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.

First global report of Hyaloperonospora brassicae in commercial broccoli and cabbage microgreens.

Microgreens are a nutrient-dense enhancement to modern diets (Choe et al. 2018), whose small production footprint in protected systems facilitates rapid crop turnover and distribution to population centers. Eleven of the 25 most broadly grown microgreens are brassicas (Choe et al. 2018). In November 2023, kale, broccoli (H009B), and cabbage (H009C) microgreen crops in Michigan were observed with downy mildew, at disease severities of 3%, 40%, and 20% foliage on 10 x 16 cm seeded blocks of plants, respectively. These crops shared a germination chamber for at least three days, which was maintained at approximately 22℃ in very humid, dark conditions. Chlorosis and grayish, sunken necrosis characterized symptoms on cotyledon surfaces (Fig. 1). In humid conditions, thick, white-light gray sporulation was present on adaxial cotyledon surfaces, accompanied by sparse sporulation on abaxial surfaces and hypocotyls. Severely diseased plants were stunted and approximately 50% gradually succumbed to downy mildew. On microscopic examination, a Hyaloperonospora spp. was tentatively identified, with long sporangiophores that dichotomously branched 3 to 6 times and hyaline sporangia borne singly on flexuous terminal sterigmata (Fig. 2). Sporangia were round to oval, with average length of 23.1 (range 16.0 to 28.3) µm; width of 20.0 (15.0 to 25.6) µm; and average length:width of 1.2 (1.0 to 1.4); (n = 97 for all). Sporangia dislodged rapidly if disturbed or as humidity decreased. Two pathogenicity tests were initiated on two sequential days. Two cotyledons from originally infected broccoli and cabbage were suspended, abaxial-side down, on coarse mesh over an open 60-mm plate of pregerminated brassica seeds on a water-saturated filter, inside a sealed, clear plastic box. Boxes contained only one type of originally diseased host, with 15 to 20 seeds of transfer varieties in unique dishes. Boxes were incubated in the dark for 2 days at 19°C with a wet paper towel atop the cotyledons. Before removal, cotyledons were lightly brushed across the surfaces of the seedlings they were just suspended above. Seedlings were grown in boxes in the presence of indirect, ambient light for 9.5 hr/day for an additional 5 days before pathogen sporulation was apparent. Filter paper was resaturated as needed. Noninoculated control plants, maintained separate from inoculated plants, were asymptomatic throughout the experiments. Total disease incidence in transfer varieties was 43.5% of 'Graffiti' cauliflower, 18.7% and 15.7% of 'Nixon' and 'Blue Vantage' cabbage; 11.8% of 'Red Russian' kale, and 6.0% of 'Ironman' broccoli, combined from two experiments. All varieties listed had at least one plant successfully infected in both pathogenicity tests. Sporulation on transfer hosts was morphologically identical to originally affected crops. Sporangiophores and sporangia were removed from H009B broccoli and H009C cabbage plants using surface sterilized forceps, placed directly into DNA extraction tubes containing buffer CD1 (Qiagen PowerSoil Pro), then kit instructions were followed. Extracts were utilized as template for ITS and cox1 PCR amplification, using DreamTaq Mastermix and ITS4/6 (45 cycles; White et al. 1990) and Levup/Levlo primers (30 cycles; Robideau et al. 2011). Cycling conditions were as published, with the number of cycles indicated by primer set. Each reaction yielded a single amplicon of approximately 1000 and 700 bp, for ITS and cox1, respectively,. Amplicons were cleaned using ExoSap-IT and submitted for Sanger sequencing, using ITS6 and Levup as sequencing primers (Robideau et al. 2011; White et al. 1990). After quality trimming, amplicons shared >98.5% identity with H. brassicae (NCBI Genbank accession MG757792 or reference genome CANTFL010000892.1). Sequences were submitted to Genbank (PP093830, PP093831, PP776812, PP776813). This is the first report of downy mildew, caused by H. brassicae, in commercial brassica microgreens, crops with vast nutritional value and expanding production.

Larger presence of ectomycorrhizae detected from pygmy pine ecotype in the fire-frequent pine barrens ecosystem.

Pine barrens ecosystem has acidic, sandy, and nutrient-poor soil and is prone to drought and fire. In the New Jersey Pine Barrens, the predominant pitch pine (Pinus rigida) consists of two ecotypes: the regular pitch pines with heights of 4.6-12 m, and the pygmy pines of low stature (1.2-1.8 m) in the New Jersey Pine Plains. Previous ecological studies suggested that the dwarf pines in the Pine Plains that are embedded within the Pine Barrens were an evolutionary adaptation to frequent fire. Pines are obligate ectomycorrhizal (EcM) mutualists, and their root mycobiota may contribute to stress protection and plant health. However, information on the mycobiota associated with plants in the pine barrens ecosystem is lacking. To have a holistic understanding of the evolution and adaptation in this stressed environment, we used both culture-independent metabarcoding and culture-based method to characterize the mycobiota from soil and root of the two ecotypes and to identify core mycobiota. We found that Agaricomycetes, Leotiomycetes, and Mucoromycotina are predominant fungi in the New Jersey Pine Barrens ecosystem, which is rich in root mutualistic fungi. We observed that the pygmy pine roots had significantly higher density of EcM tips than the regular pine roots. This was corroborated by our metabarcoding analysis, which showed that the pygmy pine trees had higher ratio of ectomycorrhiza-forming fungi than the regular-statured pines. We hypothesize that symbiotrophic EcM fungi associated with pygmy pines are capable of mitigating high fire stress in the Pine Plains.

Real-Time PCR Detection of Clarireedia spp., the Causal Agents of Dollar Spot in Turfgrasses.

Dollar spot is one of the most economically important diseases of turfgrasses. Recent taxonomic revisions have placed the dollar spot fungal pathogens in the new genus Clarireedia, with five species described. The main goal of this study was to develop a quantitative real-time PCR (qPCR) molecular detection assay based on the internal transcribed spacer (ITS) of the ribosomal RNA genes to quantify the abundance of Clarireedia spp. from environmental (field) samples. The qPCR assay was able to detect isolates of the four tested Clarireedia spp. but did not cross react with nontarget fungi, including closely related taxa, other turfgrass pathogens, or other fungal species commonly isolated from turfgrass. The assay is capable of detecting as little as 38.0 fg (3.8 × 10-14 g) of Clarireedia genomic DNA in 3 h. The qPCR assay detected Clarireedia spp. in both symptomatic and asymptomatic creeping bentgrass (Agrostis stolonifera) foliar tissue. Clarireedia spp. were rarely detected in the thatch or soil, indicating that these pathogens are not widely distributed in these areas of the environment. The fact that the pathogen was detected in asymptomatic tissue suggests that creeping bentgrass may be able to tolerate a certain quantity of the pathogens in leaves before disease symptoms appear; however, further research is needed to validate this hypothesis.