Brandon J. Horvath

DNA fingerprinting and anastomosis grouping reveal similar genetic diversity in Rhizoctonia species infecting turfgrasses in the transition zone of USA.

Rhizoctonia blight is a common and serious disease of many turfgrass species. The most widespread causal agent, Thanatephorus cucumeris (anamorph: R. solani), consists of several genetically different subpopulations. In addition, Waitea circinata varieties zeae, oryzae and circinata (anamorph: Rhizoctonia spp.) also can cause the disease. Accurate identification of the causal pathogen is important for effective management of the disease. It is challenging to distinguish the specific causal pathogen based on disease symptoms or macroscopic and microscopic morphology. Traditional methods such as anastomosis reactions with tester isolates are time consuming and sometimes difficult to interpret. In the present study universally primed PCR (UP-PCR) fingerprinting was used to assess genetic diversity of Rhizoctonia spp. infecting turfgrasses. Eighty-four Rhizoctonia isolates were sampled from diseased turfgrass leaves from seven distinct geographic areas in Virginia and Maryland. Rhizoctonia isolates were characterized by ribosomal DNA internal transcribed spacer (rDNA-ITS) region and UP-PCR. The isolates formed seven clusters based on ITS sequences analysis and unweighted pair group method with arithmetic mean (UPGMA) clustering of UP-PCR markers, which corresponded well with anastomosis groups (AGs) of the isolates. Isolates of R. solani AG 1-IB (n = 18), AG 2-2IIIB (n = 30) and AG 5 (n = 1) clustered separately. Waitea circinata var. zeae (n = 9) and var. circinata (n = 4) grouped separately. A cluster of six isolates of Waitea (UWC) did not fall into any known Waitea variety. The binucleate Rhizoctonia-like fungi (BNR) (n = 16) clustered into two groups. Rhizoctonia solani AG 2-2IIIB was the most dominant pathogen in this study, followed by AG 1-IB. There was no relationship between the geographic origin of the isolates and clustering of isolates based on the genetic associations. To our knowledge this is the first time UP-PCR was used to characterize Rhizoctonia, Waitea and Ceratobasidium isolates to their infra-species level.

Seasonal Application Timings Affect Dallisgrass (Paspalum dilatatum) Control in Tall Fescue

Abstract Field research was conducted in 2010 and 2011 to investigate the efficacy of herbicides for dallisgrass control when applied at various growing (GDD) or cooling degree day (CDD) –based application timings. Herbicide treatments included fluazifop-p-butyl (fluazifop; 105 g ai ha−1), mesotrione (280 g ai ha−1), tembotrione (92 g ai ha−1), topramezone (37 g ai ha−1), and tank mixtures of fluazifop plus mesotrione, tembotrione, or topramezone. Herbicide treatments were applied at either 75, 175, 375, 775 GDD, or 5 CDD. Treated plots were subjected to three tall fescue interseeding regimes: no seeding, seeding in spring, or seeding in fall (0, 353, and 353 kg pure live seed ha−1, respectively). In 2010, dallisgrass control from fluazifop applied at 75, 375, and 775 GDD was poor (< 50%) by 52 wk after treatment (WAT); in 2011, control from fluazifop application at these timings was higher (62 to 72%). When applied at 175 GDD or 5 CDD in 2010 and 2011, dallisgrass control from fluazifop ranged from 79 to 93% at 52 WAT. The addition of mesotrione, tembotrione, or topramezone to fluazifop did not affect dallisgrass control at any application timing, and control provided by these herbicides alone was low (< 65%). Interseeding tall fescue in the fall improved dallisgrass control from herbicides applied at 75 GDD in 2010 and 175, 375, and 775 GDD at 52 WAT in both years. Results suggest that timing of fluazifop applications at 175 GDD and 5 CDD enhances dallisgrass control. Nomenclature: Fluazifop-p-butyl; mesotrione; tembotrione; topramezone; dallisgrass; Paspalum dilatatum Poir.; tall fescue; Lolium arundinaceum (Schreb.) S.J. Darbyshire. Resumen En 2010 y 2011, se realizó un estudio de campo para investigar la eficacia de herbicidas para el control de Paspalum dilatatum, cuando estos se aplicaron en diferentes momentos basándose en grados día de crecimiento (GDD) o enfriamiento (CDD). Los tratamientos de herbicidas incluyeron fluazifop-p-butyl (fluazifop; 105 g ai ha−1), mesotrione (280 g ai ha−1), tembotrione (92 g ai ha−1), topramezone (37 g ai ha−1), y mezclas en tanque de fluazifop más mesotrione, tembotrione, o topramezone. Los tratamientos de herbicidas fueron aplicados ya fuera a 75, 175, 375, 775 GDD, o 5 CDD. Las parcelas tratadas fueron sometidas a regímenes de entre-siembra con Lolium arundinaceum: sin siembra, siembra en la primavera, o siembra en el otoño (0, 353, y 353 kg de semilla pura viva ha−1, respectivamente). En 2010, el control de P. dilatatum con fluazifop aplicado a 75, 375 y 775 GDD fue pobre (<50%) a 52 semanas después del tratamiento (WAT). En 2011, el control con la aplicación de fluazifop en estos mismos momentos fue mayor (62 a 72%). Cuando se aplicó a 175 GDD o 5 CDD en 2010 y 2011, el control con fluazifop de P. dilatatum varió entre 79 y 93% a 52 WAT. La adición de mesotrione, tembotrione, o topramezone al fluazifop no afectó el control de P. dilatatum en ninguno de los momentos de aplicación, y el control brindado por estos herbicidas aplicados solos fue bajo (<65%). La entre-siembra de L. arundinaceum en el otoño mejoró el control de P. dilatatum para herbicidas aplicados a 75 GDD en 2010 y 175, 375, y 775 GDD a 52 WAT, en ambos años. Los resultados sugieren que realizar aplicaciones de fluazifop a 175 GDD y 5 CDD mejora el control de P dilatatum.

Development of SCAR markers and UP-PCR cross-hybridization method for specific detection of four major subgroups of Rhizoctonia from infected turfgrasses

A rapid identification assay for Waitea circinata (anamorph: Rhizoctonia spp.) varieties zeae and circinata causing patch diseases on turfgrasses was developed based on the universally primed PCR (UP-PCR) products cross-blot hybridization. Tester isolates belonging to the two varieties of W. circinata were amplified with a single UP primer L21, which generated multiple DNA fragments for each variety. Probes were prepared with UP-PCR products of each tester isolate by labeling with digoxigenin. Fieldcollected W. circinata isolates and representative isolates of different R. solani anastomosis groups (AG) and AG subgroups were amplified with L21, immobilized on nylon membrane and cross hybridized with the two probes. Isolates within a W. circinata variety cross-hybridized strongly, while non-homologous isolates did not cross-hybridize or did so weakly. Closely related W. circinata varieties zeae and circinata were clearly distinguished with this assay. Sequence-characterized amplified region (SCAR) markers also were developed from UP-PCR products to identify isolates of Thanatephorus cucumeris (anamorph: R. solani) AG 1-IB and AG 2-2IIIB. These two AGs are commonly isolated from diseased, cool-season turfgrasses. The specific SCAR markers that were developed could differentiate isolates of AG 1-IB or AG 2-2IIIB groups. These SCAR markers did not amplify a product from genomic DNA of nontarget isolates of Rhizoctonia. The specificities and sensitivities of the SCAR primers were tested on total DNA extracted from several field-grown, cool-season turf species having severe brown-patch symptoms. First, the leaf samples from diseased turf species were tested for the anastomosis groups of the causal pathogen, and thereafter the total DNA was amplified with the specific primers. The specific primers were sensitive and unique enough to produce a band from total DNA of diseased turfgrasses infected with either AG 1-IB or AG 2-2IIIB.

Effect of Mowing Height and Fertility on Bermudagrass (Cynodon dactylon) Encroachment and Brown Patch Severity in Tall Fescue

Abstract Tall fescue is a commonly used turfgrass in the temperate and transition zone areas of the United States. During hot, humid summers, tall fescue is under stress and is susceptible to Rhizoctonia solani (brown patch) infection, causing turf thinning, leading to encroachment from weeds, such as bermudagrass. Field trials were established to evaluate the effect of mowing height and fertility programs on disease severity and bermudagrass encroachment in tall fescue. Mowing at 10 cm resulted in less bermudagrass encroachment than did a 6-cm mowing height. Increasing the nitrogen fertilization level from 49 to 171 and 220 kg N ha−1 generally led to more bermudagrass encroachment at the 6-cm, but not the 10-cm, mowing height. Plots receiving 220 kg N ha−1 annually at the 6-cm mowing height had the most brown patch. Turfgrass cover was greatest in plots mowed at 10 cm and receiving 220 kg N ha−1 annually. Nomenclature: Bermudagrass; Cynodon dactylon (L.) Pers.; brown patch; Rhizoctonia solani Kuhn; tall fescue; Lolium arundinaceum (Schreb.) S.J. Darbyshire (syn. Festuca arundinacea Schreb. var. arundinacea Schreb.). Resumen Lolium arundinaceum es usado comúnmente como césped en zonas templadas y de transición en los Estados Unidos. Durante veranos calientes y húmedos, L. arundinaceum sufre estrés y es susceptible a la infección de Rhizoctonia solani (mancha parda), lo que causa el raleo del césped y la consecuente colonización de malezas, tales como Cynodon dactylon. Se establecieron estudios de campo para evaluar el efecto de la altura de chapia y los programas de fertilidad en la severidad de la enfermedad y la colonización de C. dactylon en L. arundinaceum. La chapia a 10 cm resultó en menor colonización de C. dactylon que la chapia a 6 cm de altura. El incrementar el nivel de fertilización nitrogenada de 49 a 171 y 220 kg N ha−1 generalmente llevó a una mayor colonización de C. dactylon en la chapia a 6 cm, pero no en la chapia a 10 cm de altura. Las parcelas que recibieron 220 kg N ha−1 anualmente y chapia a 6 cm de altura tuvieron la mayor severidad de mancha parda. La cobertura del césped fue mayor en las parcelas con chapia a 10 cm y que recibieron 220 kg N ha−1 anualmente.

Confirmation and Control of Annual Bluegrass (Poa annua) with Resistance to Prodiamine and Glyphosate

Poor annual bluegrass control was reported in golf course roughs following treatment with prodiamine (1120 gaiha−1) and glyphosate (840 gaeha−1) during hybrid bermudagrass dormancy. Research was conducted to determine if this annual bluegrass phenotype was resistant to both prodiamine and glyphosate and to determine the efficacy of herbicide mixtures for controlling this phenotype in the field. In PRE or POST dose-response experiments, 9 to 31 times more prodiamine or glyphosate was needed to control (or reduce dry biomass of) this resistant phenotype by 50% compared to an herbicide-susceptible phenotype. Moreover, glyphosate-susceptible plants accumulated 50% more shikimic acid (898 mgkg−1) 6 d after treatment than those resistant to glyphosate (394 mgkg−1). October (fall) applications of herbicide mixtures containing trifloxysulfuron, simazine, S-metolachlor, or mesotrione controlled this resistant annual bluegrass phenotype 84 to 98% in April (spring), with no differences detected among treatments. Our findings document the second instance of annual bluegrass evolving multiple resistance in a managed turfgrass system. However, several herbicide mixtures can be used to effectively manage this resistant phenotype. Nomenclature: glyphosate; mesotrione; prodiamine; simazine; S-metolachlor; trifloxysulfuron; annual bluegrass, Poa annua L.; hybrid bermudagrass, Cynodon dactylon×Cynodon transvaalensis Burtt-Davy. Control limitado de Poa annua fue reportado en “roughs” de campos de golf después de tratamientos con prodiamine (1120 g ai ha−1) y glyphosate (840 gaeha−1) durante el período de dormancia del césped bermuda híbrido. Se realizó una investigación para determinar si este fenotipo de P. annua era resistente a prodiamine y glyphosate y para determinar la eficacia de mezclas de herbicidas para controlar este fenotipo en el campo. En experimentos de respuesta a dosis con herbicidas PRE o POST, se necesitó de 9 a 31 veces más prodiamine o glyphosate para controlar (o reducir la biomasa seca) de este fenotipo resistente en 50% en comparación a un fenotipo susceptible a estos herbicidas. Además, plantas susceptibles a glyphosate acumularon 50% más ácido shikimic (898 mg kg−1) 6 d después del tratamiento que plantas resistentes a glyphosate (394 mg kg−1). Aplicaciones en Octubre (otoño) de mezclas de herbicidas que contenían trifloxysulfuron, simazine, S-metolachlor, o mesotrione controlaron este fenotipo resistente de P. annua 84 a 98% en Abril (primavera), sin detectarse diferencias entre estos tratamientos. Nuestros resultados documentan la segunda instancia de P. annua que evoluciona resistencia múltiple en un sistema manejado de céspedes. Sin embargo, varias mezclas de herbicidas pueden ser usadas para manejar efectivamente este fenotipo resistente.