Geographic Distribution of Ophiosphaerella Species in the Mid-Atlantic United States

Spring dead spot (SDS) of bermudagrass (Cynodon dactylon) is primarily caused by Ophiosphaerella herpotricha and Ophiosphaerella korrae in North America. These two species respond differently to numerous management practices, grow optimally at different soil pH ranges, and differ in aggressiveness. Understanding the Ophiosphaerella species distribution in regions where SDS occurs will allow turfgrass managers to tailor their management practices toward the predominant species present. A survey was conducted in the Mid-Atlantic United States in which one to 14 samples of bermudagrass expressing SDS symptoms were taken from 51 athletic fields, golf courses, or sod farms across Delaware, Maryland, North Carolina, and Virginia. DNA was isolated from necrotic root and stolon tissue, amplified using species-specific primers, and detected in a real-time PCR assay. At least one isolate of O. herpotricha was recovered from 76% of the locations and O. korrae was recovered from 73% of the locations. Ophiosphaerella herpotricha was amplified from 55% of the samples while O. korrae was amplified from 37% of the samples. There were distinct regions in the Mid-Atlantic in which either O. herpotricha or O. korrae was predominant. Ophiosphaerella herpotricha was predominant in western Virginia, central North Carolina as well as Delaware and eastern Maryland. However, O. korrae was predominant in central Maryland and Virginia as well as eastern Virginia and North Carolina. Ophiosphaerella herpotricha was isolated from certain cultivars more frequently than O. korrae and vice versa. These survey results elucidate the geographic distribution of O. herpotricha and O. korrae throughout the Mid-Atlantic United States.

First report of Ophiosphaerella narmari causing spring dead spot of hybrid bermudagrass in China

Hybrid bermudagrass (Cynodon dactylon×C. transvaalensis) is widely used as turf in transition zone of China. Spring dead spot (SDS) is one of the most damaging diseases of hybrid bermudagrass. Symptoms of SDS appear when hybrid bermudagrass starts to break dormancy with warm temperature in early spring. The symptoms show sunken, circular or irregularly shaped, straw-colored patches, with 20 to 100 cm in diameter. The patches maintain dormant as the surrounding uninfected turfgrass resumes growth and turns green. SDS pathogens are soilborne fungi that colonize roots, stolons and rhizomes, infected roots or rhizomes become black and eventually collapse. Three species of fungi are reported to cause SDS: Ophiosphaerella herpotricha (Fr) J. Walker; O. korrae (J. Walker & A.M. Smith) Shoemaker & C.E. Babcock; or O. narmari (J. Walker & A.M. Smith) Wetzel, Hubert & Tisserat (Walker and Smith 1972; Walker 1980; Shoemaker and Babcock 1989; Wetzel et al. 1999). However, distribution of the three species may vary by geographical region (Cottrill et al. 2016). In October 2020, symptoms of SDS were observed on hybrid bermudagrass fairways of Taihu golf course in Wuxi, Jiangsu province. Root samples of SDS were collected, symptomatic roots with 3-4 cm length were cut, washed 2-3 times, surface sterilized in 0.6% NaOCl for 5 min, rinsed and blotted dry for 2 min and placed on potato dextrose agar (PDA) amended with 50 mg L-1 each of ampicillin, streptomycin sulfate and tetracycline. Plates were incubated in the dark at 25℃ for 5-7 days, Hyphae growing from the roots were transferred to new PDA plates. A total of 7 fungal isolates with morphology similar to SDS pathogens were obtained (Tredway et al. 2009). The genomic DNA was extracted from 2 of them (7-41, 8-6) and amplified with universal primers ITS5 and ITS4 (White et al. 1990). PCR products were sequenced (deposited as MW536995 and MW536994 in GenBank, not available yet) and showed 99.79% similarity to O. narmari (KP690979). Pathogenicity tests were performed on ‘Tifdwarf’ hybrid bermudagrass (9-week-old in 5 × 20 cm Cone-Tainers containing a sand and nutrition substrate mixture). Eight oat seeds infested with O. narmari were inserted 5 cm below the soil surface in the root zone of hybrid bermudagrass. The inoculated turfgrass grew for five weeks in the growth chamber with a 12-h day/night cycle of 25/20°C and 90% relative humidity. A control treatment was inoculated with 8 noninfested sterile oat seeds, and each treatment was replicated 3 times. The root tissues of hybrid burmudagrass inoculated with O. narmari became black and necrotic, no symptoms were observed on the roots of noninfested plants. O. narmari was consistently reisolated from symptomatic roots, and confirmed by PCR as mentioned above. To the best of our knowledge, this is the first report of O. narmari caused spring dead spot in the transition zone of China. The identification of SDS caused by O. narmari will have important implications for the management of this root-rot species on hybrid bermudagrass.

Virulence of a soil inhabiting fungus, Ophiosphaerella korrae, to rice

<p>A soil inhabiting fungus, <em>Ophiosphaerella korrae</em> (J. Walker & A.M. Sm. bis) Shoemaker & C.E. Babc. has been confirmed to be pathogenic to barley, durum wheat and bread wheat of the major crops (Hong et al., 2018; Tomioka et al., 2019ab). Foliage and spikes of the affected plants early blight with root rot and ripening disorder. In this study, we revealed virulence of the fungus to rice, which is also one of the major crops. When a rice cultivar (cv. Norin No. 22) was grown in pots in artificial climate chambers after being sowed with culture discs (6 mm in diameter) of the fungus (strains MAFF150117 and MAFF150118 from bread wheat and durum wheat, respectively) on synthetic nutrient agar (SNA) (1 disc per seed), growth delay and early foliage blight (including ripening disorder) with rotting of roots and stem bases occurred. Defect rates were 22% and 84% for the plants inoculated with strains MAFF150117 and MAFF150118, respectively. Control plants simultaneously treated with aseptic SNA discs had no symptom. The fungal strains were consistently isolated from all the inoculated plants, but not from healthy controls, demonstrating that the fungal strains were virulent to rice. Additionally, a decrease tendency of grain yield without symptom on foliage and roots was detected on a rice cultivar (cv. Koshihikari that is cv. Norin No. 1 × cv. Norin No. 22) inoculated with strain MAFF150117 in another pot experiment. <em>Ophiosphaerella korrae</em> is also known as a pathogen causing spring dead spot or necrotic ring spot of Bermudagrass (Wetzel et al., 1999ab; Camara et al., 2000; Iriarte et al., 2004; Gullino et al., 2007; Perry et al., 2010; Sasaki et al., 2010), Kentucky bluegrass (Wetzel et al., 1999a; Camara et al., 2000, 2001; Hayakawa et al., 2004; Wong et al., 2015), Louisiana grass (Wetzel et al., 1999a; Camara et al., 2000) and Zoysiagrass (Hayakawa et al., 2004; Tredway and Butler, 2007). We will investigate varietal difference against <em>O. korrae</em> as well as the fungal emergent ecology in the future.</p><p>[References] Camara et al. (2000) Mycologia 92:317-325 Camara et al. (2001) Mycol Res 105:41-56 Gullino et al. (2007) Pl Dis 91:1200 Hayakawa et al. (2004) J Jpn Soc Turf Sci 33 (Supplement 1):24-25 Hong et al. (2018) Pl Dis 103(1):158 Iriarte et al. (2004) Pl Dis 88:1341-1346 Perry et al. (2010) Mycopathologia 169:395-402 Sasaki et al. (2010) Jpn J Phytopathol 76(3):158 Tomioka et al. (2019a) Abstracts of papers presented at the 44th annual meeting of the pesticide science society of Japan, p 82 Tomioka et al. (2019b) Abstracts of papers presented at the 63th annual meeting of the mycological society of Japan, p 64 Tredway and Butler (2007) Pl Dis 91:1684 Wetzel et al. (1999a) Mycol Res 103:981-989 Wetzel et al. (1999b) Pl Dis 83:1160-1166 Wong et al. (2015) Pl Pathol 44:545-555</p><p> </p>

Multiplex End-Point PCR for the Detection of Three Species of Ophiosphaerella Causing Spring Dead Spot of Bermudagrass

A multiplex end-point polymerase chain reaction (PCR) assay was developed for identifying the three-fungal species in the genus Ophiosphaerella that cause spring dead spot (SDS), a devastating disease of bermudagrass. These fungi are difficult to identify by morphology because they seldom produce pseudothecia. To achieve species-specific diagnosis, three pairs of primers were designed to identify fungal isolates and detect the pathogen in infected roots. The internal transcribed spacer region, the translation elongation factor 1-α, and the RNA polymerase II second-largest subunit were selected as targets and served as templates for the design of each primer pair. To achieve uniform melting temperatures, three to five random nucleotide extensions (flaps) were added to the 5′ terminus of some of the designed specific primers. Temperature cycling conditions and PCR components were standardized to optimize specificity and sensitivity of the multiplex reaction. Primers were tested in multiplex on DNA extracted from axenic fungal cultures and from field-collected infected and uninfected roots. A distinct amplicon was produced for each Ophiosphaerella sp. tested. The DNA from Ophiosphaerella close relatives and other common bermudagrass pathogens did not amplify during the multiplex assay. Metagenomic DNA from infected bermudagrass produced species-specific amplicons while DNA extracted from noninfected roots did not. This multiplex end-point PCR approach is a sensitive and specific molecular technique that allows for correct identification of SDS-associated Ophiosphaerella spp. from field-collected roots.

Assessment of Nitrogen Source, Sulfur, and Fall Fungicide Applications on the Management of Spring Dead Spot of Bermudagrass

Spring dead spot, caused by species of Ophiosphaerella, is the most serious disease of bermudagrass (Cynodon spp.) in regions where cold temperatures induce winter dormancy. Previous research indicates that soil pH reduction may reduce spring dead spot severity. Of the 165 isolates collected from 16 sites in Missouri and surrounding regions, Ophiosphaerella herpotricha was the most prominent spring dead spot pathogen found, with 154 confirmed isolates. Ten isolates were identified as O. korrae, being detected at a low incidence at 6 of 16 sites. In in vitro assays, most mycelial growth of both species occurred from pH 5 to 6, with more growth on calcium-nitrate-amended media than ammonium sulfate. In a naturally infested field study, nitrogen source alone did not affect spring dead spot severity. Less spring dead spot severity (P < 0.05) was observed in plots receiving tebuconazole but no treatment provided more than 38% control after 1 year. Three sulfur applications (each at 98 kg ha−1) provided as much control as a single fall tebuconazole application (0.28 kg a.i. ha−1) in the second year; however, significant phytotoxicity was observed in sulfur-treated plots thereafter. The suppression obtained from one fall tebuconazole treatment was as effective as two.