Value-added switchgrass extractives for reduction of Escherichia coli O157:H7 and Salmonella Typhimurium populations on Formica coupons.

Recurring outbreaks linked to Escherichia coli O157:H7-contaminated lettuce and Salmonella enterica-contaminated sprouts highlight the need for improved food safety measures. The aim of this study was to determine the ability of a bio-based antimicrobial extract prepared from switchgrass, a dedicated energy crop, to reduce E. coli O157:H7 and S. Typhimurium populations on Formica coupons, a model food-contact surface. Overnight cultures of ~7 log CFU/mL E. coli O157:H7 and S. Typhimurium, air-dried on Formica coupons were treated with 0.625% NaClO, 70% ethanol, sterile water or different batches of switchgrass extractives (SE1, SE2, and SE3) for up to 30 min. E. coli O157:H7 was reduced by 4.43 log CFU/mL after 1 min by SE3, and to non-detectable levels after 1 min by all other treatments. Populations of S. Typhimurium LT2 (15-min drying) were reduced by 3.30 log CFU/mL with 70% ethanol, 5.38 log CFU/mL with SE1, and to non-detectable levels with 0.625% NaClO after 1 min, while S. Typhimurium ATCC 23564 (1-h drying) was non-detectable after 1 min by all treatments. Under soiled conditions, 10-min treatment with SE1 and 70% ethanol reduced both bacteria to non-detectable levels. Studies with concentrated switchgrass extractives combined with various other natural disinfectants or in hurdle approaches warrant further investigation.

First Report of Aerial Blight of Ruth's Golden Aster (Pityopsis ruthii) Caused by Rhizoctonia solani in the United States.

Ruth's golden aster (Pityopsis ruthii (Small) Small: Asteraceae) is an endangered, herbaceous perennial that occurs only at a few sites along the Hiwassee and Ocoee rivers in Polk County, Tennessee. This species is drought, heat, and submergence tolerant and has ornamental potential as a fall flowering landscape plant. In 2012, we vegetatively propagated various genotypes and established plantings in a landscape at Poplarville, Mississippi. In June and July of 2013, during periods of hot and humid weather, several well-established plants exhibited black or brown necrotic aerial blight symptoms including desiccation of stems and leaves. Blighted leaf samples were surface sterilized (10% commercial bleach, active ingredient 8.25% sodium hypochlorite, 1 min), rinsed in sterile water, air-dried, and plated on 2% water agar amended with 3.45 mg fenpropathrin/liter (Danitol 2.4 EC, Valent Chemical, Walnut Creek, CA) and 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO). Rhizoctonia sp. was identified based on hyphal morphology and cultures were maintained on potato dextrose agar. Colonies were fast growing, consisting of light tan to brown mycelia and tufts of crystalline aerial hyphae. Within 10 days, brown exudates were present in cultures and there was no pigmented reverse to the agar. Hyphae were a mean of 5.2 µm wide (4.6 to 6.1 µm; n = 10) and each compartment contained three or more nuclei. Hyphae were constricted at septa with right angle branching and no clamp connections, which is typical for Rhizoctonia solani (1). Light- to medium-brown, oblong to irregularly shaped sclerotia measuring 1.2 mm long (0.7 to 2.1 mm) × 0.9 mm wide (0.5 to 1.2 mm; n = 20) were formed in cultures after 3 weeks of growth. Total genomic DNA was extracted from two different colonies grown in potato dextrose broth for 7 days, amplified with PCR using ITS1 and ITS4 primers for amplification of the 18S rDNA subunit (2), the products purified, and sequenced. A consensus sequence of 657 bp was deposited in GenBank (Accession Nos. KF843729 and KF843730) and was 96% identical to two R. solani Kühn ITS sequences in GenBank (HF678125 and HF678122). R. solani was grown on twice autoclaved oats for 2 weeks at 21°C and incorporated into Pro-Mix BX, low fertility soilless medium (Premier Horticulture, Rivière-du-Loup, Quebec, Canada) at 4% (w/w) to inoculate seven P. ruthii plants grown in 10 cm-diameter pots; seven additional plants were grown in the same medium amended with 4% (w/w) sterile oats. Plants were grown in a greenhouse and covered with a plastic dome to maintain high humidity. After 2 weeks, six of the seven inoculated plants exhibited the same aerial blight symptoms as did the original infected plants from the field; none of the control plants developed disease symptoms. Colony morphology and hyphal characteristics as well as the sequence for the ITS region of rDNA from the re-isolated fungus were identical to the original isolate. To our knowledge, this is the first report of R. solani infecting Ruth's golden aster. We are not aware of the disease occurring in wild populations of the plant, but may impact plants grown in the landscape or greenhouse. References: (1) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society, St Paul, MN, 1991. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

First Report of Leaf Spot on Switchgrass Caused by Pithomyces chartarum in the United States.

There are few reports on diseases of switchgrass. In November 2009, light brown to white bleached spots (1 to 2 × 3 to 4 µm) were observed on 'Alamo' switchgrass (Panicum virgatum L.) grown in a growth chamber in Knoxville, TN, from surface-disinfested seed produced in Colorado. Symptomatic leaf tissue was surface sterilized, air dried, and plated on 2% water agar (WA) amended with 6.9 mg fenpropathrin/liter (Danitol 2.4 EC, Valent Chemical, Walnut Creek, CA) and 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO). Plates were incubated at 26°C in the dark for 5 days. A sporulating, dematiaceous, mitosporic fungus was observed and transferred to potato dextrose agar. Colonies were white to gray, with brown as conidia increased. Conidia ranged in size from 10 to 22.5 × 20 to 37.5 (average 15.2 × 26.5) µm. Conidia were golden to dark brown, broadly ellipsoidal, some pyriform, with one longitudinal septum and two to three transverse septa, sometimes constricted at the transverse septa. Based on microscopic examination, the fungus was identified as Pithomyces chartarum (Berk. & Curt.) M.B. Ellis (1); observations were consistent with the authority (2). Pathogenicity assays were conducted with 5-week-old 'Alamo' switchgrass grown from seed scarified with 60% sulfuric acid and surface-sterilized with 50% bleach. Seed were sown in 9 × 9-cm pots containing 50% (v/v) ProMix Potting and Seeding Mix (Premier Tech Horticulture, Québec, Canada) and 50% Turface ProLeague (Profile Products, Buffalo Grove, IL). Eight replicate pots with ~20 plants each were sprayed with a spore suspension of 5.7 × 105 spores/ml sterile water prepared from 6-day-old cultures grown on V8 juice agar in the dark. Two more pots were sprayed with sterile water to serve as controls. All plants were subjected to high humidity for 72 h by enclosure in a plastic bag. Plants were placed in a growth chamber at 25/20°C with a 12-h photoperiod. Leaf spot symptoms similar to the original disease were evident on plants in each of the eight replicate pots 6 to 10 days post-inoculation. Control plants had no symptoms. Lesions were excised from leaves, surface sterilized, and plated on WA. The resulting cultures were again identified as P. chartarum based on morphology. The internal transcribed spacer (ITS) region of rDNA from the original isolate and the pathogen recovered from plants in the pathogenicity tests were amplified with PCR using primers ITS4 and ITS5. PCR amplicons were obtained from both isolates, sequenced, and found to have 100% identity. A 580-bp sequence was deposited at GenBank (Accession No. JQ406588). The nucleotide sequence had 98 to 100% identity to the ITS sequences of isolates of Leptosphaerulina chartarum (anamorph: P. chartarum), including isolate Mxg-KY09-s4 (GU195649) from leaf spot on Miscanthus × giganteus in Kentucky (1), and isolates from leaf lesions on wheat (EF489400 and JX442978). To our knowledge, leaf spot caused by P. chartarum has not been described on switchgrass (3). Pithomyces chartarum is a seedborne pathogen of switchgrass, and may play a role in stand establishment. References: (1) M. O. Ahonsi et al. Plant Dis. 94:480, 2010. (2) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England. 1971. (3) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA, Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 18 January 2013.

First Report of Leaf Spot caused by Bipolaris oryzae on Switchgrass in Tennessee.

Knowledge of pathogens in switchgrass, a potential biofuels crop, is limited. In December 2007, dark brown to black irregularly shaped foliar spots were observed on 'Alamo' switchgrass (Panicum virgatum L.) on the campus of the University of Tennessee. Symptomatic leaf samples were surface-sterilized (95% ethanol, 1 min; 20% commercial bleach, 3 min; 95% ethanol, 1 min), rinsed in sterile water, air-dried, and plated on 2% water agar amended with 3.45 mg fenpropathrin/liter (Danitol 2.4 EC, Valent Chemical, Walnut Creek, CA) and 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO). A sparsely sporulating, dematiaceous mitosporic fungus was observed. Fungal plugs were transferred to surface-sterilized detached 'Alamo' leaves on sterile filter paper in a moist chamber to increase spore production. Conidia were ovate, oblong, mostly straight to slightly curved, and light to olive-brown with 3 to 10 septa. Conidial dimensions were 12.5 to 17 × 27.5 to 95 (average 14.5 × 72) µm. Conidiophores were light brown, single, multiseptate, and geniculate. Conidial production was polytretic. Morphological characteristics and disease symptoms were similar to those described for Bipolaris oryzae (Breda de Haan) Shoemaker (2). Disease assays were done with 6-week-old 'Alamo' switchgrass grown from seed scarified with 60% sulfuric acid and surface-sterilized in 50% bleach. Nine 9 × 9-cm square pots with approximately 20 plants per pot were inoculated with a mycelial slurry (due to low spore production) prepared from cultures grown on potato dextrose agar for 7 days. Cultures were flooded with sterile water and rubbed gently to loosen mycelium. Two additional pots were inoculated with sterile water and subjected to the same conditions to serve as controls. Plants were exposed to high humidity by enclosure in a plastic bag for 72 h. Bags were removed, and plants were incubated at 25/20°C with 50 to 60% relative humidity. During the disease assay, plants were kept in a growth chamber with a 12-h photoperiod of fluorescent and incandescent lighting. Foliar leaf spot symptoms appeared 5 to 14 days post-inoculation for eight of nine replicates. Control plants had no symptoms. Symptomatic leaf tissue was processed and plated as described above. The original fungal isolate and the pathogen recovered in the disease assay were identified using internal transcribed spacer (ITS) region sequences. The ITS region of rDNA was amplified with PCR and primer pairs ITS4 and ITS5 (4). PCR amplicons of 553 bp were sequenced, and sequences from the original isolate and the reisolated pathogen were identical (GenBank Accession No. JQ237248). The sequence had 100% nucleotide identity to B. oryzae from switchgrass in Mississippi (GU222690, GU222691, GU222692, and GU222693) and New York (JF693908). Leaf spot caused by B. oryzae on switchgrass has also been described in North Dakota (1) and was seedborne in Mississippi (3). To our knowledge, this is the first report of B. oryzae from switchgrass in Tennessee. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/, 28 June 2012. (2) J. M. Krupinsky et al. Can. J. Plant Pathol. 26:371, 2004. (3) M. Tomaso-Peterson and C. J. Balbalian. Plant Dis. 94:643, 2010. (4) T. J. White et al. Pages 315-322 in: PCR Protocols: a Guide to Methods and Applications. M. A. Innis et al. (eds), Acad. Press, San Diego, 1990.

First Report of Leaf Spot and Necrotic Roots on Switchgrass Caused by Curvularia lunata var. aeria in the United States.

Curvularia lunata infects many grass species; however, switchgrass (Panicum virgatum L.) has not been reported as a host (2). In June 2009, small brown leaf spots and necrotic roots were observed on stunted 2-year-old 'Alamo' switchgrass on the University of Tennessee, Knoxville campus. Symptomatic leaf and root tissues were surface-sterilized in 95% ethanol for 1 min, 20% bleach for 3 min, and 95% ethanol for 1 min, and then air dried and placed on water agar amended with 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO) and 7.5 µl/liter Danitol (Valent Chemical, Walnut Creek, CA). Cultures were incubated at 25°C for 3 days. Hyphal tips were transferred to potato dextrose agar (PDA) and incubated at 25°C. Dark brown-to-black fungal colonies with black stromata formed. Conidiophores were dark brown, unbranched, septate, polytretic, sympodial, and geniculate at the apical region with rachis conidial ontogeny. Conidia were dark brown and cymbiform with three to four septations, with one or two central cells larger than the terminal cells. Spore size ranged from 17.5 to 30.0 × 8.8 to 12.5 µm (mean 21.6 × 10.8 µm). Morphological traits matched the description of C. lunata var. aeria (1). To test pathogenicity, fungal sporulation was optimized on PDA with pieces of sterile, moistened index card placed on each plate; cultures were incubated at 25°C with a 12-h photoperiod (3). After 14 days, conidia were dislodged in sterile water and the spore concentration adjusted to 8 × 104 conidia/ml. Ten pots, with about 15 plants per pot, of 6-week-old 'Alamo' switchgrass grown from surface-sterilized seed were inoculated with the spore suspension applied to the plant crown and surrounding soil with an aerosol sprayer. Prior to inoculation, roots were wounded with a sterile scalpel. Noninoculated plants (two pots), with roots also wounded, served as controls. To maintain high humidity, each pot was covered with a plastic bag and maintained in a growth chamber at 30°C with a 16-h photoperiod. Bags were removed after 3 days; plants were maintained as described for 6 weeks. Brown leaf spots and brown-to-black necrotic roots that matched symptoms on the naturally infected plants were observed in all inoculated plants; there were no symptoms of Curvularia infection on the controls. The fungus was reisolated from inoculated plants as described above. Genomic DNA was extracted from the original isolate and the reisolate from the pathogenicity test. PCR amplification of the internal transcribed spacer (ITS) regions from ribosomal DNA was performed with primers ITS4 and ITS5. PCR products of 503 bp were sequenced. There was 100% nucleotide identity for sequences of the original isolate and the re-isolate. The sequence was submitted to GenBank (Accession No. HQ130484.1). BLAST analysis of the fungal sequence resulted in 100% nucleotide sequence identity to the ITS sequences of isolates of C. affinis, C. geniculata, and C. lunata. On the basis of the smaller spore size and abundant stromata on PDA, the isolate was identified as C. lunata var. aeria. As switchgrass is developed as a biofuels crop, identification of new pathogens may warrant development of disease management strategies. References: (1) M. B. Ellis. Mycological Papers No. 106, CMI, Surrey, 1966. (2) D. F. Farr and A. Y. Rossman, Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , August 2011. (3) R. G. Pratt. Mycopathologia 162:133, 2006.

First Report of Leaf Spot Caused by Alternaria alternata on Switchgrass in Tennessee.

Field-grown seedlings of 'Alamo' switchgrass (Panicum virgatum L.) from Vonore, TN exhibited light brown-to-dark brown leaf spots and general chlorosis in June 2009. Symptomatic leaf tissue was surface sterilized (95% ethanol for 1 min, 20% commercial bleach for 3 min, and 95% ethanol for 1 min), air dried on sterile filter paper, and plated on 2% water agar amended with 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO) and 5 µl/liter miticide (2.4 EC Danitol, Valent Chemical, Walnut Creek, CA). Plates were incubated at 26°C for 4 days in darkness. An asexual, dematiaceous mitosporic fungus was isolated and transferred to potato dextrose agar. Cultures were transferred to Alternaria sporulation medium (3) to induce conidial production. Club-shaped conidia were produced in chains with branching of chains present. Conidia were 27 to 50 × 10 to 15 µm, with an average of 42.5 × 12.5 µm. Morphological features and growth on dichloran rose bengal yeast extract sucrose agar were consistent with characteristics described previously for Alternaria alternata (1). Pathogenicity studies were conducted with 5-week-old 'Alamo' switchgrass plants grown from surface-sterilized seed. Nine pots with approximately 20 plants each were prepared. Plants were wounded by trimming the tops. Eight replicate pots were sprayed with a conidial spore suspension of 5.0 × 106 spores/ml sterile water and subjected to high humidity by enclosure in a plastic bag for 7 days. One pot was sprayed with sterile water and subjected to the same conditions to serve as a control. Plants were maintained in a growth chamber at 25/20°C with a 12-h photoperiod. Foliar leaf spot symptoms appeared 5 to 10 days postinoculation for all replicate pots inoculated with A. alternata. Symptoms of A. alternata infection were not observed on the control. Lesions were excised, surface sterilized, plated on water agar, and identified in the same manner as previously described. The internal transcribed spacer (ITS) region of ribosomal DNA and the mitochondrial small sub-unit region (SSU) from the original isolate and the reisolate recovered from the pathogenicity assay were amplified with PCR, with primer pairs ITS4 and ITS5 and NMS1 and NMS2, respectively. Resultant DNA fragments were sequenced and submitted to GenBank (Accession Nos. HQ130485.1 and HQ130486.1). A BLAST search (BLASTn, NCBI) was run against GenBank isolates. The ITS region sequences were 537 bp and matched 100% max identity with eight A. alternata isolates, including GenBank Accession No. AB470838. The SSU sequences were 551 bp and matched 100% max identity with seven A. alternata isolates, including GenBank Accession No. AF229648. A. alternata has been reported from switchgrass in Iowa and Oklahoma (2); however, this is the first report of A. alternata causing leaf spot on switchgrass in Tennessee. Switchgrass is being studied in several countries as a potentially important biofuel source, but understanding of the scope of its key diseases is limited. References: (1) B. Andersen et al. Mycol. Res. 105:291, 2001. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , September 22, 2011. (3) E. A. Shahin and J. F. Shepard. Phytopathology 69:618, 1979.

First Report of Dollar Spot Caused by Sclerotinia homoeocarpa on Switchgrass in the United States.

Sclerotinia homoeocarpa causes dollar spot on many grass species; however, it has not been described on switchgrass (Panicum virgatum L.) as a host. In August 2010, bleached, tan-to-straw-colored leaf spots with dark brown-to-reddish brown margins were found in patchy distribution in small field plots of 'Alamo' switchgrass at the East Tennessee Research and Education Center, Knoxville, TN. The plots had been planted to switchgrass for the past 21 years. Disease lesions covered 75 to 80% of leaf tissue per patch and were also evident on stems. To identify the pathogen, center portions of diseased leaves were cut into 20- to 30-cm segments, surface disinfested (95% ethanol for 30 s, 10% bleach for 1 min, and 95% ethanol for 30 s), and dried. Disinfested leaves (5-cm sections that included a leading edge of a lesion) were plated on potato dextrose agar (PDA). Plates were incubated at 22°C. Within 12 h, white, fluffy, aerial mycelium developed. Viewed from above, colonies were tan to cinnamon in color with a dark brown-to-black substratal stroma on and in the agar, which appeared brown as viewed from below the petri dish. No spores were observed. Morphological characteristics of colony and hyphal growth were identical to those of S. homoeocarpa F.T. Bennett (1). Pathogenicity studies were conducted with 6-week-old 'Alamo' switchgrass grown from scarified (2), surface-disinfested seed. Nine (9 × 9-cm2) pots with 18 plants each were inoculated with 20 mycelial plugs (6-mm diameter) per pot, taken from 3-to-5-day-old fungal cultures. Two control pots were inoculated with sterile PDA plugs and subjected to the same conditions. Plugs were placed on leaf surfaces and around the plant crowns. Plants were subjected to high humidity by enclosure in a plastic bag and incubated in a growth chamber at 25/20°C with a 12-h photoperiod. Plastic bags were removed after 48 h. Leaf spots appeared as early as 2 days postinoculation, with full symptoms after 2 weeks for eight of nine replicates. Control plants had no symptoms. The fungus was cultured from leaf spots and stem lesions of inoculated plants as described above. The same disease and fungus were observed, completing Koch's postulates. The internal transcribed spacer (ITS) regions of ribosomal DNA from the original isolate used for inoculation and from the isolate recovered from plants in the pathogenicity assay were amplified with PCR with primers ITS4 and ITS5 (4). PCR amplicons of ~565 bp were sequenced; sequences of amplicons from the original isolate and reisolate were identical and submitted to GenBank (Accession No. HQ850151). The sequence had 99% homology with several S. homoeocarpa isolates in GenBank, including three isolates from buffalograss in Oklahoma (Accession Nos. EU123800, EU123802, and EU123803). The mitochondrial small subunit region was amplified from the original isolate with primers NMS1 and NMS2 (3). The resultant 536-bp fragment was sequenced and submitted to GenBank (Accession No. HQ850152), but no S. homoeocarpa sequences were available for comparison. To our knowledge, this is the first confirmed report of switchgrass as a natural host for S. homoeocarpa, extending the known host range for the pathogen. References: (1) F. T. Bennett. Ann. Appl. Biol. 24:236, 1937. (2) K. D. Gwinn et al. Crop Sci. 31:1369, 1991. (3) K. N. Li et al. Appl. Environ. Microbiol. 60:4324, 1994. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, NY, 1990.

First Report of Leaf Spot Caused by Bipolaris spicifera on Switchgrass in the United States.

Light-to-dark brown leaf spots and general chlorosis were observed on 'Alamo' switchgrass (Panicum virgatum L.) grown in ornamental plantings on the campus of the University of Tennessee in Knoxville in December 2007. Disease distribution was patchy, infecting ~10% of plants. Patches had mild to severely infected plants with stunting in areas of severe infection. Symptomatic leaf tissue was surface sterilized, air dried on sterile filter paper, and plated on 2% water agar amended with 10 mg/liter of rifampicin (Sigma-Aldrich, St. Louis, MO) and 10 µl/liter of 2.4 EC Danitol miticide (Valent Chemical, Walnut Creek, CA). Plates were incubated at 26°C in darkness for 5 days. A sporulating, dematiaceous mitosporic fungus was observed and transferred to potato dextrose agar (PDA). Conidiophores were single, light brown, multiseptate, mostly straight, polytretic, geniculate, and sympodial. Conidia were 17.5 × 12 (22) to 30 × 14 (12.5) µm, oval, light brown, and distoseptate, with one to three septa and a flattened hilum on the basal cell. Conidia germinated from both poles. The causal agent was identified as Bipolaris spicifera (Bainier) Subram. Morphological features were as described for B. spicifera (2). Pathogenicity studies were conducted with 5-week-old 'Alamo' switchgrass plants grown from surface-sterilized seed in 9 × 9-cm pots containing 50% ProMix Potting and Seeding Mix (Premier Tech Horticulture, Rivière-du-Loup, Québec, Canada) and 50% Turface ProLeague (Profile Products, Buffalo Grove, IL) (vol/vol). Ten replicate pots with ~20 plants each were sprayed with a spore suspension of 4.5 × 106 spores/ml of sterile water prepared from 6-day-old cultures grown on PDA. Plants were subjected to high humidity for 45 h then incubated at 25/20°C with a 12-h photoperiod in a growth chamber. Leaf spot symptoms similar to the original disease appeared on plants in each of the 10 replicate pots 6 days postinoculation. Lesions were excised from leaves, surface sterilized, plated on water agar, and the resulting cultures were again identified as B. spicifera. The internal transcribed spacer (ITS) region of ribosomal DNA from the original isolate used for inoculation and the reisolated culture recovered from plants in the pathogenicity studies were amplified with PCR using primers ITS4 and ITS5 (3). PCR amplicons of ~560 bp were obtained from both isolates and sequenced. Amplicon sequences were identical and the sequence was submitted to GenBank (Accession No. HQ015445). The DNA sequence had 100% homology to the ITS sequence of B. spicifera strain NRRL 47508 (GenBank Accession No. GU183125.1) that had been isolated from sorghum seed. To our knowledge, leaf spot caused by B. spicifera has not been described on switchgrass (1). B. spicifera can be seedborne and has been reported on turfgrass seed exported from the United States to Korea (2). As switchgrass is transitioned from a prairie grass to a biofuels crop planted in large acreages, disease incidences and severities will likely increase, necessitating rapid disease identification and cost effective management strategies. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 4 August 2010. (2) H.-M. Koo et al. Plant Pathol. J. 19:133, 2003. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds, Academic Press, San Diego, 1990.

First Report of Spot Blotch and Common Root Rot Caused by Bipolaris sorokiniana on Switchgrass in Tennessee.

Light-to-dark brown, irregular-shaped leaf spots, chlorosis, necrotic roots, and severe stunting were observed on 'Alamo' switchgrass (Panicum virgatum L.) grown on the campus of the University of Tennessee in December 2007. Symptomatic leaf and root samples were surface sterilized, air dried on sterile filter paper, and plated on 2% water agar amended with 10 mg/liter of rifampicin (Sigma-Aldrich, St. Louis, MO) and 10 µl/liter of 2,4 EC Danitol miticide (Valent Chemical, Walnut Creek, CA). Plates were incubated at 25°C in darkness for 4 days. A sporulating, dematiaceous mitosporic fungus was noted and transferred to potato dextrose agar (PDA). Conidia were ovate, oblong, mostly straight, and olive to brown with three to nine septa. Conidial dimensions were 12.5 × 27.5 (17.5) to 20 × 77.5 (57) µm. Conidia were produced on single, light brown, multiseptate conidiophores that were polytretic, geniculate, and sympodial. Morphological features were as described for Bipolaris sorokiniana (Sacc.) Shoemaker (teleomorph = Cochliobolus sativus) (2,3). Disease assays were conducted with 5-week-old 'Alamo' switchgrass grown from surface-sterilized seed. Ten 9 × 9-cm2 with ~20 switchgrass seedlings were sprayed with 2.4 × 105 spores/ml of sterile water. Plants were subjected to high humidity created by enclosure in a plastic bag for 45 h. The bag was removed and plants were incubated at 25/20°C with 50 to 60% relative humidity. During the incubation, plants were maintained in growth chamber with a 12-h photoperiod of fluorescent and incandescent lighting. Foliar leaf spot symptoms appeared 6 to 10 days postinoculation for plants in all 10 replicates and necrotic lesions were observed on roots. Foliar lesions and diseased roots were surface sterilized, plated on water agar, and resultant fungal colonies were identified as B. sorokiniana. The internal transcribed spacer (ITS) and mitochondrial small subunit (SSU) regions of ribosomal DNA from the original isolate, and the isolate recovered from plants in the pathogenicity assay, were amplified with PCR, with primer pairs ITS4 and ITS5 and NMS1 and NMS2. PCR amplicons of ~551 and 571 bp were obtained with the two primer pairs, respectively. Both amplicons were obtained from both isolates and sequenced. Amplicon sequences from the original isolate and re-isolate were identical and the sequences were submitted to GenBank (Accession Nos. HQ611957 and HQ611958). The ITS sequences had 98% homology to 23 B. sorokiniana isolates, including B. sorokiniana strain DSM 62608 (GenBank Accession No. EF187908); SSU sequences had 99% homology to Cochliobolus sativus isolate AFTOL-ID 271 (GenBank Accession No. FJ190589). Spot blotch caused by B. sorokiniana has been reported on switchgrass in Iowa, Nebraska, Pennsylvania, and Virginia (1). To our knowledge, this is the first report of B. sorokiniana causing spot blotch or common root rot of switchgrass in Tennessee, which extends the current known distribution of these diseases. More recently, we isolated B. sorokiniana from switchgrass seed received from commercial sources in the United States, indicating a seedborne transmission. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 15 November 2010. (2) R. F. Nyvall and J. A. Percich. Plant Dis. 83:936, 1999. (3) A. Sivanesan and P. Holliday. CMI Descr. Pathog. Fungi bact. 71:701, 1981.

Analysis of genetic diversity in flowering dogwood natural stands using microsatellites: the effects of dogwood anthracnose.

Flowering dogwood (Cornus florida L.) populations recently have experienced severe declines caused by dogwood anthracnose. Mortality has ranged from 48 to 98%, raising the concern that genetic diversity has been reduced significantly. Microsatellite data were used to evaluate the level and distribution of genetic variation throughout much of the native range of the tree. Genetic variation in areas affected by anthracnose was as high as or higher than areas without die-offs. We found evidence of four widespread, spatially contiguous genetic clusters. However, there was little relationship between geographic distance and genetic difference. These observations suggest that high dispersal rates and large effective population sizes have so far prevented rapid loss of genetic diversity. The effects of anthracnose on demography and community structure are likely to be far more consequential than short-term genetic effects.

Co-infection of Soybean with Soybean mosaic virus and Alfalfa mosaic virus Results in Disease Synergism and Alteration in Accumulation Level of Both Viruses.

Co-infection of potyviruses with taxonomically diverse plant viruses results in disease synergism and elevation in the level of accumulation of non-potyviruses involved. In the majority of cases, however, the accumulation level of potyviruses remains essentially unaltered. A few potyviruses, such as Soybean mosaic virus (SMV), naturally infect soybean (Glycine max). Soybean is also a natural host to a number of non-potyviruses including Alfalfa mosaic virus (AMV), which causes mild symptoms often associated with symptom remission. We have now studied the interactions between AMV and SMV on symptom severity and accumulation level of each of the two viruses in soybean. Co-infection of soybean with AMV and SMV was established following mechanical inoculation, irrespective of simultaneous or sequential introduction of the two viruses. In multiple experiments, co-infection of soybean resulted in severe symptoms in doubly infected plants in a strain-independent manner, with enhancement in the level of AMV indicating that the interaction of AMV with SMV is synergistic. Conversely, the level of SMV accumulation was reduced. This suggests that in co-infection with AMV, SMV interacts antagonistically. The observation that co-infection of AMV and SMV results in disease synergism suggests enhancement of potential that AMV may become a serious viral disease of soybean.

First Report of Rust on Switchgrass (Panicum virgatum) Caused by Puccinia emaculata in Tennessee.

In the spring of 2007, switchgrass accessions and cultivars Alamo, Kanlow, SL-93-2001, and NSL 2001-1 (lowland), Blackwell (upland), and Grenville, Falcon, and Miami (unknown ploidy levels) were sown at the East Tennessee Research and Extension Center in Knoxville for evaluation and controlled hybridizations. In July and August of 2007, uredinia were observed primarily on the upper leaf surfaces, and to a lesser extent on the undersides of leaves, of switchgrass cvs. Alamo, Blackwell, Grenville, Falcon, Kanlow, and Miami. Uredinia were observed on all cultivars and accessions in 2008. Dimensions of spores are reported as mean ± standard deviation. Uredinia were epiphyllous, adaxial, caulicolous, oblong, and the color of cinnamon brown. Urediniospores were globose to broadly ellipsoid, 26.0 ± 3.0 × 23.2 ± 2.4 µm, with a wall that was cinnamon brown, 1.5 to 2.0 µm thick, finely echinulate with three to four equatorial pores, corresponding to Puccinia emaculata Schw. (3). Abundant teliospores were isolated from Grenville, Falcon, and Blackwell, with fewer teliospores isolated from Alamo. Telia were epiphyllous, adaxial, and caulicolous, densely crowded to scattered, oblong, and dark brown to black. Teliospores were dark brown, two-celled, ellipsoid to oblong, 33.6 ± 4.8 µm long with an apical cell width of 17.5 ± 1.2 µm and basal cell width of 15.9 ± 2.5 µm. Teliospore walls were 1.5 to 2.0 µm wide at the sides and 4 to 6 µm apically. Pedicels were brown or colorless and up to approximately one length of the teliospore, 28.5 ± 7.4 µm. Teliospore morphology confirmed the identification of this rust as P. emaculata (3), which has been reported to infect upland and lowland populations of switchgrass (2). A 2,109-bp fragment containing the internal transcribed spacer (ITS) 1, 5.8S, ITS 2, and D1/D2 region of the large subunit ribosomal DNA was sequenced for a specimen on 'Falcon' (GenBank Accession No. EU915294 and BPI No. 878722) from two overlapping PCR fragments amplified with primers PRITS1F (L. A. Castlebury, unpublished data) and ITS4B (1) for one fragment and Rust5.8SF (L. A. Castlebury, unpublished data) and LR7 (4) for the second fragment. No sequences of P. emaculata were available for comparison; however, BLAST searches of the ITS resulted in hits to P. asparagi DC (527 of 576, 91%) and P. andropogonis Schw. (523 of 568, 92%) placing this fungus in the genus Puccinia Pers. The alternate hosts of this rust are species of the Euphorbiaceae (2,3), which are ubiquitous in this area although the aecial stage has not been observed. To our knowledge, this is the first report of P. emaculata on switchgrass in Tennessee. Given the highly susceptible response of certain varieties of switchgrass to this rust in field plots, reduction in total biomass in large acreages is likely and long-standing fields of this perennial grass will compound the problem. References: (1) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (2) D. M. Gustafson et al. Crop Sci. 43:755, 2003. (3) P. Ramachar and G. Cummins. Mycopathol. Mycol. Appl. 25:7, 1965. (4) R. Vilgalys and M. Hester. J. Bacteriol. 172:4238, 1990.

Viability and stability of biological control agents on cotton and snap bean seeds.

Cotton and snap bean were selected for a multi-year, multi-state regional (south-eastern USA) research project to evaluate the efficacy of both commercial and experimental bacterial and fungal biological control agents for the management of damping-off diseases. The goal for this portion of the project was to determine the viability and stability of biological agents after application to seed. The biological seed treatments used included: (1) Bacillaceae bacteria, (2) non-Bacillaceae bacteria, (3) the fungus Trichoderma and (4) the fungus Beauveria bassiana. Seed assays were conducted to evaluate the following application factors: short-term (< or = 3 months) stability after seed treatment; quality (i.e. isolate purity); compatibility with chemical pesticides and other biocontrol agents; application uniformity between years and plant species. For the bacterial treatments, the Bacillaceae genera (Bacillus and Paenibacillus) maintained the greatest population of bacteria per seed, the best viability over time and the best application uniformity across years and seed type. The non-Bacillaceae genera Burkholderia and Pseudomonas had the least viability and uniformity. Although Beauveria bassiana was only evaluated one year, the seed fungal populations were high and uniform. The seed fungal populations and uniformity for the Trichoderma isolates were more variable, except for the commercial product T-22. However, this product was contaminated with a Streptomyces isolate in both the years that it was evaluated. The study demonstrated that Bacillaceae can be mixed with Trichoderma isolates or with numerous pesticides to provide an integrated pest control/growth enhancement package.

Effect of delivery method and population size of Trichoderma harzianum on growth response of unrooted chrysanthemum cuttings.

In a previous study, addition of Trichoderma harzianum Rifai isolate T-12 to a propagative medium resulted in improved performance of chrysanthemum cuttings. However, root and shoot growth of one cultivar, 'Dark Bronze Charm', were more responsive to a lower (5 g T-12/kg medium) than higher (25 g T-12/kg medium) rate of fungal propagules, suggesting potential phytotoxicity at higher concentrations. The objectives of this study were to investigate higher rates of T-12 medium amendment for phytotoxicity, and to examine an alternative method of delivering the fungus to the propagative medium in order to obtain a more uniform response from cuttings. Isolate T-12 was added to the propagative medium as either a powdered peat-bran amendment (0, 5, or 50 g T-12/kg medium) or as alginate prills (80 or 800 g T-12/kg medium). There were no differences among treatments on day seven, but by day 21, shoot fresh weight and heights were significantly greater for plants treated with prills at 800 g T-12/kg medium. Both prill treatments resulted in greater shoot height on day 14 and 21 than all other treatments, which were similar to controls. Amendment with T-12 powder at 50 g/kg increased root length, but 80 g/kg medium added as prills decreased root dry weight compared to the control. The highest rate of T-12 (800 g prills/kg medium) had no effect on root growth. This suggests that moderate, rather than high rates of T-12 are more effective in promoting rooting of unrooted chrysanthemum, and that there is a potential for phytotoxic effects on root growth with higher rates.

Soil Chemical and Physical Properties Associated with Suppression of Take-all of Wheat by Trichoderma koningii.

ABSTRACT Trichoderma koningii, originally isolated from a take-all-suppressive soil in Western Australia, has been shown to protect wheat against take-all disease and increase grain yield in field trials in Australia, China, and the United States. However, within a region, the level of protection provided by T. koningii can dramatically vary between field sites. We evaluated suppression of take-all by this fungus in eight silt loams from the Pacific Northwest of the United States and the influence of 21 abiotic soil parameters on biocontrol activity. While T. koningii significantly increased plant growth and reduced disease severity in all eight silt loams, the level of protection varied significantly among the soils. Disease suppression was not associated with the conduciveness of a soil to take-all, but rather to the supportiveness of a soil to biocontrol activity. Biocontrol activity was positively correlated with iron, nitrate-nitrogen, boron, copper, soluble magnesium, and percent clay, and negatively correlated with soil pH and available phosphorus. Principal component factor analysis using these eight variables resulted in a three-component solution that accounted for 95% of the variation in disease rating. Least squares regression analysis (R(2) = 0.992) identified a model that included nitrate-nitrogen, soil pH, copper, and soluble magnesium, and described the variance in take-all suppression by T. koningii. Potential applications of these results include amending soil or inoculants with beneficial factors that may be lacking in the target soil and customizing biocontrol treatments for sites that have parameters predictive of a favorable environment for disease suppression.