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DNA Fingerprinting and Anastomosis Grouping Reveal Similar Genetic Diversity in Rhizoctonia Species Infecting Turfgrasses in the Transition Zone of USA B

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DNA Fingerprinting and Anastomosis Grouping Reveal Similar Genetic Diversity in Rhizoctonia Species Infecting Turfgrasses in the Transition Zone of USA B University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Agronomy & Horticulture -- Faculty Publications Agronomy and Horticulture Department 2013 DNA fingerprinting and anastomosis grouping reveal similar genetic diversity in Rhizoctonia species infecting turfgrasses in the transition zone of USA B. S. Amaradasa University of Nebraska-Lincoln B. J. Horvath University of Tennessee, Knoxville D. K. Lakshman Floral and Nursery Plants Research Unit, Beltsville Agricultural Research Center-West, Beltsville, Maryland, [email protected] S. E. Warnke Floral and Nursery Plants Research Unit, Beltsville Agricultural Research Center-West, Beltsville, Maryland, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/agronomyfacpub Part of the Agricultural Science Commons, Agriculture Commons, Agronomy and Crop Sciences Commons, Botany Commons, Horticulture Commons, Other Plant Sciences Commons, and the Plant Biology Commons Amaradasa, B. S.; Horvath, B. J.; Lakshman, D. K.; and Warnke, S. E., "DNA fingerprinting and anastomosis grouping reveal similar genetic diversity in Rhizoctonia species infecting turfgrasses in the transition zone of USA" (2013). Agronomy & Horticulture -- Faculty Publications. 851. https://digitalcommons.unl.edu/agronomyfacpub/851 This Article is brought to you for free and open access by the Agronomy and Horticulture Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Agronomy & Horticulture -- Faculty Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Mycologia, 105(5), 2013, pp. 1190–1201. DOI: 10.3852/12-368 # 2013 by The Mycological Society of America, Lawrence, KS 66044-8897 DNA fingerprinting and anastomosis grouping reveal similar genetic diversity in Rhizoctonia species infecting turfgrasses in the transition zone of USA B.S. Amaradasa nia solani AG 2-2IIIB was the most dominant Department of Agronomy and Horticulture, University pathogen in this study, followed by AG 1-IB. There of Nebraska, Lincoln, Nebraska 68583 was no relationship between the geographic origin of B.J. Horvath the isolates and clustering of isolates based on the Department of Plant Sciences, University of Tennessee, genetic associations. To our knowledge this is the first Knoxville, Tennessee 37996 time UP-PCR was used to characterize Rhizoctonia, 1 Waitea and Ceratobasidium isolates to their infra- D.K. Lakshman species level. S.E. Warnke Key words: binucleate Rhizoctonia,rDNA-ITS, Floral and Nursery Plants Research Unit, Beltsville Rhizoctonia solani, turfgrass, UP-PCR, Waitea circinata Agricultural Research Center-West, Beltsville, Maryland 20705 INTRODUCTION Abstract: Rhizoctonia blight is a common and serious Multiple Rhizoctonia species are responsible for disease of many turfgrass species. The most wide- causing leaf blight on turfgrasses. Both warm-season spread causal agent, Thanatephorus cucumeris (ana- and cool-season turfgrasses are susceptible to Rhizoc- morph: R. solani), consists of several genetically tonia blight with the warm humid and warm tropical different subpopulations. In addition, Waitea circi- climatic zones highly favoring the disease (Burpee nata varieties zeae, oryzae and circinata (anamorph: and Martin 1992). Rhizoctonia spp.) also can cause the disease. Accurate In the United States, cool-season turfgrasses dom- identification of the causal pathogen is important for inate northern states while warm-season turfgrasses effective management of the disease. It is challenging are adapted to the warm-humid or warm-arid condi- to distinguish the specific causal pathogen based on tions of the southern states. The central part of the disease symptoms or macroscopic and microscopic country that is at the northern range of adaptation of morphology. Traditional methods such as anastomo- the warm-season grasses and at the southern range of sis reactions with tester isolates are time consuming adaptation of the cool-season grasses is known as the and sometimes difficult to interpret. In the present transition zone. Although none of the turfgrass types study universally primed PCR (UP-PCR) fingerprint- are well adapted to the transition zone, both are ing was used to assess genetic diversity of Rhizoctonia routinely grown and managed in this region. spp. infecting turfgrasses. Eighty-four Rhizoctonia Rhizoctonia blight poses a major threat to successful isolates were sampled from diseased turfgrass leaves growth and maintenance of several important turf- from seven distinct geographic areas in Virginia and grass species in both southern and transition zones Maryland. Rhizoctonia isolates were characterized by (Burpee and Martin 1992). The most common ribosomal DNA internal transcribed spacer (rDNA- Rhizoctonia species, R. solani Ku¨hn (teleomorph: ITS) region and UP-PCR. The isolates formed seven Thanatephorus cucumeris (Frank) Donk), is responsi- clusters based on ITS sequences analysis and un- ble for causing brown patch on cool-season grasses weighted pair group method with arithmetic mean and large patch on warm-season grasses. Both R. zeae (UPGMA) clustering of UP-PCR markers, which Voorhees (teleomorph: Waitea circinata var. zeae corresponded well with anastomosis groups (AGs) Warcup and Talbot) and R. oryzae Ryker and Gooch of the isolates. Isolates of R. solani AG 1-IB (n 5 18), (teleomorph: W. circinata var. oryzae Warcup and AG 2-2IIIB (n 5 30) and AG 5 (n 5 1) clustered Talbot) are responsible for leaf and sheath spot separately. Waitea circinata var. zeae (n 5 9) and var. disease. Unlike other Rhizoctonia diseases, yellow circinata (n 5 4) grouped separately. A cluster of six patch caused by R. cerealis van der Hoeven (tele- isolates of Waitea (UWC) did not fall into any known omorph: Ceratobasidium cereale Murray and Burpee; Waitea variety. The binucleate Rhizoctonia-like fungi anastomosis group: AG-D) favors cool and wet (BNR) (n 5 16) clustered into two groups. Rhizocto- weather (Smiley et al. 2005, de la Cerda et al. 2007). Another recently identified causal agent, Waitea Submitted 2 Nov 2012; accepted for publication 8 Apr 2013. circinata var. circinata, is responsible for brown ring 1 Corresponding author. E-mail: [email protected] patch in golf courses (de la Cerda et al. 2007). All 1190 AMARADASA ET AL.: UP-PCR ANALYSIS OF RHIZOCTONIA ISOLATES 1191 aforementioned species have multinucleate cells (Boysen et al. 1996, Kuninaga et al. 1997, Kuninaga et al. except R. cerealis, which is a binucleate species. There 2000, Gonzalez et al. 2001, Hsiang and Dean 2001, are reports of other binucleate Rhizoctonia-like fungi Pannecoucque and Ho¨fte 2009). Although analysis of the (BNR) being isolated from turfgrass swards or soils ITS region is a well tested method for identifying (Hurd and Grisham 1983, Burpee and Goulty 1984, Rhizoctonia species, it can be cost prohibitive for Burpee and Martin 1992). Those isolates differ from investigating a large number of samples. Some isolates R. cerealis in physiological characters and anastomosis are difficult to sequence without cloning, which adds time reactions. In general, diseased turfgrasses develop and cost to the analysis. The advancement of technology circular areas of blighted brown leaves. Therefore, and automated sequencing has made the analysis of DNA reliance on field symptoms to identify Rhizoctonia fingerprinting profiles significantly less expensive than ITS causal agents can be misleading. Microscopically all sequencing. Various PCR-based DNA fingerprinting Rhizoctonia species look similar in the asexual state methods have been used for identification and classifica- and typically characterized by nonsporulating mycelia tion of Rhizoctonia species (Lu¨beck and Poulsen 2001, with 90 degree branches having dolipore septa Sharon et al. 2006). Some fingerprinting techniques are (Smiley et al. 2005). The sexual stage of the causal more suited for characterizing individuals within an AG pathogens is rare in nature and difficult to induce in rather than among different AGs (Cubeta and Vilgalys vitro. 1997, Sharon et al. 2006). The universally primed PCR Rhizoctonia species and anastomosis groups (AGs) (UP-PCR) is a fingerprinting technique that generates vary in sensitivity to commonly applied fungicides multiple DNA fragments from an organism without prior (Martin et al. 1984a, b; Carling et al. 1990; Kataria et knowledge of its DNA sequence. The main difference al. 1991; Kataria and Gisi 1996; Campion et al. 2003). between UP-PCR and the well known random amplified In addition, different Rhizoctonia species have differ- polymorphic DNA (RAPD) technique is the length of the ent temperature ranges most conducive for causing primers used. Universal primers (UP) are longer (15– disease. Therefore accurate identification of the 21 nt) than conventional RAPD primers (typically 10 nt) causal pathogen is important to predict disease and designed to anneal under more stringent conditions progression and make future disease management (52–60 C) ensuring higher reproducibility of banding decisions. Moreover, knowledge on the prevalence profiles (Trigiano et al. 2004, Lu¨beck and Lu¨beck 2005). and distribution of pathogenic Rhizoctonia isolates is Lu¨beck and Poulsen (2001) found considerable variation important for selecting appropriate turfgrass germ- in UP-PCR banding profiles among isolates of R. solani plasm with resistance to Rhizoctonia blight. from AGs and AG subgroups. However they
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