James P. Kerns

Molecular Characterisation and Diagnosis of Root-Knot Nematodes (Meloidogyne spp.) from Turfgrasses in North Carolina, USA.

Root-knot nematodes (Meloidogyne spp.) are the most common and destructive plant-parasitic nematode group worldwide and adversely influence both crop quality and yield. In this study, a total of 51 root-knot nematode populations from turfgrasses were tested, of which 44 were from North Carolina, 6 from South Carolina and 1 from Virginia. Molecular characterisation was performed on these samples by DNA sequencing on the ribosomal DNA 18S, ITS and 28S D2/D3. Species-specific primers were developed to identify turfgrass root-knot nematode through simplex or duplex PCR. Four species were identified, including M. marylandi Jepson & Golden in Jepson, 1987, M. graminis (Sledge & Golden, 1964) Whitehead, 1968, M. incognita (Kofoid & White, 1919) Chitwood, 1949 and M. naasi Franklin, 1965 through a combined analysis of DNA sequencing and PCR by species-specific primers. M. marylandi has been reported from North Carolina and South Carolina for the first time. Molecular diagnosis using PCR by species-specific primers provides a rapid and cheap species identification approach for turfgrass root-knot nematodes.

Sclerotinia homoeocarpa Overwinters in Turfgrass and Is Present in Commercial Seed

Dollar spot is the most economically important disease of amenity turfgrasses in the United States, yet little is known about the source of primary inoculum for this disease. With the exception of a few isolates from the United Kingdom, Sclerotinia homoeocarpa, the causal agent of dollar spot, does not produce spores. Consequently, it was assumed that overwintering of this organism in soil, thatch, and plant debris provides primary inoculum for dollar spot epidemics. Overwintering of S. homoeocarpa in roots and shoots of symptomatic and asymptomatic creeping bentgrass turfgrass was quantified over the course of a three-year field experiment. Roots did not consistently harbor S. homoeocarpa, whereas S. homoeocarpa was isolated from 30% of symptomatic shoots and 10% of asymptomatic shoots in the spring of two out of three years. The presence of stroma-like pathogen material on leaf blades was associated with an increase in S. homoeocarpa isolation and colony diameter at 48 hpi. Commercial seed has also been hypothesized to be a potential source of initial inoculum for S. homoeocarpa. Two or more commercial seed lots of six creeping bentgrass cultivars were tested for contamination with S. homoeocarpa using culture-based and molecular detection methods. A viable, pathogenic isolate of S. homoeocarpa was isolated from one commercial seed lot and contamination of this lot was confirmed with nested PCR using S. homoeocarpa specific primers. A sensitive nested PCR assay detected S. homoeocarpa contamination in eight of twelve (75%) commercial seed lots. Seed source, but not cultivar or resistance to dollar spot, influenced contamination by S. homoeocarpa. Overall, this research suggests that seeds are a potential source of initial inoculum for dollar spot epidemics and presents the need for further research in this area.

Plant Growth Regulator Effects on Bacterial Etiolation of Creeping Bentgrass Putting Green Turf Caused by Acidovorax avenae

Bacterial etiolation, caused by Acidovorax avenae, is a widespread problem in creeping bentgrass putting green turf. The symptoms normally appear as abnormally elongated turfgrass stems and leaves. Observations at multiple field sites suggest the involvement of plant growth regulators (i.e., GA-biosynthesis inhibitors) commonly applied to turf, alluding to a phytohormone imbalance caused by the bacterium. A 2-year field study examined the effects of trinexapac-ethyl, flurprimidol, and paclobutrazol on bacterial etiolation severity caused by A. avenae. Trinexapac-ethyl applied at 0.05 kg a.i. ha-1 every 7 days and 0.10 kg ha-1 every 14 days increased etiolation compared with all other treatments in both years. Flurprimidol and paclobutrazol were not different from the control but high-rate applications caused phytotoxicity that lowered turf quality early in 2014. When the etiolated turfgrass was removed with mowing, turfgrass treated with trinexapac-ethyl exhibited the highest turfgrass quality on most rating dates. Results from this work illustrate that using plant growth regulator materials with different modes of action is a solution to managing creeping bentgrass growth while limiting the potential for bacterial etiolation outbreaks.

Molecular Characterization and Phylogenetic Relationships of Plant-Parasitic Nematodes Associated with Turfgrasses in North Carolina and South Carolina, United States

The near-full-length 18S ribosomal DNA (rDNA) gene and internal transcribed spacer 1 region were amplified and sequenced from 52 nematode populations belonging to 28 representative species in 13 families recovered from turfgrasses in North Carolina (38 populations) and South Carolina (14 populations). This study also included 13 nematode populations from eight other plant hosts from North Carolina for comparison. Nematodes were molecularly characterized and the phylogenetic relationships were explored based on 18S rDNA sequences. Phylogenetic analysis using Bayesian inference was performed using five groups of the plant-parasitic nematode populations Tylenchids, Criconematids, Longidorids, Xiphinematids, and Trichodorids. The 65 nematode populations were clustered correspondingly within appropriate positions of 13 families, including Belonolaimidae, Caloosiidae, Criconematidae, Dolichodoridae, Hemicycliophoridae, Hoplolaimidae, Heteroderidae, Longidoridae, Meloidogynidae, Paratylenchidae, Pratylenchidae, Telotylenchidae, and Trichodoridae. This study confirms previous morphological-based identification of the plant-parasitic nematode species found in turfgrasses and provides a framework for future studies of plant-parasitic nematodes associated with turfgrasses based upon DNA sequences and phylogenetic relationships.

Brachypodium: A Potential Model Host for Fungal Pathogens of Turfgrasses

Brachypodium distachyon is a C3 grass that is an attractive model host system for studying pathogenicity of major turfgrass pathogens due to its genetic similarity to many cool-season turfgrasses. Infection assays with two or more isolates of the casual agents of dollar spot, brown patch, and Microdochium patch resulted in compatible interactions with B. distachyon inbred line Bd21-3. The symptoms produced by these pathogens on Bd21-3 closely resembled those observed on the natural turfgrass host (creeping bentgrass), demonstrating that B. distachyon is susceptible to the fungal pathogens that cause dollar spot, brown patch, and Microdochium patch on turfgrasses. The interaction between Sclerotinia homoeocarpa isolates and Brachypodium ecotypes was also investigated. Interestingly, differential responses of these ecotypes to S. homoeocarpa isolates was found, particularly when comparing B. distachyon to B. hybridum ecotypes. Taken together, these findings demonstrate that B. distachyon can be used as a model host system for these turfgrass diseases and leveraged for studies of molecular mechanisms contributing to host resistance.

Development of a semi-selective medium for improved isolation of the turfgrass dollar spot pathogen Sclerotinia homoeocarpa from host tissues

Abstract Dollar spot, caused by Sclerotinia homoeocarpa, is one of the most economically devastating diseases of amenity turfgrasses worldwide. The pathogen is readily isolated from active lesions, but detection from seed, dormant host tissue and other plant debris that may serve as a source of primary inoculum is difficult. A semi-selective medium was developed to enhance isolation of S. homoeocarpa. Various fungicides used on turfgrass, the pH indicator dye bromophenol blue, and two pH levels, were assessed for their effects on, and in the case of bromophenol blue, response to growth of S. homoeocarpa and contaminant fungi frequently isolated from field and seed samples. Amendment of the medium to pH 4 promoted growth of S. homoeocarpa in the absence of fungicides and enhanced pathogen growth relative to contaminant isolates on medium amended with 0.1 μg mL−1 triticonazole or 5 μg mL−1 azoxystrobin. The growth rate of S. homoeocarpa on these three media, as determined by in vitro radial growth assays, was consistent for many isolates representing the majority of S. homoeocarpa vegetative compatibility groups. Isolation efficiency on the pH-amended media was tested against antibiotic-amended potato dextrose agar from field samples and artificially inoculated turfgrass seed. In both cases, the medium amended to pH 4 and containing 5 μg mL−1 azoxystrobin significantly decreased contaminant growth with no adverse effects on recovery of S. homoeocarpa in comparison with antibiotic-amended PDA. The enhanced specificity of this medium will be a useful tool for selective detection of S. homoeocarpa and identification of sources of initial inoculum for dollar spot development.

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.