Search

Mango anthracnose, caused by Colletotrichum spp., is the most significant disease of mango ( Mangifera indica L.) in almost all production areas around the world. In Mexico, mango anthracnose has only been attributed to C. asianum and C. gloeosporioides . The aims of this study were to identify the Colletotrichum species associated with mango anthracnose symptoms in Mexico by phylogenetic inference using the ApMat marker, to determine the distribution of these species, and to test their pathogenicity and virulence on mango fruits. Surveys were carried out from 2010 to 2012 in 59 commercial orchards in the major mango growing states of Mexico, and a total of 118 isolates were obtained from leaves, twigs, and fruits with typical anthracnose symptoms. All isolates were tentatively identified in the C. gloeosporioides species complex based on morphological and cultural characteristics. The Bayesian inference phylogenetic tree generated with Apn2 /MAT intergenic spacer sequences of 59 isolates (one per orchard) revealed that C. alienum , C. asianum , C. fructicola , C. siamense , and C. tropicale were associated with symptoms of mango anthracnose. In this study, C. alienum , C. fructicola , C. siamense , and C. tropicale are reported for the first time in association with mango tissues in Mexico. This study represents the first report of C. alienum causing mango anthracnose worldwide. The distribution of Colletotrichum species varied among the mango growing states from Mexico. Chiapas was the only state in which all five species were found. Pathogenicity tests on mango fruit cultivar Manila showed that all Colletotrichum species from this study could induce anthracnose lesions. However, differences in virulence were evident among species. C. siamense and C. asianum were the most virulent, whereas C. alienum and C. fructicola were considered the least virulent species.

Anthracnose is one of the most important diseases of lima bean in Brazil. Previously, the disease was attributed exclusively to Colletotrichum truncatum . Therefore, this work aimed to characterize the diversity, prevalence, and virulence of Colletotrichum spp. associated with anthracnose in lima bean in Brazil. Here, we report the species C. truncatum, C. brevisporum , C. lobatum , C. plurivorum , and C. musicola in association with anthracnose of lima bean. All species were pathogenic to lima bean. In addition, several strains were found that represent novel lineages, presented here as Colletotrichum lineages 1 to 5. C. truncatum is the prevailing species and more virulent than all other species studied.

Anthracnose, caused by Colletotrichum musae, is the most important postharvest disease of banana and is widely distributed among the banana production regions in Brazil. Although thiophanate-methyl is a fungicide frequently used in Brazilian banana orchards to control Sigatoka leaf spot, Collettotrichum populations are also exposed, resulting in the evolution of fungicide resistance and the inability to manage banana anthracnose. We investigated 139 Brazilian isolates of C. musae for thiophanate-methyl sensitivity in vitro. The 50% mycelial growth inhibition (EC 50 ) values varied between 0.003 and 48.73 μg/ml. One-hundred and thirty isolates were classified as sensitive, with EC 50 values ranging from 0.003 to 4.84 μg/ml, while the remaining nine isolates were considered moderately resistant, with EC 50 values ranging between 10.43 and 48.73 μg/ml. Resistant or highly resistant isolates (EC 50 > 100 μg/ml) were not found. A substitution of TAC for TTC at codon 200 in a coding region of the β-tubulin gene was associated with the moderately resistant phenotype. Applications of thiophanate-methyl formulation to detached banana fruit at the label rate (500 μg/ml) showed low efficacy in controlling the moderately resistant isolates on banana fruits. However, there is no indication of a reduction in fitness associated with fungicide resistance as sensitive and moderately resistant isolates do not differ with respect to mycelial growth rate (P = 0.098), spore production (P = 0.066), spore germination (P = 0.366), osmotic sensitivity (P = 0.051), and virulence (P = 0.057). Our results revealed absence of adaptability cost for the moderately resistant isolates, suggesting that they can be dominant in population if the fungicide continue to be applied.

In January 2011, leaves of several daylily (Hemerocallis flava L.) plants in nurseries in Vitória da Conquista, northeastern Brazil, showed typical anthracnose symptoms. Reddish brown lesions with a yellow halo were first observed at the tip leaves. As the disease progressed, the lesions rapidly expanded down the leaves, resulting in severe blight. Small pieces up to 5 mm in diameter were removed from the lesion margins, surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterile distilled water, and plated onto potato dextrose agar (PDA) amended with 0.5 g liter -1 streptomycin sulfate. Macroscopic colony characters and microscopic morphology characteristics of two isolates were developed after growth on PDA for 7 days at 25°C under a 12-h light/dark cycle. Colonies presented effuse mycelium, initially white and becoming pale gray, with numerous black structures like sclerotia, setae, and acervuli absent in culture media. Conidia were hyaline, aseptate, curved or slightly curved, round or somewhat acute apex, base truncate, 13.4 to 22.7 (18.2 ± 2.16) μm length, and 3.2 to 5.8 (4.24 ± 0.62) μm width, length/width ratio 4.37, and were typical of Colletotrichum spp. DNA sequencing of partial sequence of actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes and the internal transcribed spacer (ITS1-5.8S-ITS2 rRNA gene cluster) were conducted to accurately identify the species. Sequences of two daylily isolates were highly similar to those of C. spaethianum (Allesch.) Damm, P.F. Cannon & Crous. A phylogenetic analysis using Bayesian inference and including published ACT, CHS-1, GPDH, and ITS data for C. spaethianum and other Colletotrichum species associated with daylily anthracnose (1,3) showed that the isolated fungi belong to the C. spaethianum clade. Sequences of the isolates obtained in this study were deposited in GenBank (ACT Accession Nos. KC598114 and KC598115; CHS-1 Accession Nos. KC598116 and KC598117; GPDH Accession Nos. KC598118 and KC598119; ITS Accession Nos. KC598120 and KC598121). Cultures are deposited in the Culture Collection of Phytopathogenic Fungi of the Universidade Federal Rural de Pernambuco, Recife, Brazil (CMM1224 and CMM1225). Pathogenicity tests were conducted with the two C. spaethianum strains on daylily leaves. Mycelial plugs taken from the margin of actively growing colonies (PDA) of each isolate were applied in shallow wounds near the tip leaves. Four detached leaves were inoculated for each isolate, and PDA discs without fungal growth were used as controls. The leaves were maintained in humid chamber for 2 days at 25°C under a 12-h photoperiod. Anthracnose symptoms that closely resembled those observed in the affected nurseries were developed up to 5 days after inoculation. No symptoms developed on the control plants. C. spaethianum was successfully re-isolated from symptomatic plants to fulfill Koch's postulates. C. spaethianum was described from H. fulva and H. citrina in China, Hosta sielbodiana in Germany, and Lilium sp. in South Korea (3), and from Peucedanum praeruptorum in China (2). To our knowledge, this is the first report of C. spaethianum in Brazil and the first report on H. flava. References: (1) U. Damm et al. Fungal Divers. 39:45, 2009. (2) M. Guo et al. Plant Dis. 97:1380, 2013. (3) Y. Yang et al. Trop. Plant Pathol. 37:165, 2012.

Papaya fruits (Carica papaya L.) (cv. Golden) showing post-harvest anthracnose symptoms were observed during surveys of papaya disease in northeastern Brazil from 2008 to 2012. Fruits affected by anthracnose showed sunken, prominent, dark brown to black lesions. Small pieces (4 to 5 mm) of necrotic tissue were surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterile distilled water, and plated onto potato dextrose agar (PDA) amended with 0.5 g liter -1 streptomycin sulfate. Macroscopic colony characters and microscopic morphology characteristics of four isolates were observed after growth on PDA (2) for 7 days at 25°C under a 12-hr light/dark cycle. Colonies varied between colorless and pale brown in reverse, with orange conidial mass. Conidia were hyaline, aseptate, cylindrical with round ends, slightly flattened, smooth-walled, guttulate, and 13.5 (10.5 to 17.1) μm × 3.8 (2.1 to 4.8) μm (l/w ratio = 3.5, n = 50), typical of Colletotrichum spp. DNA sequencing of partial sequences of actin (ACT) gene and the internal transcribed spacer (ITS1-5.8S-ITS2 rRNA) were conducted to accurately identify the species. Sequences of the papaya isolates were 99% similar to those of Colletotrichum brevisporum (GenBank Accession Nos. JN050216, JN050217, JN050238, and JN050239). A phylogenetic analysis using Bayesian inference and including published ACT and ITS data for C. brevisporum and other Colletotrichum species was carried out (1). Based on morphological and molecular data, the papaya isolates were identified as C. brevisporum. Conidia of the papaya isolates were narrower than those described for C. brevisporum (2.9 to 4.8 μm and 5 to 6 μm, respectively) (1), which may be due to differences in incubation temperature or a typical variation in conidial size in Colletotrichum species (3). Sequences of the isolates obtained in this study are deposited in GenBank (ACT Accession Nos. KC702903, KC702904, KC702905, and KC702906; ITS Accession Nos. HM163181, HM015851, HM015854, and HM015859). Cultures are deposited in the Culture Collection of Phytopathogenic Fungi of the Universidade Federal Rural de Pernambuco, Recife, Brazil (CMM 1672, CMM 1702, CMM 1822, and CMM 2005). Pathogenicity testing was conducted with all four strains of C. brevisporum on papaya fruits (cv. Golden). Fruits were wounded at the medium region by pushing the tip of four sterile pins through the surface of the skin to a depth of 3 mm. Mycelial plugs taken from the margin of actively growing colonies (PDA) of each isolate were placed in shallow wounds. PDA discs without fungal growth were used as control. Inoculated fruits were maintained in a humid chamber for 2 days at 25°C in the dark. After 6 days, anthracnose symptoms developed that were typical of diseased fruit in the field. C. brevisporum was successfully reisolated from symptomatic fruits to fulfill Koch's postulates. C. brevisporum was described from Neoregalia sp. and Pandanus pygmaeus in Thailand (1). To our knowledge, this is the first report of C. brevisporum in Brazil and the first report of this species causing papaya fruit anthracnose. References: (1) P. Noireung et al. Cryptogamie Mycol., 33:347, 2012. (2) B. C. Sutton. The Genus Glomerella and its anamorph Colletotrichum. CAB International, Wallingford, UK, 1992. (3) B. S. Weir et al. Stud. Mycol. 73:115, 2012.

From April to June 2010, mango fruits (Mangifera indica L.) (cv. Tommy Atkins) showing post-harvest anthracnose symptoms were collected during a survey conducted in São Francisco Valley, northeastern Brazil. Fruits affected by anthracnose showed sunken, prominent, dark brown to black decay spots. Small pieces (4 to 5 mm) of necrotic tissues were surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterile distilled water, and plated onto potato dextrose agar (PDA) amended with 0.5 g liter -1 streptomycin sulfate. Plates were incubated at 25°C in the dark for 5 to 7 days and colonies that were morphologically similar to species of Colletotrichum were transferred to PDA (1). Identification was made using morphological characteristics and phylogenetic analysis. Two isolates (CMM 4101 and CMM 4102) presented colonies that had white aerial mycelia and orange conidial mass, varying between colorless and pale orange in reverse. Conidia were hyaline, cylindrical, and aseptate 14.52 (10.40 to 20.20) μm long and 4.90 (3.80 to 6.50) μm wide, length/width ratio = 3.0. Mycelial growth rate was 5.20 mm per day at 25°C. Morphological and cultural characterizations were consistent with the description of Colletotrichum karstii (3). PCR amplification by universal primers (ITS1/ITS4) and DNA sequencing of the internal transcribed spacer (ITS1-5.8S-ITS2 rRNA gene cluster) were conducted to confirm the identifications. Analysis of representative sequences (GenBank Accession Nos. HM585409 and HM585406) suggested that the isolated pathogen was C. karstii. Using published ITS data for C. karstii (3), a phylogenetic analysis was made via Bayesian inference, which shows that the isolated fungi belong to the C. karstii clade. Sequences of the isolates obtained in this study were deposited in GenBank (KC295235 and KC295236), and cultures were deposited in the Culture Collection of Phytopathogenic Fungi of the Universidade Federal Rural de Pernambuco (CMM, Recife, Brazil). Pathogenicity tests were conducted with the C. karstii strains on mango fruits cv. Tommy Atkins. Mycelial plugs taken from the margin of actively growing colonies (PDA) of each isolate were applied in shallow wounds (0.4 cm in diameter) at the medium region of the each fruit. PDA discs without fungal growing were used as controls. Inoculated fruits were placed in plastic containers lined with paper towels wetted in distilled water. The containers were partially sealed with plastic bags to maintain high humidity and incubated at 25°C in the dark. The plastic bags and paper towels were removed after 24 h, and fruits were kept at the same temperature. The experiment was arranged in a completely randomized design with four replicates per treatment (isolate) and four fruits per replicate. Typical anthracnose symptoms were observed after 10 days in mango fruits. C. karstii was successfully reisolated from symptomatic mango fruits to fulfill Koch's postulates. C. karstii was previously described from Orchidaceae in southwest China and the United States (2,3). To our knowledge, this is the first report of C. karstii causing mango anthracnose in Brazil and worldwide. References: (1) U. Damm et al. Stud. Mycol. 73:1, 2012. (2) I. Jadrane et al. Plant Dis. 96:1227, 2012. (3) Yang et al. Cryptogamie Mycol. 32:229, 2011.

In October 2010, 2-year-old papaya (cv. Hawaii) trees with high incidence of stem rot were observed during a survey conducted in Rio Grande do Norte state, northeastern Brazil. Stems showing reddish brown-to-dark brown symptoms were collected and small pieces (4 to 5 mm) of necrotic tissues were surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterile distilled water, and plated onto potato dextrose agar (PDA) amended with 0.5 g liter -1 streptomycin sulfate. Plates were incubated at 25°C with a 12-h photopheriod for 4 days. Pure cultures with white, fluffy aerial mycelia were obtained by subculturing hyphal tips onto PDA. Identification was made using morphological characteristics and DNA based molecular techniques. Colonies grown on PDA and Spezieller Nährstoffarmer agar (SNA) for 10 days at 25°C with a 12-h photoperiod were used for morphological identification (3). The fungus produced cream sporodochia and two types of spores: microconidia were thin-walled, hyaline, ovoid, one-celled, and 6.8 to 14.6 × 2.3 to 4.2 μm; macroconidia were thick walled, hyaline, slightly curved, 3- to 5-celled, and 25.8 to 53.1 × 3.9 to 5.7 μm. Fifty spores of each type were measured. Rounded, thick-walled chlamydospores were produced, with two to four arranged together. On the basis of morphological characteristics (1), three fungal isolates (CMM-3825, CMM-3826, and CMM-3827) were identified as Fusarium solani (Mart.) Sacc. and were deposited in the Culture Collection of Phytopathogenic Fungi of the Universidade Federal Rural de Pernambuco (Recife, Brazil). Single-spore isolates were obtained and genomic DNA of the isolates was extracted and a portion of the translation elongation factor 1-alpha (EF1-α) gene of the isolates was amplified and sequenced (2). When compared with sequences available in the GenBank and Fusarium-ID databases, DNA sequences of the three isolates shared 99 to 100% sequence identity with F. solani species complex (GenBank Accession Nos. JF740784.1, DQ247523.1, and DQ247017.1). Representative sequences of the isolates were deposited in GenBank (Accession Nos. JQ808499, JQ808500, and JQ808501). Pathogenicity tests were conducted with four isolates on 3-month-old papaya (cv. Hawaii) seedlings. Mycelial plugs taken from the margin of actively growing colonies (PDA) of each isolate were applied in shallow wounds (0.4 cm in diameter) on the stem (center) of each plant. Inoculation wounds were wrapped with Parafilm. Control seedlings received sterile PDA plugs. Inoculated and control seedlings (10 each) were kept in a greenhouse at 25 to 30°C. After 2 weeks, all inoculated seedlings showed reddish brown necrotic lesions in the stems. No symptoms were observed in the control plants. The pathogen was successfully reisolated from symptomatic plants to fulfill Koch's postulates. To our knowledge, this is the first report of F. solani species complex causing papaya stem rot in Brazil. Papaya is an important fruit crop in the northeastern Brazil and the occurrence of this disease needs to be taken into account in papaya production. References: (1) C. Booth. Fusarium Laboratory Guide to the Identification of the Major Species. CMI, Kew, England, 1977. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.

From September to December 2010, mango (Mangifera indica L.) stems showing dieback symptoms were collected during a survey conducted in São Francisco Valley, northeastern Brazil. Small pieces (4 to 5 mm) of necrotic tissues were surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterile distilled water, and plated onto potato dextrose agar (PDA) amended with 0.5 g liter -1 streptomycin sulfate. Plates were incubated at 25°C in the dark for 14 to 21 days and colonies that were morphologically similar to species of Botryosphaeriaceae were transferred to PDA. Colonies developed a compact mycelium that was initially white, but becoming gray dark after 4 to 6 days of incubation at 25°C in darkness. Identification was made using morphological characteristics and DNA based molecular techniques. Pycnidia were obtained on 2% water agar with sterilized pine needles as substratum after 3 weeks of incubation at 25°C under near-UV light. Pycnidia were large, multilocular, eustromatic, covered with hyphae; locule totally embedded without ostioles, locule walls consisting of a dark brown textura angularis, becoming thinner and hyaline toward the conidiogenous region. Conidia were hyaline, thin to slightly thickened walled, aseptate, with granular contents, bacilliform, straight to slightly curved, apex and base both bluntly rounded or just blunt, 15.6 to 25.0 (20.8) μm long, and 2.7 to 7.9 (5.2) μm wide, length/width = 4.00. According to these morphological characteristics, three isolates (CMM1364, CMM1365, and CMM1450) were identified as Pseudofusicoccum stromaticum (1,3,4). PCR amplification by universal primers (ITS4/ITS5) and DNA sequencing of the internal transcribed spacer (ITS1-5.8S-ITS2 rRNA gene cluster) were conducted to confirm the identifications through BLAST searches in GenBank. The isolates were 100% homologous with P. stromaticum (3) (GenBank Accession Nos. AY693974 and DQ436935). Representative sequences of the isolates were deposited in GenBank (Accession Nos. JF896432, JF966392, and JF966393). Pathogenicity tests were conducted with the P. stromaticum strains on 5-month-old mango seedlings (cv. Tommy Atkins). Mycelial plugs taken from the margin of actively growing colonies (PDA) of each isolate were applied in shallow wounds (0.4 cm in diameter) on the stem (center) of each plant. Inoculation wounds were wrapped with Parafilm. Control seedlings received sterile PDA plugs. Inoculated and control seedlings (five each) were kept in a greenhouse at 25 to 30°C. After 5 weeks, all inoculated seedlings showed leaf wilting, drying out of the branches, and necrotic lesions in the stems. No symptoms were observed in the control plants. P. stromaticum was successfully reisolated from symptomatic plants to fulfill Koch's postulates. P. stromaticum was described from Acacia, Eucalyptus, and Pinus trees in Venezuela (3,4), and there are no reports of this fungus in other hosts (2). To our knowledge, this is the first report of P. stromaticum causing mango dieback in Brazil and worldwide. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 18 May 2011. (3) S. Mohali et al. Mycol. Res. 110:405, 2006. (4) S. R. Mohali et al. Fungal Divers. 25:103, 2007.

Species of the genus Colletotrichum are commonly reported as pathogens of fruits in tropical regions. Papaya fruits (Carica papaya L.), cv. Golden, with typical lesions of anthracnose, chocolate spot, and/or stem-end rot were collected from 18 papaya-producing areas of northeast Brazil in 2007. One hundred and fifty-five isolates of Colletotrichum spp. were obtained from the fruit lesions and cultured on potato dextrose agar. Pathogenicity tests were conducted by placing a 20-μl drop of 10 5 conidia ml -1 suspension on a wounded area of two healthy fruits of cv Golden at the climacteric stage. Inoculated fruits were placed in a moist chamber at 26°C (±2) for 48 h. After this period, the plastic covers of the trays used to form the moist chamber were removed and the trays were kept at 26°C (±2) for 98 h when symptoms were assessed. The causal agents of fruit rot were recovered from inoculated fruits showing symptoms of anthracnose and chocolate spot. Conidia from fresh lesions were collected and measured. Conidia dimensions were 13.49 × 3.80 μm, length/width ratio = 3.55 μm. Conidia were predominantly cylindrical to bluntly rounded ends and slightly flattened. All isolates were morphologically similar to Colletotrichum gloeosporioides Penz (1). Molecular analyses of the isolates were carried out with taxon-specific primers for C. acutatum J.H Simmonds and C. gloeosporioides (3). Only one amplicon was detected for eight isolates with the C. gloeosporioides primer. All isolates were genotyped using inter-simple sequence repeat (ISSR) primers. Three groups of isolates were found, one containing the eight C. gloeosporioides isolates, a second group comprised of 141 isolates, and a third contained six isolates. The second and third groups were more similar to each other than to the first C. gloeosporioides group. Thirty two representative isolates of the three ISSR groups were sequenced for the internal transcribed spacer (ITS) and glutamine synthetase (GS) (GenBank Nos. HM163181 and HM015847) regions. With molecular phylogenetic analyses, two well-supported clades were formed, one with the C. gloeosporioides isolates and the other with sequences highly similar (99% similarity) to the two ITS sequences available in GenBank (DQ003310 and GU358453) and the GS region of G. magna Jenkins & Winstead (DQ792873). The latter was reported in the United States and Taiwan (2,4). Isolates of C. magna and C. gloeosporioides are morphologically similar and identification needs to be based on molecular analyses. To our knowledge, this is the first report of C. magna causing rot of papaya fruit in Brazil. References: (1) P. F. Cannon et al. Mycotaxon 104:189, 2008. (2) M. Z. Du et al. Mycologia 97:641, 2005. (3) P. Talhinhas et al. Appl. Environ. Microbiol. 71:2987, 2005. (4) J. G. Tsay et al. Plant Dis. 94:787, 2010.

Colletotrichum boninense was isolated from pepper (Capsicum annuum) fruits (cv. Amanda) with preharvest anthracnose symptoms collected in the Brazilian states of Rio Grande do Sul and São Paulo in July of 2005. In the field, the disease affected mature fruits and leaves with an incidence near 25%. Typical symptoms in fruits were circular, sunken lesions with orange spore masses in a dark center. Three single conidia isolates were obtained from infected fruits. When grown on potato dextrose agar at 25°C with a 12-h photoperiod, these isolates produced white colonies with a cream-to-orange color in the opposite side, but no sclerotia. Conidia were cylindrical, had obtuse ends and a hilum-like low protuberance at the base, and measured 13.5 to 15.5 × 4.6 to 5.1 μm. Conidial length/width ratio was 2.8 to 3.0. These morphological characteristics are consistent with the description of C. boninense (1). To confirm pathogen identity, the internal transcribed spacer rRNA region was sequenced (GenBank Accession Nos. FJ010199, FJ010200, and FJ010201) and compared with the same region of C. boninense (GenBank Accession No. DQ286160.1). Similarity between these sequences was 98 to 99%. The pathogenicity of the three isolates was determined on pepper fruits cv. Amanda. Attached as well as detached fruits from potted plants were inoculated. Inoculation was performed by depositing 40-μl droplets of a suspension (10 5 conidia per ml) on the surfaces of nonwounded (detached n = 5; attached n = 5) and wounded (detached n = 5; attached n = 5) fruits with a sterilized hypodermic needle. Incubation took place in a moist chamber for 12 days at 25°C with a 12-h photoperiod. Inoculation of control fruits was similar in procedure and number to that of test fruits, except sterile distilled water was used instead of the conidial suspension. Symptoms, observed in wounded and nonwounded test fruits 3 to 5 days after inoculation, were characterized by necrotic, sunken zones containing acervuli, black setae, and orange spore masses. Control fruits presented no symptoms. Pathogens reisolated from infected fruits showed the same morphological and molecular characteristics of the isolates previously inoculated. To our knowledge, this is the first report of C. boninense infecting pepper in Brazil. Reference: (1) J. Moriwaki et al. Mycoscience 44:47, 2003.

Creeping bentgrass (Agrostis palustris; syn. Agrostis stolonifera) is widely used on golf course putting greens. In September and October 1998, samples of diseased creeping bentgrass were received from golf courses in Maryland, Virginia, and Ohio. Disease symptoms developed in August or September 1998, and appeared initially as 1.0- to 2.0-cm-diameter, reddish brown spots that enlarged to about 8.0 cm in diameter. Leaves of plants in the center of diseased patches were tan and those on the periphery were reddish brown. Dark, ectotrophic hyphae were not observed on roots. Numerous pseudothecia were embedded in necrotic leaf and stolon tissues. A fungus was isolated from leaves, stems, and roots, and single-spore isolates were obtained from pseudothecia. Colonies of all isolates were identical in appearance and were initially rose-quartz to pinkish brown, developing a gray color as they aged. Inoculum was prepared by placing mycelium from a single-spore isolate on an autoclaved medium consisting of 50% tall fescue (Festuca arundinacea) seed, and 50% wheat (Triticum aestivum) bran (vol/vol) and grown at 28°C for 8 days. Putter and Crenshaw creeping bentgrass seedlings were grown for 14 days in 12 cm 2 pots containing an autoclaved topdressing mix with a mechanical analysis of 95% sand, 1% silt, and 4% clay. The inoculum (200 mg) was mixed into the upper 5 mm of the sandy soil. Pots were placed in plastic bags and incubated during the daytime on a windowsill bench (20 to 24°C), and were maintained at 25°C at night in a darkened growth chamber. After 7 days, 2.0-cm-diameter patches of blighted leaves were observed on both cultivars in nearly all pots, and pseudothecia were found on the inoculum or on blighted foliage in some pots after 20 days. Blighted leaves were covered with a pale pinkish white mycelium and newly infected leaves at the periphery of the dead spot were a pale reddish brown. Most plants were dead 20 days after inoculation. The fungus was reisolated from blighted leaves of both cultivars and all isolates produced colonies identical in appearance and growth rate to those produced by the single-spore isolate. Pseudothecia produced in vivo were sectioned with a freezing microtome and examined microscopically. Bitunicate asci were observed and contained light-brown, 6- to 15-septate, filiform ascospores that were usually spirally twisted in the ascus and measured 70 to 150 × 2.0 to 2.5 μm. Characteristics of the pseudothecia and the ascospores fit those of the genus Ophiosphaerella Speg. (1). Based on morphometric studies of 12 collections from three different states, this fungus can be distinguished from O. graminicola by the lack of periphyses and fewer septa in ascospores (i.e., 12 to 20 septa in O. graminicola). It was distinguished from O. herpotricha by characteristics of the pseudothecia neck, ascospores, and colony color. Because of these differences, we suggest that this fungus represents a new species attacking creeping bentgrass, which will be described after further morphometric and molecular analyses. Reference: (1) J. Walker. Mycotaxon 11:1, 1980.