Environmental and Edaphic Factors that Influence Spring Dead Spot Epidemics.

Spring dead spot (SDS) (Ophiosphaerella spp.) is a soilborne disease of warm-season turfgrasses grown where winter dormancy occurs. The edaphic factors that influence where SDS epidemics occur are not well defined. A study was conducted during the spring of 2020 and repeated in the spring of 2021 on four 'TifSport' hybrid bermudagrass (Cynodon dactylon × transvaalensis) golf course fairways expressing SDS symptoms in Cape Charles, VA, U.S.A. SDS within each fairway was mapped from aerial imagery collected in the spring of 2019 with a 20 MP CMOS 4k true color sensor mounted on a DJI Phantom 4 Pro drone. Three disease intensity zones were designated from the maps (low, moderate, high) based on the density of SDS patches in an area. Disease incidence and severity, soil samples, surface firmness, thatch depth, and organic matter measurements were taken from 10 plots within each disease intensity zone from each of the four fairways (n = 120). Multivariate pairwise correlation analyses (P < 0.1) and best subset stepwise regression analyses were conducted to determine which edaphic factors most influenced the SDS epidemic within each fairway and each year. Edaphic factors that correlated with an increase in SDS or were selected for the best fitting model varied across holes and years. However, in certain cases, soil pH and thatch depth were predictors for an increase in SDS. No factors were consistently associated with SDS occurrence, but results from this foundational study of SDS epidemics can guide future research to relate edaphic factors to SDS disease development.

Soil surfactants influence fungicide movement in United States Golf Association putting green soil.

The management of root and crown diseases of turfgrasses is challenging. To manage these diseases, golf course superintendents and other turfgrass managers often spray fungicides at a high carrier volume and irrigate after application to move fungicides into the root zone. Furthermore, previous research has demonstrated that soil surfactants can increase fungicide movement and distribution in soil. Two laboratory experiments were conducted using lysimeters, which were coated with sand on their inner walls to prevent preferential flow and contained 90/10% sand/peat moss (v/v), to determine the effects of soil surfactants on movement of selected fungicides in soil. The soil surface in the first experiment was treated three times at 2-wk intervals with one of three soil surfactants: Aquifer (propoxylated polyethylene glycols), Fleet (polyoxyalkylene polymers), and Revolution (modified alkylated polyol). The soil in the second experiment was treated with only Revolution four times at 2- to 3-wk intervals. Immediately after the final surfactant application, soil columns were treated with 14 C-labeled fungicide. 14 C-Myclobutanil was applied in the first experiment, and 14 C-azoxystrobin and 14 C-propiconazole were applied in the second experiment. In the first experiment, 14 percent units more of the recovered 14 C-myclobutanil was detected in the 5- to 7.6-cm sampling depth, and >4 percent units more was detected in the 7.6- to 10.2-cm depth after soil surfactant application compared with the fungicide-alone treatment. Each soil surfactant also yielded >28% more leachate than the nontreated control. In the second experiment, the total recovered 14 C-azoxystrobin and 14 C-propiconazole in the 7.6- to 10.2-cm depth increased by 2.8 and 1.9 percent units, respectively, compared with soil treated with fungicide alone. These data indicate that soil surfactant inclusion may increase fungicide distribution in soil, which may enhance the efficacy of fungicides in suppressing root and crown diseases.

Fitness Attributes of Pythium aphanidermatum with Dual Resistance to Mefenoxam and Fenamidone.

Pythium aphanidermatum is the predominant species causing Pythium root rot on commercially grown poinsettias in North Carolina. Resistance to mefenoxam is common in populations of P. aphanidermatum but resistance to fenamidone and other quinone outside inhibitor fungicides has only just been reported in greenhouse floriculture crops. The in vitro sensitivity to the label rate of mefenoxam (17.6 µl active ingredient [a.i.]/ml) and fenamidone (488 µl a.i./ml) was determined for 96 isolates of P. aphanidermatum. Isolates were assigned to four fungicide phenotypes: mefenoxam-sensitive/fenamidone-sensitive (MefS, FenS), mefenoxam-sensitive/fenamidone-insensitive (MefS, FenR), mefenoxam-insensitive/fenamidone-sensitive (MefR, FenS), and mefenoxam-insensitive/fenamidone-insensitive (MefR, FenR). In all, 58% of isolates were insensitive to one (MefR, FenS = 36% and MefS, FenR = 16%) or both fungicides (MefR, FenR = 6%). A single point mutation in the cytochrome b gene (G143A) was identified in fenamidone-insensitive isolates. Mycelial growth rate at three temperatures (20, 25, and 30°C), in vitro oospore production, and aggressiveness on poinsettia were evaluated to assess relative fitness of sensitive and insensitive isolates. Isolates with dual insensitivity to mefenoxam and fenamidone had reduced radial hyphal growth at 30°C and produced fewer oospores than isolates sensitive to one or both fungicides. Isolates sensitive to both fungicides produced greater numbers of oospores. Aggressiveness on poinsettia varied by isolate but fungicide phenotype was not a good predictor of aggressiveness. These results suggest that populations of P. aphanidermatum with dual resistance to mefenoxam and fenamidone may be less fit than sensitive populations under our imposed experimental conditions but populations of P. aphanidermatum should continue to be monitored in poinsettia production systems for mefenoxam and fenamidone insensitivity.

Development and validation of a weather-based warning system to advise fungicide applications to control dollar spot on turfgrass.

Dollar spot is one of the most common diseases of golf course turfgrass and numerous fungicide applications are often required to provide adequate control. Weather-based disease warning systems have been developed to more accurately time fungicide applications; however, they tend to be ineffective and are not currently in widespread use. The primary objective of this research was to develop a new weather-based disease warning system to more accurately advise fungicide applications to control dollar spot activity across a broad geographic and climactic range. The new dollar spot warning system was developed from data collected at field sites in Madison, WI and Stillwater, OK in 2008 and warning system validation sites were established in Madison, WI, Stillwater, OK, Knoxville, TN, State College, PA, Starkville, MS, and Storrs, CT between 2011 and 2016. A meta-analysis of all site-years was conducted and the most effective warning system for dollar spot development consisted of a five-day moving average of relative humidity and average daily temperature. Using this model the highest effective probability that provided dollar spot control similar to that of a calendar-based program across the numerous sites and years was 20%. Additional analysis found that the 20% spray threshold provided comparable control to the calendar-based program while reducing fungicide usage by up to 30%, though further refinement may be needed as practitioners implement this warning system in a range of environments not tested here. The weather-based dollar spot warning system presented here will likely become an important tool for implementing precision disease management strategies for future turfgrass managers, especially as financial and regulatory pressures increase the need to reduce pesticide usage on golf course turfgrass.

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.

Preventive Control of Pythium Root Dysfunction in Creeping Bentgrass Putting Greens and Sensitivity of Pythium volutum to Fungicides.

Pythium root dysfunction (PRD), caused by Pythium volutum, has been observed on golf course putting greens established with creeping bentgrass in the southeastern United States since 2002. To evaluate preventative strategies for management of this disease, a 3-year field experiment was conducted in Pinehurst, NC on a 'G-2' creeping bentgrass putting green. Fungicide treatments were applied twice in the fall (September and October) and three times in the spring (March, April, and May) in each of the 3 years. Applications of pyraclostrobin provided superior preventative control compared with the other fungicides tested. Azoxystrobin and cyazofamid provided moderate control of PRD in two of three seasons. Experiments were conducted to determine whether the disease suppression provided by pyraclostrobin was due to fungicidal activity or physiological effects on the host. In vitro sensitivity to pyraclostrobin, azoxystrobin, fluoxastrobin, cyazofamid, mefenoxam, propamocarb, and fluopicolide was determined for 11 P. volutum isolates and 1 P. aphanidermatum isolate. Isolates of P. volutum were most sensitive to pyraclostrobin (50% effective concentration [EC50] value = 0.005), cyazofamid (EC50 = 0.004), and fluoxastrobin (EC50= 0.010), followed by azoxystrobin (EC50 = 0.052), and mefenoxam (EC50 = 0.139). P. volutum isolates were not sensitive to fluopicolide or propamocarb. Applications of pyraclostrobin did not increase the foliar growth rate or visual quality of creeping bentgrass in growth-chamber experiments. This work demonstrates that fall and spring applications of pyraclostrobin, azoxystrobin, and cyazofamid suppress the expression of PRD symptoms during summer and that field efficacy is related to the sensitivity of P. volutum to these fungicides.

Influence of Temperature on Pathogenicity of Pythium volutum Toward Creeping Bentgrass.

Symptoms of Pythium root dysfunction (PRD) in creeping bentgrass (Agrostis stolonifera) are most common in the summer during periods of heat and drought stress. However, recent observations in North Carolina indicate that Pythium volutum, a causal agent of PRD, is most active during the fall and spring. Soil temperature thresholds for this pathogen are needed so that preventive fungicide applications can be timed accurately. A mycelial growth assay was performed by incubating 11 P. volutum isolates at 10 temperatures ranging from 10 to 31°C. To determine the optimal temperature range for infection by P. volutum, five isolates of P. volutum were used to inoculate 5-week-old 'Penn A-1' creeping bentgrass plants. Inoculated plants were transferred to growth chambers at constant 12, 16, 20, 24, 28, or 32°C (12-h day/night cycles) for 4 weeks to permit root infection, then the temperature in all chambers was increased to 32/26°C day/night to induce foliar symptoms. P. volutum grew most rapidly in vitro when temperatures were between 18 and 26°C. Typical PRD foliar symptoms developed in the 12, 16, 20, and 24°C treatments 2 weeks after the temperature was elevated to 32/26°C day/night. Disease severity was greatest when plants were incubated at 16°C after inoculation. Reductions in root depth and/or root mass were observed prior to raising the temperature to 32/26°C in the 12, 16, and 20°C temperature treatments. Once exposed to 4 weeks of heat treatment, extensive root dieback occurred in the 12, 16, 20, and 24°C treatments. These results demonstrate that P. volutum is most active at temperatures prevalent during the fall and spring in North Carolina, supporting the hypothesis that the majority of root infection occurs during this time and that fungicides should be applied when soil temperatures are between 12 and 24°C to achieve preventative control of PRD symptoms in the summer.

Pathogenicity of Pythium Species Associated with Pythium Root Dysfunction of Creeping Bentgrass and Their Impact on Root Growth and Survival.

Symptoms resembling Pythium root dysfunction have been observed on golf course putting greens established with creeping bentgrass across the southeastern United States since 2002. Root isolations from 14 golf courses yielded 59 isolates of Pythium volutum and 16 isolates of Pythium torulosum. Pathogenicity of five isolates of P. volutum, two isolates of P. torulosum, and a combination of the two species was determined by inoculating mature 'A-1' creeping bentgrass plants. Inoculated plants were incubated for 4 weeks at 24/16°C (day/night) to permit root infection, then temperatures were increased to 32/26°C to induce foliar symptoms. No isolates impacted root depth, root mass, or foliar disease severity after 4 weeks at 24/16°C. After increasing the temperature to 32/26°C, isolates of P. volutum induced foliar disease severity ranging from 60 to 84%, whereas isolates of P. torulosum induced only 14 to 35% disease. Isolates of P. volutum consistently reduced root mass and root depth after 4 weeks at 32/26°C, but P. torulosum exhibited no effect. These results demonstrate that P. volutum is a pathogen of mature creeping bentgrass plants. Infections that occur during cool weather reduce the growth and survival of creeping bentgrass roots during hot weather and give rise to foliar symptoms.

First Report of Pythium Root Dysfunction of Creeping Bentgrass Caused by Pythium volutum in North Carolina.

In July and August of 2002 and 2003, a disease of unknown etiology was observed in Charlotte, NC on 'A-1' creeping bentgrass (CRB; Agrostis stolonifera L.) putting greens that were constructed in 2000. Symptoms appeared in irregular patches ranging from 15 to 30 cm in diameter. Grass in the affected areas was initially wilted and chlorotic, but later exhibited a yellow-to-orange foliar decline. Similar symptoms were observed in Durham, NC in July and August of 2003 on CRB greens established in 2001 with a 1:1 blend of 'A-1' and 'A-4'. The disease was initially diagnosed as take-all patch, but attempts to isolate Gaeumannomyces graminis var. avenae and other ectotrophic root pathogens were unsuccessful. Symptoms of the disease reappeared during periods of warm, dry weather in the fall of 2003 and spring of 2004. At that time, examination of affected root tissue revealed bulbous root tips, loose cortical structure, absence of root hairs, and abundant Pythium oospores and hyphae. These signs and symptoms are typical of Pythium root dysfunction (PRD) as described by Hodges and Coleman (2) in 1985 and Feng and Dernoeden (3) in 1999. Isolation of Pythium spp. was performed by plating directly on V8 agar (4) or baiting with 'A-4' CRB seedlings. Eleven Pythium isolates were obtained from Charlotte (seven via baiting) and 10 were obtained from Durham (all via baiting). All isolates were transferred to grass leaf-blade cultures (4) to induce development of sporangia, oospores, and antheridia for identification using the keys and descriptions of Dick (1). All isolates produced lobate sporangia, large oospores (27 to 33 ± 2.8 µm), and three to nine diclinous antheridia typical of Pythium volutum. Cone-Tainers (3.8 × 20 cm) containing sand meeting USGA specifications were seeded with 'A-1' CRB and grown for 6 weeks in the greenhouse. Each Cone-Tainer was inoculated by cutting the root system at a 5 cm depth, placing five to seven infested grass blades onto the surface of fresh sand, and then replacing the turf. Cone-Tainers inoculated with one of three P. volutum isolates and an uninoculated control (six reps each) were placed in a growth chamber with 12 h of light/dark periods at 24/16°C for 4 weeks to allow pathogen infection and disease development. After 4 weeks, the chamber temperature was raised to 32/26°C to induce symptom development. Two weeks after raising the temperature, all P. volutum isolates caused significant (P = <0.0001) foliar chlorosis and dieback (70 to 100% disease) and reduced root depth and mass by 25 to 65% compared with the uninoculated control. Roots of inoculated plants were colonized with Pythium hyphae, contained numerous oospores, and consistently yielded P. volutum in isolations. To our knowledge, this is the first reported occurrence of PRD in North Carolina and provides further support for the importance of P. volutum as a pathogen of creeping bentgrass. On the basis of our observations, the majority of pathogen activity and disease development occurs in the fall and spring, with foliar symptoms being induced by heat or other stresses. References: (1) M. W. Dick. Keys to Pythium. University of Reading Press, Reading, UK, 1990. (2) C. F. Hodges and L. W. Coleman. Plant Dis. 69:336, 1985. (3) Y. Feng and P. H. Dernoeden. Plant Dis. 83:516, 1999. (4) F. N. Martin. Pythium. Pages 39-49 in: Methods for Research on Soilborne Phytopathogenic Fungi. L. L. Singleton et al., eds. The American Phytopathological Society, St. Paul, MN, 1992.