Paul L. Koch

Phase II study of second-line gemcitabine in sensitive or refractory small cell lung cancer

UNLABELLED Small cell lung cancer (SCLC) is highly sensitive to chemotherapy. Despite a dramatic initial response, however, most patients relapse. Given the activity of gemcitabine in non-small cell lung cancer (NSCLC), and early clinical trials suggesting activity of gemcitabine in chemo-naive SCLC patients, we conducted a phase II study to determine the efficacy and toxicities of gemcitabine in SCLC patients who have failed first-line chemotherapy. Gemcitabine 1250 mg/m(2) was given intravenously on days 1 and 8, every 3 weeks. Eligibility criteria included prior treatment with only one chemotherapy regimen and Eastern Cooperative Oncology Group (ECOG) performance status of 0-2. Patients with brain metastases were eligible. RESULTS Between April 1998 and October 2001, 27 patients were enrolled: 15 patients with sensitive (S) disease (recurred>3 months after first-line chemotherapy) and 12 patients with refractory (R) disease (failed<3 months after first-line chemotherapy). Median age was 61 (range 45-74). All patients had received prior platinum-based therapy involving etoposide and either cisplatin or carboplatin. There were one early death and two early withdrawals because of toxicity. No responses were observed. Of 24 patients who received at least two cycles of gemcitabine, only three achieved stable disease after six cycles while 21 progressed. The median time to progression (TTP) was 6 weeks in S group, 5.6 weeks in R group, and 6 weeks overall. After a minimum potential follow-up of almost 1 year for all patients, the median survival was 8.8 months in S group, 4.2 months in R group, and 6.4 months for the whole group. One-year survival rate was 33.3% in S group, 16.7% in R group, and 25.4% for all patients. Myelosuppression was the most commonly observed adverse effect, with grade 3/4 neutropenia in 30%, and grade 3 thrombocytopenia in 30%. One patient (3.7%) developed neutropenic fever. Respiratory failure and death, possibly related to pulmonary toxicity, was observed in one patient (3.7%). CONCLUSION monotherapy gemcitabine as second-line agent has limited activity in previously treated SCLC.

Thiophanate-Methyl and Propiconazole Sensitivity in Sclerotinia homoeocarpa Populations from Golf Courses in Wisconsin and Massachusetts

Management of dollar spot, caused by the fungus Sclerotinia homoeocarpa, is dependent upon repeated fungicide applications in intensively managed turfgrass such as golf course putting greens and fairways. Repeated fungicide applications could potentially select for fungicide-resistant isolates and result in a reduction of disease control. The objectives of this study were to determine the degree of S. homoeocarpa in vitro sensitivity to the fungicides thiophanate-methyl and propiconazole using isolates collected from golf course putting greens, fairways, and roughs; and to determine the relationships of golf course age and fungicide history to the frequency of fungicide-insensitive isolates within the population. More than 1,400 S. homoeocarpa isolates were collected from putting greens, fairways, and roughs at six Wisconsin golf courses and one Massachusetts golf course and subjected to in vitro fungicide sensitivity assays with single discriminatory concentrations of thiophanate-methyl and propiconazole. Five of seven pathogen populations from rough areas were not significantly different from one another in propiconazole sensitivity. These populations were collectively the most sensitive to both fungicides and therefore, served as baseline populations for comparison with fungicide-exposed populations from putting greens and fairways. Greater propiconazole insensitivity was observed in populations collected from fairways and putting greens that received more frequent applications of the fungicide than those isolated from the roughs. In nearly all the golf courses, the frequency of thiophanate-methyl insensitivity was higher among isolates of S. homoeocarpa collected from fairways than from roughs regardless of the age of the golf course or history of benzimidazole use. Thus, while the development of resistance to propiconazole can be predicted in part by the relative frequency of demethylation inhibitor fungicide applications, the occurrence of populations resistant to thiophanate-methyl appears to be unrelated to recent use of the benzimidazole class of fungicides.

Detection of root-infecting fungi on cool-season turfgrasses using loop-mediated isothermal amplification and recombinase polymerase amplification

Root-infecting fungal pathogens such as Gaeumannomyces avenae, Ophiosphaerella korrae, and Magnaporthiopsis poae cause extensive damage to amenity turfgrasses in temperate climates. The diseases they cause are difficult to diagnose by visual symptoms or microscopic inspection, and traditional polymerase chain reaction-based assays require large financial investments in equipment such as thermal cyclers and highly trained staff. The primary objective of this research was to develop fast and accurate detection assays for the three pathogens listed above that did not require the use of thermal cycling equipment. Loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) assays were developed for each pathogen based on known fungal cultures. The assays were tested on 27 samples received at the University of Wisconsins Turfgrass Diagnostic Laboratory in 2016 and 2017 and both methods provided accurate diagnoses within about 30 min with minimal sample preparation. However, the RPA assays had lower levels of false positive contamination relative to the LAMP assays and are more likely to be effective in a field or diagnostic laboratory for improved turf root-pathogen detection.

Data for designing two isothermal amplification assays for the detection of root-infecting fungi on cool-season turfgrasses

Loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) are two rapid isothermal amplification methods for detecting three common fungal root pathogens of cool-season turfgrass: Gaeumannomyces avenae, Magnaporthiopsis poae and Ophiosphaerella korrae, “Detection of root-infecting fungi on cool-season turfgrasses using loop-mediated isothermal amplification and recombinase polymerase amplification” (Karakkat et al., 2018) [1]. The data provided here describe the information for designing primers and probes for LAMP and RPA, how specific they are for each of the three fungi, and the evaluation of RPA on field samples.

Temperature Impacts on Soil Microbial Communities and Potential Implications for the Biodegradation of Turfgrass Pesticides

Maintaining healthy turfgrass often results in the use of pesticides to manage weed, insect, and disease pests. To identify and understand potential nontarget impacts of pesticide usage while still maintaining attractive and functional turfgrass sites, it is important to improve our understanding of how pesticides degrade in various environments throughout the growing season. Temperature heavily influences microbial community composition and activity, and the microbial community often heavily influences pesticide degradation in soil ecosystems. Pesticide transformation products generated through the action of soil microbial degradation networks can vary in their toxicity, with the potential result that a pesticide applied in the spring at 10°C could produce different transformation products with different toxicological impacts than the sample pesticide applied to the same site at 22°C. The objective of this review is to examine past research surrounding soil microbial activity related to pesticide degradation and provide a foundation for how the soil microbiome interacts with pesticides and how seasonal temperature variations may influence those interactions.

First report of brown ring patch caused by Waitea circinata var. circinata on Poa annua in Wisconsin and Minnesota.

In summer of 2008, two turfgrass samples were submitted to the Turfgrass Diagnostic Lab at the University of Wisconsin-Madison. The samples were from golf courses in Beaver Dam, WI on 12 June and Minneapolis, MN on 14 July. Both samples were collected from 40-year-old native soil putting greens mowed at 3.2 mm that had received annual sand topdressing since 1992. The putting greens were a mixture of approximately 75% annual bluegrass (Poa annua L.) and 25% creeping bentgrass (Agrostis stolonifera L.) Stand symptoms observed in the field were bright yellow, sunken rings that were approximately 5 cm thick and 15 to 35 cm in diameter. Some rings were incomplete, giving a scalloped appearance. Affected plants were severely chlorotic and lacked any discrete lesions or spots. Symptoms were more prominent on annual bluegrass than creeping bentgrass. Upon incubation of samples at room temperature in a moist chamber for 24 h, fungal mycelia with septations and right-angle branching were observed in the foliage and thatch layer. Two isolates were obtained from affected annual bluegrass in each sample. Isolations were performed by washing affected leaves in 0.5% NaOCl solution for 2 min, blotting the tissue dry, and plating the tissue on potato dextrose agar (PDA) amended with chloramphenicol (0.05 g/liter), streptomycin (0.05 g/liter), and tetracycline (0.05 g/liter). After incubation for 2 days at 23°C, isolates were transferred and maintained on PDA. All four isolates had multinucleate hyphae and displayed sclerotial characteristics similar to those reported for Waitea circinata var. circinata (2). Sequencing the ITS1F/ITS4-amplified rDNA internal transcribed spacer (ITS) region confirmed the isolates as W. circinata var. circinata, with ≥99% sequence similarity to published W. circinata var. circinata ITS sequences (GenBank Accession No. FJ755849) (1,2,4). To confirm pathogenicity, isolates were inoculated onto 6-week-old annual bluegrass (True Putt/DW184) grown in 10-cm-diameter pots containing calcined clay (Turface; Profile Products LLC., Buffalo Grove, IL). Two 4-mm-diameter agar plugs for each isolate were removed from the margins of 3-day-old colonies grown on PDA and placed near the soil surface to ensure contact with the lower leaf blades. Each isolate was placed in four separate pots to have four replicated tests per isolate, and four noninfested pots were utilized as negative controls. All pots were placed in moist chambers at 28°C with a 12-h light/dark cycle. Within 4 to 6 days, inoculated plants exhibited severe chlorosis and a minor amount of aerial mycelium was observed. Inoculated plants became necrotic after 15 to 20 days, while the noninoculated plants remained healthy. W. circinata var. circinata was reisolated from inoculated plants and its identity was confirmed by morphological and molecular characteristics. This pathogen was previously reported as a causal agent of brown ring patch of creeping bentgrass in Japan and annual bluegrass in the western United States (2,4). To our knowledge, this is the first report of brown ring patch in Minnesota and Wisconsin. Intensive fungicide practices are needed to control brown ring patch; therefore, this disease could have significant economic impact throughout the Upper Midwest (3). References: (1) C. M. Chen et al. Plant Dis. 93:906, 2009 (2) K. de la Cerda et al. Plant Dis. 91:791, 2007. (3) J. Kaminski and F. Wong. Golf Course Manage. 75(9):98, 2007. (4) T. Toda et al. Plant Dis. 89:536, 2005.