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.

Occurrence of QoI Fungicide Resistance in Cercospora sojina from Mississippi Soybean.

Frogeye leaf spot, caused by Cercospora sojina Hara, is a foliar disease affecting soybean (Glycine max (L.) Merr.), often managed by applications of quinone outside inhibitor (QoI) fungicides. In 2013 and 2014, 634 C. sojina monoconidial isolates were collected from soybean fields throughout Mississippi. Initially, in vitro bioassays were performed to evaluate the sensitivity of 14 of 634 isolates plus a baseline. Resistant and sensitive isolates were characterized by determining the effective fungicide concentrations at which 50% of conidial germination was inhibited (EC50). The molecular mechanism of resistance was determined for all 634 isolates, using a PCR-RFLP method and comparing nucleotide sequences of the cytochrome b gene. The state of Mississippi was divided into five distinct geographical regions (the Hills, Delta, Pines, Capital, and Coast) based on estimates of total soybean hectares. The greatest proportion (16.7%) of QoI-sensitive isolates was collected in the Hills while the Coast had no QoI-sensitive isolates. QoI-sensitive isolates from the Pines, Capital, and Delta ranged from 1.6 to 7.0%. Results of this study determined that more than 93% of C. sojina isolates collected in Mississippi carried the G143A amino acid substitution, indicating a shift to a QoI-resistant population throughout Mississippi soybean fields.

First Report of Bipolaris oryzae Causing Leaf Spot of Switchgrass in Mississippi.

'Alamo' switchgrass (Panicum virgatum L.) seedlings growing in a soilless mix exhibiting dark brown, irregular-shaped foliar lesions with black borders were submitted to the Mississippi State University Plant Disease Diagnostic Laboratory in the summer of 2009 from a local forest products company. Symptomatic tissues were plated onto water agar (WA) and incubated for 21 days on a laboratory bench top with a 12-h photoperiod at 22°C. An asexual, sporulating, dematiaceous hyphomycete identified as Bipolaris oryzae (Breda de Haan) Shoemaker was observed. Conidiophores were single, mostly straight, multiseptate, brown, and ranging from 138 to 306 × 7.7 to 15.3 µm and averaged 221.6 × 10.7 µm. Conidia were golden brown, multiseptate, ranging from 3 to 10 septa, straight to slightly curved to fusoid, wider midway, and tapering toward the terminal cells. Conidia measured 40.8 to 109.7 × 10.2 to 20.4 µm and averaged 75.8 × 13.8 µm. Morphological characteristics of B. oryzae were similar to those described by Drechsler (1) and Sivanesan (3). The internal transcribed spacer (ITS) region of ribosomal DNA from four pure culture colonies derived from single conidia was amplified by PCR using ITS1 and ITS4 primers. The resultant 572 bp was sequenced for isolates 86 through 89 (GenBank Accession Nos. GU222690-93). The sequences were 99% similar to the sequence of B. oryzae strain ATCC-MYA 3330 (GenBank No. FJ746665) isolated from P. virgatum. Pathogenicity of isolates 86 and 88 was confirmed by inoculating sterile potting mix with a fungal slurry. Sterile Alamo switchgrass seeds were sown into the infested soil in Magenta boxes and incubated for 6 weeks in a growth chamber with a 14-h photoperiod at 30°C. Leaf lesions and leaf blight were observed in seedling stands growing in B. oryzae-infested soil. Lesions were excised and plated onto WA. Sporulation of B. oryzae was observed on symptomatic tissue. In the interim, 300 nonsterilized Alamo switchgrass seeds of the same seed lot as the original symptomatic seedlings and originating from Oklahoma were plated onto WA (10 seed per plate). The seeds were incubated on the bench top as previously described. The experiment was repeated and B. oryzae colonized 1.4% of the total switchgrass seeds evaluated, indicating seed transmission and subsequent seedling infection as previously observed in the original seedlings. Leaf spot, caused by B. oryzae, was first reported as a new disease of switchgrass in North Dakota (2). In the summer of 2009, the authors observed leaf spot in four cultivars of switchgrass, including Alamo, growing in research plots in Webster County, MS. Twenty-two isolates of B. oryzae were recovered from diseased leaves of these switchgrass cultivars. To our knowledge, this is the first report of B. oryzae causing leaf spot of switchgrass in Mississippi, which broadens the natural distribution of this disease. References: (1) C. Drechlser. J. Agric. Res. 24:641, 1923. (2) J. M. Krupinsky et al. Can. J. Plant Pathol. 26:371, 2004. (3) A. Sivanesan. Mycol. Pap. 158:201, 1987.

First Report of Colletotrichum cereale Causing Anthracnose Foliar Blight of Creeping Bentgrass in Mississippi and Alabama.

Colletotrichum cereale Manns, formerly C. graminicola (Ces.) G.W. Wils., is the causal agent of anthracnose foliar blight (AFB) of creeping bentgrass (Agrostis stolonifera L.) and other grass species (1). AFB is most prevalent on creeping bentgrass during summer heat stress (2). Symptoms of AFB progress from older to younger leaves with leaf tips becoming chlorotic and eventually developing complete leaf necrosis. Symptoms in turf stands appear as yellow-to-bronze, irregularly shaped patches often associated with a loss of turf density (2). When C. cereale is actively infecting the foliar tissue, appressoria can be observed microscopically in the leaf sheaths of creeping bentgrass. C. cereale colonizes the foliar tissue, producing abundant acervuli, where conidia and setae develop. Creeping bentgrass samples exhibiting symptoms of AFB were collected from West Point, MS and Birmingham, AL in July 2006. Symptomatic plants with signs of C. cereale were surface disinfested and plated onto one-quarter-strength potato dextrose agar (PDA). Monoconidial C. cereale isolates were grown on full-strength PDA for 21 days at 25°C under fluorescent lights. Single-celled conidia were mostly falcate, ranged from 13.1 to 25.6 µm long × 3.6 to 6.3 µm wide, and averaged 22.2 × 4.7 µm. Hyphal appressoria were irregularly shaped and heavily lobed, ranging from 5.6 to 16.1 µm long × 4 to 10.6 µm wide, and averaged 12.1 × 7.9 µm. In culture, setae were acicular, five to seven septate, thick walled, ranged from 74 to 213.5 µm long, and averaged 151.3 µm. The morphological characteristics of 44 AFB isolates were similar to those of C. cereale reported by Crouch et al. (1). Nucleotide sequences were generated for the internal transcribed spacer rDNA for isolates OO7-T42, OW15-H32, and 04-111 (GenBank Accession Nos. EU859957, EU859958, and EU859959). Maximum likelihood-based phylogenetic analyses of these sequences with authentic isolates of Colletotrichum species from grass hosts (2) indicated that all three isolates were C. cereale. 'Penn A-1' creeping bentgrass seedlings (10.16-cm pots) were inoculated with the C. cereale isolates OO7-T42, OW15-H32, or 04-111 by spraying a conidial suspension (1.5 × 105 conidia/ml) on plants until water droplets were evident within the canopy. An uninoculated control sprayed with distilled water only was used for comparison. Three replicates per C. cereale isolate were included simultaneously when performing Koch's postulates. The inoculated creeping bentgrass seedlings were placed in covered plastic boxes to maintain humidity and incubated under 12 h of fluorescent light with day/night temperatures at 35/28°C. After 4 days, the covers were removed and creeping bentgrass was maintained an additional 14 days until symptoms and signs were observed on the foliage. C. cereale was reisolated from inoculated creeping bentgrass exhibiting symptoms of AFB for all three isolates used. No acervuli, setae, or conidial masses were observed on uninoculated control plants. To our knowledge, this is the first report of C. cereale causing AFB on creeping bentgrass in Mississippi and Alabama. References: (1) J. A. Crouch et al. Phytopathology 96:46, 2006. (2) R. W. Smiley et al. Compendium of Turfgrass Diseases. The American Phytopathological Society, St. Paul, MN, 2005.

First Report of Ophiosphaerella herpotricha Causing Spring Dead Spot of Bermudagrass in Mississippi.

Spring dead spot (SDS) is the most destructive disease of bermudagrass (Cynodon dactylon (L.) Pers.). Symptoms of SDS appear in the spring when bermudagrass transitions out of winter dormancy. These symptoms include depressed, straw-colored patches that range from several centimeters to a meter in diameter. Infected roots and rhizomes are black, brittle, and necrotic. The disease is caused by three species of fungi: Ophiosphaerella herpotricha (Fr: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. However, O. korrae is the most prevalent causal organism of SDS in the southeastern United States and was the only species reported in Mississippi (1). In April of 2006, root samples were collected from a bermudagrass putting green in Booneville, MS with a high level of SDS incidence and severity. Symptomatic roots were collected and surface disinfested in 0.6% NaOCl and plated on one-quarter-strength potato dextrose agar (PDA) amended with streptomycin sulfate and chloramphenicol. Hyphae growing from the roots were transferred to full-strength PDA after 5 to 7 days. Mycelium from five pure-culture colonies plus an O. herpotricha control was harvested after 4 weeks of growth and the genomic DNA was extracted. The genomic DNA of the Booneville isolates and the O. herpotricha control were amplified by PCR using species-specific primers OHITS1 and OHITS2 for O. herpotricha (2). Amplification of a 454-bp fragment of DNA confirmed one of the five unknown isolates as O. herpotricha. The other four isolates were not identified. 'Sahara' bermudagrass (4 weeks old in 3.8 × 20 cm Cone-tainers containing a sand and soil mixture) was inoculated with the Booneville-O. herpotricha isolate and the O. herpotricha control. One gram of oat seed infested with O. herpotricha isolates was inserted 2 cm below the crowns in the root zone of bermudagrass plugs. The inoculated bermudagrass plants were incubated for 4 weeks in the greenhouse. A control consisting of noninfested sterile oats was included. Following incubation, black, necrotic roots were observed on the plants inoculated with both O. herpotricha isolates. No symptoms were observed on roots of noninfested plants. Symptomatic roots were disinfested and plated on one-quarter-strength PDA. Koch's postulates were completed after O. herpotricha was reisolated from roots of plants inoculated with both O. herpotricha isolates and confirmed by PCR as mentioned above. The identification of O. herpotricha as a causal organism of SDS in Mississippi clarifies the involvement of multiple causal agents in this state and broadens the geographic distribution of this root-rot species. References: (1) F. B. Iriarte et al. Plant Dis. 88:1341, 2004. (2) N. A. Tisserat et al. Phytopathology 84:478, 1994.

Characterization of Rhizoctonia-Like Fungi Isolated from Agronomic Crops and Turfgrasses in Mississippi.

Twenty-three isolates of Rhizoctonia spp. from agronomic crops and turfgrasses were characterized by cytological and pathological methods in order to establish the identity, pathogenicity, and virulence of Rhizoctonia spp. and anastomosis groups that occur on these hosts in Mississippi. Twelve isolates were identified as R. solani, including the five anastomosis groups (AGs) AG-1-IB, AG-2-2, AG-4, AG-5, and AG-13. Rhizoctonia zeae, R. oryzae, and eight binucleate Rhizoctonia sp., including R. cerealis, also were identified. R. solani AG-4 isolates were consistently the most virulent isolates on all hosts in pathogenicity evaluations. Pathogenicity of AG-2-2 and AG-5 isolates, binucleate Rhizoctonia spp., and R. oryzae varied between hosts. Two AG-2-2 isolates from bermudagrass or wheat were determined to be clonal isolates, with numerous self-anastomosis reactions. R. solani (AG-1-IB) was pathogenic on all graminaceous hosts. R. cerealis produced sharp eyespot symptoms on wheat and corn and minimal symptoms on cotton and soybean. This is a first report of R. cerealis as a pathogen of corn. R. zeae isolates were pathogenic on all hosts, including cotton and soybean. These results indicate that a diverse group of Rhizoctonia spp. occurs as pathogens on a wide variety of agronomic crops and turfgrasses in Mississippi.

Rhizoctonia solani AG-13 Isolated from Corn in Mississippi.

Rhizoctonia solani K¨hn anastomosis group (AG) 13 was isolated from asymptomatic root tissue of a corn (Zea mays L.) seedling grown at the Black Belt Branch Experiment Station, Brooksville, MS. Rhizoctonia solani AG-13 was recently reported from cotton grown in Georgia (2). Rhizoctonia solani isolate MS-168 was successfully anastomosed with tester isolate AG-13 (courtesy of D. E. Carling, University of Alaska). The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and cell death of adjacent cells was observed. These results were confirmed by D. E. Carling and are consistent with C2 anastomosis hyphal reactions (1). Rhizoctonia solani isolate MS-168 was cultured on potato dextrose agar (PDA) and incubated at 27 ± °C with a 12-h photoperiod for 28 days. Mycelium was buff-brown to beige with diurnal zonation throughout the colony. Aggregates of bulbils developed in the center of the colony that were dark brown, dome shaped, and accompanied by brown exudate. Bulbils were submerged in the medium and scattered across the surface of the colony. The optimal growing temperature of MS-168 was 27°C. Two pathogenicity evaluations were conducted on 170 corn seedlings planted into soilless potting medium. Four-day-old corn seedlings were inoculated with 7-day-old PDA hyphal plugs (2 mm in diameter) of R. solani isolate MS-168 by placing the mycelium side of the hyphal plug in contact with the mesocotyl tissue beneath the soil surface. The hyphal plugs were covered with soil. The nontreated corn seedlings were inoculated with PDA plugs minus the fungus. Corn seedlings were maintained under environmentally controlled conditions at 27 ±2 °C with a 12-h photoperiod and watered to prevent wilting. Disease symptoms on mesocotyl tissue were rated from 1 to 4 in which 1 = no symptoms, 2 = a few pinpoint lesions and diffuse discolored areas, 3 = distinct necrotic lesions, and 4 = girdling lesions (3). Fourteen days postinoculation, treated seedlings had a significantly higher disease rating (1.5) than the nontreated control (1.0). Thirty of eighty-seven corn seedlings inoculated with MS-168 expressed symptoms of discoloration and pinpoint and necrotic lesions on the mesocotyl tissue at the site of inoculation. On the basis of the results of the pathogenicity evaluation, MS-168 can be characterized as weakly virulent on seedling corn when grown under controlled environmental conditions. The identification of R. solani isolate MS-168 (AG-13) from corn in Mississippi broadens the natural distribution of occurrence and host range of this anastomosis group. References: (1) D. E. Carling. Grouping in Rhizoctonia solani by hyphal anastomosis reactions. Pages 37-47 in: Rhizoctonia Species: Taxonomy, Molecular, Biological, Ecological, Pathology, and Disease Control. B. Sneh et al., eds. Kluwer Academic Publishers, The Netherlands, 1996. (2) D. E. Carling et al. Phytopathology 92:893, 2002. (3) C. S. Rothrock. Report of the cottonseed treatment committee for 1993. Page 216-217 in: Proc. Beltwide Cotton Conf. Natl. Cotton Counc. Am., Memphis, TN, 1994.

Fungal occurrence, disease incidence and severity, and yield of maize symptomatic for seedling disease in Mississippi.

A study was conducted in Mississippi from 1995 to 1997 comparing soil rhizosphere fungi isolated from Pioneer 3167 hybrid maize (Zea mays L.) planted on Brooksville silty clay and Memphis silt loam soils. Maize seedlings were collected over four sampling dates from conventional and no-tillage plots. Eleven fungal genera consisting of nineteen species were isolated from these plants; Trichoderma spp. were most frequently isolated, followed by Fusarium spp. The highest disease incidence occurred in tilled plots of the latest planting date on Brooksville silty clay when samples were collected 17 days after planting. Root disease was most severe in 1996 from seedlings planted on the last planting date in tilled plots sampled 17 days after planting. Yields were significantly (P < or = 0.05) higher on Brooksville silty clay soil than on Memphis silt loam in both 1995 and 1996. Yields were highest from no-tillage plots and from maize planted on the earliest date. There was a significant correlation between incidence of root infection and disease severity. There was no correlation between the incidence of root infection and yield or between disease severity and yield at either location.

Gaeumannomyces graminis var. graminis Isolated from Emerald Zoysiagrass in Texas.

Wilkinson and Kane (3) previously reported diseased zoysiagrass infected by Gaeumannomyces graminis (Sacc.) Arx & Olivier var. graminis in the spring in Illinois. Emerald zoysiagrass (Zoysia japonicum Steud. × Zoysia matrella (L.) Merr var. tenufolia (Willd. ex Thiele) established by sod in a home lawn for one year in Austin, TX, developed irregular, chlorotic, and, subsequently, necrotic patches 30 cm in diameter and larger in late summer of 1999. Patches were restricted to areas of the lawn receiving full sun. The lawn was fertilized, mowed at 2.5 cm, and watered daily during active growth. A thatch layer in excess of 1.9 cm was present. Tillers within diseased patches were removed easily from stolons. Crowns were rotted and colonized by dark brown septate hyphae (4.5 µm wide) and olivaceous brown lobed hyphopodia (25 × 21 µm). Diseased tillers were desiccated and newly developed leaves were chlorotic. Stolons were also chlorotic and developed water-soaked lesions adjacent to crowns. Diseased roots appeared light brown and brittle with strands of dark brown septate runner hyphae along the surface of the root axis and olivaceous brown growth cessation structures within the cortical tissue. Overall, symptoms were more severe on crowns and nodes than roots. A Gaeumannomyces fungus was isolated from root, sheath, and bud tissues. Taxonomy of the fungus was consistent with the description of G. graminis var. graminis by Walker (1,2). Diseased plants were washed free of soil and other debris and maintained in a moist chamber for 14 days. Perithecia were formed on leaf sheaths. Morphology of perithecia, asci, and ascospores was consistent with Walker's description of perithecia, asci, and ascospores of G. graminis var. graminis (2). Leaf buds and root tissue, colonized by G. graminis var. graminis, were plated directly onto potato-dextrose agar containing streptomycin sulfate and rifampicin (100 ppm, respectively). Colonies of sparse white, slightly aerial mycelium turning olive brown with age and producing lobed hyphopodia, developed from plated plant material. Hyphae at the margin of colonies curled back, characteristic of G. graminis var. graminis. Symptoms reported here are similar to those described by Wilkinson and Kane (3); however, the season and prevailing environmental conditions were different. References: (1) J. Walker. Trans. Br. Mycol. Soc. 58:427, 1972. (2) J. Walker. Mycotaxon 11:1, 1980. (3) H. T. Wilkinson and R. T. Kane. Plant Dis 77:100, 1993.

Take-All Root Rot of St. Augustinegrass: First Report in Mississippi.

Take-all root rot has been reported as a destructive disease of St. Augustinegrass home lawns in Florida and Alabama (1). In June 1998 and 1999, St. Augustinegrass home lawns in central Mississippi developed chlorotic, thinning patches ranging from 0.5 to 4.5 m in diameter. By August of each summer, plants within affected patches were necrotic and dead. Roots of affected St. Augustinegrass were necrotic and shorter than those of unaffected plants; nodes on stolons were necrotic, and lesions developed on internodes. Ectotrophic runner hyphae and dark brown, lobed hyphopodia were visible on roots and aboveground plant parts, respectively. Symptomatic tissues collected from St. Augustinegrass home lawns were plated onto potato dextrose agar (PDA); the incitant of take-all root rot, Gaeumannomyces graminis(Sacc.) Arx & Olivier var. graminis, was isolated. Verification of G. graminis var. graminis was based on colony morphology and taxonomic identification consistent with the description by Walker (2). G. graminis var. graminis isolated from symptomatic St. Augustinegrass was grown on sterile tall fescue seed and incorporated into sterile sand/peat moss mix. Asymptomatic St. Augustinegrass sprigs were washed, and roots were removed prior to planting in infested and noninfested soil. Plants were cultured in the greenhouse for 60 days. St. Augustinegrass planted into noninfested soil was asymptomatic while plants collected from G. graminis var. graminis-infested soil were symptomatic for take-all root rot. Crowns and roots of affected plants were necrotic; leaves were chlorotic and necrotic. Both runner hyphae and lobed hyphopodia were visible. G. graminis var. graminis was reisolated from symptomatic tissues and confirmed as the incitant of take-all root rot. This is the first report of take-all root rot of St. Augustinegrass in Mississippi. References: (1) M. Elliott. Plant Dis. 77:206, 1993. (2) J. Walker. Trans. Br. Mycol. Soc. 58:427, 1972.