Horizontal and vertical distribution of Clarireedia spp., in asymptomatic and symptomatic creeping bentgrass cultivars.

Dollar spot is an important disease of both cool- and warm-season turfgrasses caused by six fungal species in the genus Clarireedia, yet the ecology and epidemiology of these pathogens remains poorly understood. The goal of this study was to determine the distribution of Clarireedia in asymptomatic and symptomatic creeping bentgrass (Agrostis stolonifera) in the field using a previously developed qPCR assay. To determine the horizontal distribution of the pathogen, the abundance of Clarireedia spp. was measured in leaf and crown tissue from 90, 1-cm diameter cores spaced 10-cm apart in May 2019 and 2020 (asymptomatic tissue) and August 2019 and July 2020 (symptomatic tissue). Thirty-seven to 69% of cores sampled from asymptomatic turfgrass and 77 to 95% of cores taken from symptomatic turfgrass yielded positive detections for Clarireedia. Spatial analysis indicated that Clarireedia was randomly distributed in the field in both asymptomatic and symptomatic turfgrass. To assess the vertical distribution of the pathogen, the abundance of Clarireedia was measured in the foliar, crown, and thatch layers of 39, 1-cm dia. x 2.5-cm deep cores of creeping bentgrass maintained at fairway height (9.5 mm) during 2019 and 2020. Clarireedia was most abundant in foliar tissue, followed by crown tissue, and thatch (lowest abundance) throughout the two-year study. Both studies provide evidence that Clarireedia is widely distributed in turfgrass swards prior to symptom development and that it can persist within turfgrass as an endophyte. These findings will improve our understanding of Clarireedia epidemiology and may lead to more sustainable dollar spot management.

First Report of Phialocephala bamuru Causing Root Rot on Hard Fescue (Festuca brevipila) in the United States.

Turfgrasses are susceptible to a wide variety of ectotrophic root-infecting (ERI) fungi that cause root rot (Tredway et al., 2023). Among the root rot diseases, fairway patch, caused by Phialocephala bamuru P.T.W. Wong & C. Dong sp. nov., was recently identified and characterized in Australia infecting bermudagrass (Cynodon dactylon) and kikuyu (Pennisetum clandestinum) grass (Wong et al., 2015). Symptoms begin as small, 5-10 cm diameter patches of yellowed turf that may coalesce into larger areas of diseased grass. A characteristic sign of fairway patch is roots colonized by dark brown to black, ectotrophic mycelium. In June 2020, many tan colored, irregular-shaped patches ranging from 10-30 cm in diameter developed on a hard fescue (Festuca brevipila) cultivar 'Beacon' turfgrass field in North Brunswick, New Jersey, USA. The centers of these patches later died and became sunken or filled in partially by recovering hard fescue. The patches grew into tan irregular-shaped rings with diameters up to 3 m by Aug 2023. Symptoms were indicative of a root disease. Five 'Beacon' hard fescue soil cores at the interface of the symptomatic and non-symptomatic area were sampled in Aug 2023. Root and crown samples were observed under a dissecting microscope and dark ectotrophic hyphae were observed on both. Roots with visible ectotrophic mycelium were removed, rinsed in sterile water three times, cut into 5 mm pieces, and plated onto 10% potato dextrose agar amended with streptomycin and gentamicin at 100 mg/L (PDA+). The plates were incubated at 25°C in the dark for 5 days. The most abundant colonies being characteristic long, septate hyphae that were hyaline at the edge and dark brown to black in the center and resembled the fungus described in Wong et al., 2015. These colonies were subcultured onto PDA+ medium and selected for molecular identification. Other less abundant colonies could be identified using morphology after subcultured and had no record being pathogenic to turfgrass. To confirm the isolate's identity, its internal transcribed spacer (ITS) region was amplified in PCR using the ITS1F/ITS4 primers (Bellemain et al., 2010). The amplicon was then sequenced with both ITS1 and ITS4 primers by Sanger sequencing. Sequences were assembled (GenBank #PP000819). The consensus sequence was then BLASTn analyzed with default settings, and the result showed 99.64% sequence identity with P. bamuru (GenBank #MG195534.1). Koch's postulate was conducted in an environmentally controlled growth chamber. Six healthy 'Beacon' hard fescue plugs were sampled from the field. Three of the six plugs (treatment) were each inoculated with P. bamuru by placing 20 g of P. bamuru colonized millets beneath and around the plug before filling the pots with sand. The other three plugs (control) received the same treatment except the P. bamuru colonized millets were autoclaved. The pots were incubated in the growth chamber with a 16 h light period and 25/20°C day/night temperatures. Symptoms resembling those observed in the field appeared on the treatment pots after 21 days of incubation while the control pots remained healthy. The roots from the treatment pots were examined under the dissecting microscope to confirm the colonization of P. bamuru on the roots, and P. bamuru was reisolated and confirmed using the aforementioned morphological traits and molecular assays (GenBank #PP000820). This is the first report of a turfgrass root rot disease caused by P. bamuru in the United States. Further epidemiological, disease ecological, and pathogen biological studies are required to clarify the importance of this disease in the United States and establish proper disease containment or control measures.

Rapid Detection of the Recently Identified Turfgrass Pathogen Magnaporthiopsis meyeri-festucae Using Recombinase Polymerase Amplification.

Magnaporthiopsis meyeri-festucae is a recently identified root-infecting pathogen of fine fescue (Festuca spp.) turfgrasses. Although it is phylogenetically similar to other root-infecting turfgrass pathogens such as M. poae, management of M. meyeri-festucae is distinct and highlights the need for fast and accurate identification. The objective of this study was to develop a rapid detection method for M. meyeri-festucae using recombinase polymerase amplification (RPA) to assist turfgrass managers in identifying the disease in the field and facilitate further epidemiological research on the pathogen. Three isolates of M. meyeri-festucae and eight isolates from four related Magnaporthiopsis species were used to test the specificity of the RPA assay targeting M. meyeri-festucae. Rapid visualization of the RPA assay results using a mixture of purified amplicon and SYBR-Safe fluorescence emitting asymmetrical cyanine dye showed that the assay was effective at detecting M. meyeri-festucae on turfgrass roots with no observed incidence of false positives or false negatives. The assay also differentiated between M. meyeri-festucae and other Magnaporthiopsis species, although overall sensitivity was lower compared with a PCR-based method. The RPA assay successfully detected M. meyeri-festucae following inoculation onto and grinding of turfgrass roots, indicating possible use as a rapid field diagnostic tool for turfgrass managers. The fast and accurate RPA M. meyeri-festucae detection method presented here will be used for additional field and laboratory applications that will help improve the management of this emerging pathogen.

Anthracnose Disease on Annual Bluegrass as Affected by Foot Traffic and Sand Topdressing.

Sand topdressing is applied to maintain or enhance playability of the turf surface of putting greens. Anthracnose is a devastating disease of annual bluegrass (ABG; Poa annua) putting green turf, caused by Colletotrichum cereale. The disease is more severe on weakened turf and reputed to be exacerbated by management practices that wound turf. A 2-year field study was initiated in 2007 to evaluate the effects of foot traffic (0 versus 327 footsteps m-2, equivalent to 200 rounds day-1) and sand topdressing (0 and 0.3 liter m-2 every week) on anthracnose severity of ABG mowed at 3.2 mm. Surprisingly, foot traffic reduced anthracnose severity as much as 28%, regardless of sand topdressing, during both years. Although sand topdressing initially increased disease severity (up to 7%) in 2007, continued applications decreased severity by 9% later in August 2007 and again in 2008. The treatment combination of foot traffic 5 days week-1 and weekly sand topdressing resulted in the best turf quality by the end of both seasons. Results indicate that the practice of sand topdressing can be continued even under conditions of intense foot traffic and anthracnose disease development on ABG putting greens.

Magnaporthiopsis meyeri-festucae, sp. nov., associated with a summer patch-like disease of fine fescue turfgrasses.

Summer patch is a common and destructive root disease of turfgrasses. In this study, a new Magnaporthiopsis species, M. meyeri-festucae, was identified from the roots of fine fescue (Festuca spp.) turfgrasses with summer patch-like symptoms. It is described and illustrated on the basis of phenotypic characteristics and partial sequences of rDNA 18S, internal transcribed spacer (ITS), and 28S regions, and of MCM7, RPB1, and TEF1 genes. A key for all seven described species in the genus Magnaporthiopsis is provided. Distinctions between the new species and related species are discussed. Fulfillment of Koch's postulates confirmed Magnaporthiopsis meyeri-festucae as a pathogen causing summer patch-like symptoms of fine fescue turfgrasses. This work is the basis for future studies on biogeography, host range, and impact of summer patch pathogens on a broader scale.