 2004, we received beet samples from seven fields from Minnesota and Wyoming that had foliar interveinal yellowing symptoms and vascular discoloration frequently associated with Fusarium yellows. Isolations were made from the vascular and cortical tissue. Hyphal tip isolates of Fusarium were obtained from beets, including eight isolates of Fusarium graminearum. F. graminearum was isolated from beets from three fields in Minnesota and one field in northern Wyoming. F. graminearum isolates were tested for pathogenicity by dipping roots of 5-week-old sugar beet plants (FC716) in a suspension of 104 spores per ml for 8 min, 10 plants per isolate. Spore suspensions were shaken periodically to aid mixing. A known moderately virulent isolate of F. oxysporum f. sp. betae (Fob13) (3), the causal agent of Fusarium yellows of sugar beet, was used as a positive control. For a negative control, plants were dipped in sterile water. Dipped plants were planted in Cone-tainers (3.8 cm diameter × 21 cm; Stuewe and Sons, Inc., Corvallis, OR) containing pasteurized potting mix and placed in a greenhouse at 24 to 27°C. Plants were fertilized with 15-30-15 fertilizer biweekly. After 2 weeks, plants were rated weekly for 5 weeks using a 0 to 4 scale in which 0 = no disease and 4 = complete plant death (2). After the final rating, plants were removed from soil and the tap root was examined for symptoms. Root segments were surface disinfested with 0.5% sodium hypochlorite and plated on potato dextrose agar to confirm presence of the pathogen. The experiment was done twice. Three of the eight isolates of F. graminearum caused mild to moderate foliar symptoms (rating 2 to 3), including interveinal yellowing, wilting, and stunting of inoculated plants, and mild vascular discoloration was observed in some root sections. Pathogenic isolates were originally from different beets. Foliar symptoms were similar to those caused by Fob13, but the F. oxysporum f. sp. betae caused more vascular discoloration than did the F. graminearum isolates. No interveinal yellowing or wilting was observed on foliage of the control plants, and no vascular discoloration was observed in a cross section of the root. Cultures of F. graminearum or F. oxysporum recovered from inoculated plants were morphologically similar to isolates used for the inoculations. No Fusarium was isolated from the roots of plants soaked in sterile water. An interesting note is that no isolates of F. graminearum were recovered among more than 100 Fusarium isolates collected from sugar beet roots from Colorado over a 4-year period. F. graminearum was recovered in one sample from Wyoming in 2004. However, in the 2004 samples from Minnesota, this species was isolated at the same frequency as F. oxysporum. While F. graminearum has been isolated from beets in the Red River Valley (1), it has not previously been reported to cause symptoms on growing sugar beet. References: (1) U. Bosch and C. J. Mirocha. Appl. Environ. Microbiol. 58:3233, 1992. (2) L. E. Hanson and A. L. Hill. J. Sugar Beet Res. 41:139, 2004. (3) C. E. Windels et al. Plant Dis. 89:341, 2005.

Beet Root-Rot Inducing Isolates of Fusarium oxysporum from Colorado and Montana.

A root-tip rot of sugar beet (Beta vulgaris L.) caused by Fusarium oxysporum Schlecht. emend. Snyd. & Hans. has been reported from Texas (2). This disease is typified by yellowing of the foliage, vascular discoloration, and a rot of the root tip. During 2002 and 2003, sugar beet samples from several fields in Colorado and Montana, some with tip rot symptoms, were received by the authors. Isolations were made from the root vascular tissue and tissue adjacent to the rot in Colorado and from the rot tissue in Montana. Isolates of Fusarium were obtained and identified as Fusarium oxysporum. At the ARS laboratory in Colorado, F. oxysporum isolates were tested for pathogenicity by dipping roots of 5-week-old sugar beet plants (FC716) in a suspension of 104 spores per ml for 8 min, 10 plants per isolate. One known isolate of F. oxysporum f. sp. betae that causes Fusarium yellows, Fob13, was used for comparison. For a negative control, plants were dipped in sterile water. Beets were planted in Cone-Tainers (3.8 cm in diameter × 21 cm) containing pasteurized potting mix. Plants were placed in a greenhouse at 24 to 27°C and fertilized with 15-30-15 fertilizer every 2 weeks to avoid chlorosis from nutrient deficiency. Plants were rated weekly for foliar symptoms for 6 weeks with a Fusarium yellows rating scale of 0 to 4, in which 0 = no disease and 4 = complete plant death (1). After the final rating, plants were removed from the soil and the taproot was examined for rot symptoms. Root segments were surface disinfested with 0.5% sodium hypochlorite and cultured on potato dextrose agar to confirm the presence of the pathogen. The experiment was done twice. Six of ten F. oxysporum isolates tested caused root vascular discoloration and foliar symptoms, including interveinal yellowing and wilting, of inoculated plants. A rot of the root tip was observed on the roots of plants inoculated with three of the six pathogenic isolates. Isolate Fob13 caused only vascular discoloration and foliar symptoms with no rot. Similar experiments were done in Montana with the exception that 3-week-old plants (cv. Monohikari) were used and planted in 10-cm plastic pots with five seedlings per pot. Inoculum levels were 105 spores per ml of F. oxysporum f. sp. betae (isolate 216C) or tip rot isolates (3 isolates), and the experiments were terminated 4 weeks after planting. The root rot isolates caused foliar symptoms, vascular discoloration, and root rot similar to that seen in the field, whereas isolate 216C caused only foliar wilt symptoms and vascular discoloration. Isolations from inoculated plants in Colorado and Montana resulted in F. oxysporum cultures similar to those used in inoculation. To our knowledge, this is the first report of F. oxysporum causing root rot of sugar beet outside of Texas. References: (1) L. E. Hanson and A. L. J. Hill. Sugarbeet Res. 41:163, 2004. (2) R. M. Harveson and C. M. Rush. Plant Dis. 82:1039, 1998.

Elicitors of Plant Defense Responses from Biocontrol Strains of Trichoderma viren.

ABSTRACT Effective biocontrol strains of Trichoderma virens can induce the production of defense-related compounds in the roots of cotton. Ineffective strains do not induce these compounds to significant levels. This elicittation was found to be heat stable, insoluble in chloroform, passed through a 5K molecular weight cut-off (MWCO) filter, but not a 3K MWCO filter, and was sensitive to treatment by proteinase K. When the active material was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, several bands were present in the material from biocontrol-active strains that were lacking in inactive strains. When eluted and tested for elicitation activity, with or without renaturation, four bands stimulated cotton terpenoid production. One band showed cross-reaction with an antibody to the ethylene-inducing xylanase from T. viride. Another band of approximately 18 kDa, gave significant stimulation of cotton terpenoid production and increased peroxidase activity in cotton radicles in all tests, with or without renaturation. The 18-kDa protein was subjected to amino-terminal sequence analysis, and the first 19 amino acids at the amino terminus were determined to be DTVSYDTGYDNGSRSLNDV. A database homology search using the BLASTp algorithm showed the highest similarity to a serine proteinase from Fusarium sporotrichioides.

The Perfect Stage of Powdery Mildew of Sugar Beets Found in Idaho and Colorado.

Powdery mildew (Erysiphe polygoni DC [synonym E. betae {Vanha} Weltzien]) of sugar beet (Beta vulgaris L.) has been a significant problem in many sugar beet growing areas of the United States since the first serious epidemic in 1974. Disease has been attributed solely to the asexual stage of the pathogen in the United States, except for one report of the perfect stage in a single field in Washington coincidental with the 1974 epidemic (1). In August 2001, ascomata were observed in several fields in Owyhee County in southwestern Idaho near Grand View. The perfect stage was widespread and easily found, and in one field the surfaces of leaves collected from 50 randomly sampled plants were between 10 and 90% covered with ascomata. Subsequently, the ascigerous stage was found in September and October in multiple fields in three additional counties in southwestern and south-central Idaho and two counties in northern Colorado. Ascomata were found on 12 commercial varieties in the two states and six breeding lines in Colorado. Asci contained one to four hyaline or yellow-to-golden pigmented ascospores per ascus. Ascomata observed in Idaho and Colorado are similar to those described from Europe (2). Ascospores appeared intact after leaves were dried and stored at 4 to 7°C more than 4 weeks. However, after leaves with ascomata were dried and stored at 24 to 27°C for 1 week or more, ascomata and asci appeared intact microscopically, but ascospores were no longer delineated and appeared desiccated or degraded. Because the ascigerous stage provides a means of genetic recombination, there is the potential for races of the pathogen to arise with greater frequency. This has serious implications for managing fungicide resistance and breeding for disease resistance to sugar beet powdery mildew. References: (1) D. L. Coyier et al. (Abstr.) Proc. Am. Phytopathol. Soc. 2:112, 1975. (2) S. Francis. Mol. Plant Pathol. 3:119, 2002.

Induction of Terpenoid Synthesis in Cotton Roots and Control of Rhizoctonia solani by Seed Treatment with Trichoderma virens.

ABSTRACT Research on the mechanisms employed by the biocontrol agent Trichoderma virens to suppress cotton (Gossypium hirsutum) seedling disease incited by Rhizoctonia solani has shown that mycoparasitism and antibiotic production are not major contributors to successful biological control. In this study, we examined the possibility that seed treatment with T. virens stimulates defense responses, as indicated by the synthesis of terpenoids in cotton roots. We also examined the role of these terpenoid compounds in disease control. Analysis of extracts of cotton roots and hypocotyls grown from T. virens-treated seed showed that terpenoid synthesis and peroxidase activity were increased in the roots of treated plants, but not in the hypocotyls of these plants or in the untreated controls. Bioassay of the terpenoids for toxicity to R. solani showed that the pathway intermediates desoxyhemigossypol (dHG) and hemigossypol (HG) were strongly inhibitory to the pathogen, while the final product gossypol (G) was toxic only at a much higher concentration. Strains of T. virens and T. koningii were much more resistant to HG than was R. solani, and they thoroughly colonized the cotton roots. A comparison of biocontrol efficacy and induction of terpenoid synthesis in cotton roots by strains of T. virens, T. koningii, T. harzianum, and protoplast fusants indicated that there was a strong correlation (+0.89) between these two phenomena. It, therefore, appears that induction of defense response, particularly terpenoid synthesis, in cotton roots by T. virens may be an important mechanism in the biological control by this fungus of R. solani-incited cotton seedling disease.

Nursing students' perspectives: experiences of caring and not-so-caring interactions with faculty.

The purpose of this study was to describe the meaning of baccalaureate nursing students' lived experience of caring and not-so-caring interactions with faculty. The research questions explored the phenomenon of being cared-for or not-cared-for by asking, "What does being cared-for by faculty mean to baccalaureate nursing students?" Students were also asked to describe not-so caring interactions with faculty. Seventeen baccalaureate nursing students at a private liberal arts college and 15 baccalaureate nursing students at a public university were interviewed. Transcripts of the tape-recorded interviews were analyzed using Giorgi's technique. After reflection upon the content of the interviews, significant statements were identified, meaning units were developed, and themes were extracted which were then abstracted into categories of Recognition, Connection, and Confirmation/Affirmation. Finally, general structural descriptions of phenomena of a caring and of a not-so-caring interaction were constructed. Implications for teaching, research, and practice are described.

Leptospira interrogans serovar bratislava infection in two dogs.

Two dogs with clinical histories suggestive of leptospirosis were examined serologically and culturally for evidence of leptospiral infection. Antibodies to Leptospira interrogans serovar bratislava were detected in serum from one dog, and the organism was isolated from urine of that dog. In a serologic survey of dogs in the state of Illinois, reactor rates to bratislava were higher than those to canicola or icterohaemorrhagiae. In cases of suspect canine leptospirosis, serovars such as bratislava, not contained in canine vaccines, should be considered in a differential diagnosis.

Effect of vaccination with a bacterin containing Leptospira interrogans serovar bratislava on the breeding performance of swine herds.

Swine herds suspected to be infected with Leptospira interrogans serovar bratislava were vaccinated with bacterins containing 5 or 6 leptospiral serovars in which serovar bratislava was the unique component. The principal diagnostic feature indicating an infection by this organism was demonstration of antibody against serovar bratislava in sera from stillborn pigs. For 1 breeding cycle after vaccination of herds on 3 farms, 255 of 266 (95.9%) sows and gilts given the 6-serovar bacterin farrowed. In contrast, 233 of 311 (74.9%) sows and gilts given the 5-serovar bacterin farrowed. These results, as evaluated by analysis of variance techniques, showed a significant improvement (P less than 0.01) in reproductive performance for groups vaccinated against serovar bratislava.

Lighting and sex ratio for breeding ringnecked pheasants in confined housing.

In two trials with breeding pheasants, pheasants were assigned to two male:female sex ratios (1:12 and 1:18) arranged factorially with two lighting regimens (14 or 16 hr of light) in a 24-hr period. In each trial, 1644 or 1680 pheasant hens divided in two replicates were assigned to each experimental treatment and placed in controlled environment housing. Hen mortality, percent culled eggs, total eggs per hen-housed, fertility, hatchability, and usable chicks per hen-housed were determined over a 9-week production period. Although mortality and percent hatch were not affected by either sex ratio or lighting, percent culled eggs increased with increased light (16 hr light) and usable chicks per hen-housed decreased with increased light. Increasing the sex ratio from 1:12 to 1:18 increased egg production but decreased fertility. These data demonstrate that pheasants reared in a controlled environment require less than 16 hr light for maximizing usable chick production per hen-housed and there is a trade-off between fertility and egg production.