Baseline Sensitivity of Cercospora zeae-maydis to Quinone Outside Inhibitor Fungicides.

Cercospora zeae-maydis, the causal agent of gray leaf spot on corn (Zea mays), can cause severe yield loss in the United States. Quinone outside inhibitor (QoI) fungicides are effective tools that can be used to manage gray leaf spot, and their use has increased in corn production in the United States. In total, 61 C. zeae-maydis isolates collected from fields in which QoI fungicides had never been applied were tested in vitro using azoxystrobin-, pyraclostrobin-, or trifloxystrobin-amended medium to determine the effective fungicide concentration at which 50% of the conidial germination was inhibited (EC50). The effect of salicylhydroxamic acid (SHAM) also was evaluated for seven isolates to determine whether C. zeae-maydis is capable of using alternative respiration in azoxystrobin-amended medium. All seven C. zeae-maydis isolates tested had significantly greater (P < 0.02) EC50 values when SHAM was not included in medium amended with azoxystrobin, indicating that C. zeae-maydis has the potential to utilize alternative respiration to overcome QoI fungicide inhibition in vitro. Baseline EC50 values of azoxystrobin ranged from 0.003 to 0.031 µg/ml, with mean and median values of 0.018 and 0.019 µg/ml, respectively. Baseline EC50 values of pyraclostrobin ranged from 0.0003 to 0.0025 µg/ml, with mean and median values of 0.0010 and 0.0010 µg/ml, respectively. Baseline EC50 values of trifloxystrobin ranged from 0.0004 to 0.0034 µg/ml, with mean and median values of 0.0023 and 0.0024 µg/ml, respectively. These baseline sensitivity values will be used in a fungicide resistance monitoring program to determine whether shifts in sensitivity to QoI fungicides are occurring in C. zeae-maydis populations.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Sunflower in Illinois.

In September 2009, sunflower (Helianthus annuus L.) plants (cv. Mycogen 8C451) from a University of Illinois field research trial in Fayette County, Illinois exhibited silvery gray girdling lesions on the lower stems and premature death. When lower stems and roots were split open, the pith tissue was compressed into layers. Black microsclerotia (90 to 180 µm) were present on the outside of the lower stem tissue and in the stem vascular tissue. Five pieces (approximately 1 cm long) of symptomatic stem tissue from five different affected plants (25 pieces total) were soaked in a 0.5% solution of NaOCl for 30 s, rinsed with sterile distilled water, and placed on potato dextrose agar (PDA; Becton, Dickinson, and Company, Franklin Lakes, NJ). Gray hyphae grew from all of the stem pieces, which subsequently turned black and formed black microsclerotia (75 to 175 µm). On the basis of plant symptoms and size and color of the microsclerotia, the disease was diagnosed as charcoal rot caused by Macrophomina phaseolina (Tassi) Goid (2). To confirm that the isolated fungus was M. phaseolina, DNA was extracted from the pure culture, and PCR amplification of a subunit rDNA and internal transcribed spacer (ITS) region with primers EF3RCNL and ITS4 was performed (3). The Keck Biotechnology Center at the University of Illinois, Urbana sequenced the PCR product. The resulting nucleotide sequence shared the highest homology (99%) with sequences of M. phaseolina when compared with the subunit rDNA and ITS sequences in the nucleotide database ( http://www.ncbi.nlm.nih.gov ). A greenhouse experiment was conducted to confirm pathogenicity; the greenhouse temperature was approximately 27°C and sunflower plants (cv. Cargill 270) were grown in pots and watered daily to maintain adequate soil moisture for growth. Sterile toothpicks were infested with M. phaseolina and placed through the stems (10 cm above the soil surface) of five 40-day-old sunflower plants that were approximately at growth stage R4 (1,4). Five sterile, noninfested toothpicks were similarly placed through sunflower plants to act as controls. Parafilm was used to hold the toothpick in the stem and seal the stem injury. Thirty-five days after inoculation, the mean lesion length on stems inoculated with M. phaseolina was 595 mm and no lesions developed on the control plants. M. phaseolina-inoculated plants also began to wilt and die. Cultures identical to the original M. phaseolina isolate were reisolated from stem lesions of the M. phaseolina-inoculated plants. This is the first report of charcoal rot on sunflower in Illinois to our knowledge. Sunflower is currently not a major crop grown in Illinois, but on-going research is focused on evaluating sunflower as a potential late-planted crop to follow winter wheat. If sunflower production increases in Illinois, growers may need to take precautions to manage charcoal rot. References: (1) L. K. Edmunds. Phytopathology 54:514, 1964. (2) T. Gulya et al. Page 263 in: Sunflower Technology and Production. American Society of Agronomy, Madison, WI, 1997. (3) N. S. Lord et al. FEMS Microbiol. Ecol. 42:327, 2002. (4) A. A. Schneiter and J. F. Miller. Crop Sci. 21:901, 1981.

Occurrences of Diplodia Leaf Streak Caused by Stenocarpella macrospora on Corn (Zea mays) in Illinois.

In August 2008, long and narrow lesions were observed on leaves of corn (Zea mays L.) growing in a field in Pope County, Illinois. Lesions were 10 to 35 × 50 to 250 mm and were cream to tan. Dark pycnidia inside the lesions were immersed and approximately 350 µm in diameter. Affected leaves were collected and placed into a moist chamber to encourage the development of conidia. Conidia developed in cirri and were dark, one septate, and 7 to 11 × 59 to 87 µm. Cirri were streaked onto potato dextrose agar (PDA; Becton, Dickinson, and Company, Franklin Lakes, NJ) and cultures arising from single conidia were transferred and maintained. On the basis of the corn leaf symptoms and the morphological characteristics of the pycnidia and conidia, the fungus was tentatively identified as Stenocarpella macrospora (Earle) Sutton (1). To complete Koch's postulates, 'Garst 84H80-3000GT' corn was inoculated in the greenhouse. Conidia were produced by placing a S. macrospora isolate from Pope County, IL onto water agar containing autoclaved corn leaves and incubating at room temperature until pycnidia and conidia were produced (approximately 3 weeks). A conidial suspension was used to inoculate the leaf whorls of corn plants (approximately at the V4 growth stage). Control plants were mock inoculated with sterile water. The experiment was repeated once over time. Twenty days after inoculation, all plants inoculated with S. macrospora conidia developed lesions similar to those observed in the field, and mock-inoculated plants remained symptomless. The fungus was reisolated on PDA from the symptomatic leaves. In August 2009, symptomatic leaves similar to those observed in Pope County, IL in 2008 were observed and collected from corn fields in Gallatin and Vermillion counties. Pycnidia and conidia from these lesions were similar to those described above, and isolates from single conidia were obtained from these samples. To confirm the identity of all isolates collected, PCR amplification of the small subunit rDNA and internal transcribed spacer (ITS) region with primers EF3RCNL and ITS4 was conducted (3). The PCR product was sequenced with these primers at the Keck Biotechnology Center at the University of Illinois, Urbana. The resulting nucleotide sequence was compared with small subunit rDNA and ITS sequences deposited in the GenBank nucleotide database, which revealed 99% homology to sequences of S. macrospora. In total, six of our S. macrospora isolates from Gallatin, Pope, and Vermillion counties were submitted to the United States Department of Agriculture-Agriculture Research Service Culture Collection in Peoria, IL, where they have received NRRL Accession Nos. 54190-54195. To our knowledge, this is the first report of S. macrospora affecting corn in Illinois. Although not observed in the Illinois corn fields described above, S. macrospora has been reported to infect stalks and ears (2). Because of the large leaf lesions caused by S. macrospora and its reported aggressiveness in causing disease on leaves, ears, and stalks, this pathogen has the potential to cause severe yield and quality losses to corn in the United States (2). References: (1) M. L. Carson. Diseases of minor importance or limited occurrence. Page 23 in: Compendium of Corn Diseases. 3rd ed. The American Phytopathological Society, St. Paul, MN, 1999. (2) F. M. Latterell and A. E. Rossi. Plant Dis. 67:725, 1983. (3) N. S. Lord et al. FEMS Microbiol. Ecol. 42:327, 2002.

First Report of Phomopsis Stem Canker of Sunflower in Illinois Caused by Phomopsis helianthi.

Stem cankers were observed on confection sunflower (Helianthus annuus) plants growing in a field in Champaign County, Illinois in August 2008. Lesions were brown to reddish brown, elongated (approximately 10 to 15 cm long), and centered over the area where leaf petioles connected to the stems. Stem tissues underneath the lesions were degraded. Lesions from diseased stems were cut into 5- to 7-mm pieces and immersed in a 0.5% NaOCl solution for 1 min, rinsed with sterilized distilled water, and placed into petri dishes containing acidified potato dextrose agar (APDA; 4 ml of 25% lactic acid per liter). Fungal colonies that grew from the stem lesion pieces on APDA were white, floccose, and dense with dark colored substrate mycelia. On the basis of the symptoms on sunflower plants and the growth characteristics on APDA, the fungus was tentatively identified as Phomopsis helianthi (1). To confirm the identity of the fungus, PCR amplification of the small subunit rDNA and internal transcribed spacer (ITS) region with primers EF3RCNL and ITS4 was done (2). The PCR product was sequenced with these primers at the Keck Biotechnology Center at the University of Illinois, Urbana. The resulting nucleotide sequence was compared with small subunit rDNA and ITS sequences deposited in the nucleotide database ( http://www.ncbi.nlm.nih.gov ) and showed highest homology to sequences of Diaporthe helianthi, teleomorph of P. helianthi. To confirm pathogenicity of the fungus, sunflower plants (cv. Cargill 270) were grown in the greenhouse and inoculated with the isolated fungus. The stems of sunflower plants between the V2 and V4 growth stages (3) were excised just below the uppermost node. Mycelia plugs of the fungus were placed into the large end of disposable micropipette tips (200 µl). The micropipette tip containing the fungus was subsequently placed over a cut sunflower stem. The fungal isolate was used to inoculate five stems. To serve as controls, five cut sunflower stems were inoculated with micropipette tips containing plugs of noninfested PDA and five cut stems were not inoculated. Mean lesion length on the stem was measured from the inoculated tip toward the soil line 7 days after inoculation. The experiment was replicated over time. Mean lesion length over both replications averaged 24 mm on the fungus-inoculated plants, 2 mm on the noninfested PDA-inoculated control plants, and no lesions were present on the noninoculated control plants. The fungus was reisolated on PDA from the inoculated plants in the greenhouse. To our knowledge, this is the first report of P. helianthi causing a stem canker of sunflower in Illinois. Although commercial sunflower production in Illinois is currently limited, it is being evaluated as a potential crop to follow winter wheat in portions of the state. If sunflower production were to increase in the state, growers may have to monitor for and manage Phomopsis stem canker. References: (1) T. Gulya et al. Sunflower diseases. Page 263 in: Sunflower Technology and Production. American Society of Agronomy, Madison, WI, 1997. (2) N. S. Lord et al. FEMS Microbiol. Ecol. 42:327, 2002. (3) A. A. Schneiter and J. F. Miller, Crop Sci. 21:901, 1981.

First Report of Gray Leaf Spot Caused by Pyricularia grisea on Lolium perenne in Illinois.

Each year from 1991 to 1999, a disease matching the description of gray leaf spot (1) was observed in the central and north central regions of Illinois. Disease severity was low (<10% blight) from 1991 to 1994 and 1999 and was severe (>50% blight in some areas) from 1995 to 1998. The disease was observed on Lolium perenne (perennial ryegrass) golf course fairways and sports fields. Isolations of Pyricularia grisea were made from L. perenne collected from golf courses in Bloomington, Decatur, Kankakee, Pekin, Urbana, and Moline, IL. All isolates were collected from surface-sterilized, symptomatic leaves. Cultures were maintained on one-fifth strength potato-dextrose agar (PDA) and induced to sporulate on full-strength oatmeal agar. All isolates in culture displayed vegetative and conidial characteristics similar to those previously described for P. grisea (1). Twenty-five different L. perenne germ plasms were inoculated with isolate WF9826 (Kankakee) using a suspension of 1 × 105 conidia per milliliter. The 4-week-old lawns (100 plants per 3-cm-diameter cone-tainer) of each ryegrass germ plasm were inoculated by spraying foliage with the conidial suspension until runoff. Inoculated and uninoculated lawns were enclosed in plastic bags and placed in an incubator (16 h light; 28°C) for 7 days. Disease severity was rated using a scale of 0 to 10 (10 = 100% blight). Each treatment was replicated three times, and all experiments were repeated four times. Small blue-gray, water-soaked lesions with dark brown borders were observed on leaves of all inoculated ryegrass germ plasms. Advanced symptoms included blighting of much of the leaves. The mean disease severity rating was 3.8 (range 2 to 7) for all experimental units and all 25 germ plasms. P. grisea was isolated from leaves that were inoculated with WF9826. This is the first report of gray leaf spot of perennial ryegrass caused by P. grisea in Illinois. Reference: (1) P. J. Landschoot et al. Plant Dis. 76:1280, 1992.

Effect of an intramammary polyethylene device in primiparous dairy cows.

A sterilized polyethylene intramammary device (IMD) was inserted through the papillary duct into a front and a rear mammary quarter of 12 primiparous dairy cows, which were mastitis-free. This study was conducted during lactation from month 1 postpartum to completion (generally month 9). After the device was inserted, milk somatic cell counts (SCC) increased. Milk scc in the quarters fitted with IMD from foremilk, primary, and stripping samples were 5.2, 2.6, and 5.9 X 10(5) cells/ml, respectively. Comparable counts for control quarters were 2.1, 1.4, and 3.6 X 10(5) cells/ml. The SCC in foremilk from the quarters fitted with the device were 2 to 3 times greater than the counts in foremilk from control quarters during month 2 and subsequent months of lactation. The SCC were significantly increased in primary and stripping samples from quarters fitted with IMD; however, the SCC of 2.6 X 10(5) cells/ml in primary milk would not affect market milk quality. Milk production for the lactation was decreased (0.6 kg/quarter/day) in the quarters fitted with IMD in comparison with control quarters throughout the lactation. There were no differences in percentages of milk fat or protein between deviced and control quarters. Conductivity values were increased in foremilk and stripping samples, but not in primary samples, for the quarters with IMD. Bovine serum albumin concentrations were also increased.