J. AMER.SOC.HORT.SCI. 132(4):534–540. 2007. Distribution of Rose Black Spot (Diplocarpon rosae) Genetic Diversity in Eastern North America Using Amplified Fragment Length Polymorphism and Implications for Resistance Screening Vance M. Whitaker and Stan C. Hokanson1 Department of Horticultural Science, University of Minnesota, 258 Alderman Hall, 1970 Folwell Avenue, St. Paul, MN 55108 James Bradeen Department of Plant Pathology, University of Minnesota, 495 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108 ADDITIONAL INDEX WORDS. AMOVA, dendrogram, fungal isolate, Jaccard’s coefficient, pathogenic race, principal component analysis ABSTRACT. Black spot, incited by the fungus Diplocarpon rosae Wolf, is the most significant disease problem of landscape roses (Rosa hybrida L.) worldwide. The documented presence of pathogenic races necessitates that rose breeders screen germplasm with isolates that represent the range of D. rosae diversity for their target region. The objectives of this study were to characterize the genetic diversity of single-spore isolates from eastern North America and to examine their distribution according to geographic origin, host of origin, and race. Fifty isolates of D. rosae were collected from roses representing multiple horticultural classes in disparate locations across eastern North America and analyzed by amplified fragment length polymorphism. Considerable marker diversity among isolates was discovered, although phenetic and cladistic analyses revealed no significant clustering according to host of origin or race. Some clustering within collection locations suggested short-distance dispersal through asexual conidia. Lack of clustering resulting from geographic origin was consistent with movement of D. rosae on vegetatively propagated roses. Results suggest that field screening for black spot resistance in multiple locations may not be necessary; however, controlled inoculations with single-spore isolates representing known races is desirable as a result of the inherent limitations of field screening. The fungus Diplocarpon rosae (anamorph = Marssonina (Horst, 1983), although frequency of sexual recombination in rosae Lind.) is the causal organism of rose black spot, which D. rosae populations has not been determined. is the most damaging disease of rose worldwide (Dobbs, 1984). Phenotypic variability among single-conidial isolates sug- Symptoms include lesions on leaves and stems as well as gests that D. rosae is a genetically diverse pathogen (Wenefrida frequent leaf yellowing and defoliation that significantly and Spencer, 1993). Pathogenicity tests have revealed the compromise plant growth and appearance. Consequently, presence of numerous races. Five races were discovered in numerous topical and systemic fungicides are used by home- Germany (Debener et al., 1998) among 15 isolates, four have owners and landscapers to combat this disease. Diplocarpon been found in England (Yokoya et al., 2000), four were rosae is classified as an ascomycete in the family Dermateaceae described in Ontario, Canada (Svejda and Bolton, 1980), and and is a hemibiotrophic fungus that is restricted to the genus seven races were described in Mississippi (Spencer and Wood, Rosa L. (Blechert and Debener, 2005). It is spread primarily 1992a, 1992b). Although it is not known whether any of these through waterborne, two-celled asexual spores (conidia) that races from different studies are the same, the occurrence of require free water to germinate. Visual symptoms may develop multiple races in each location suggests a large total number of in susceptible cultivars in as little as 4 d after spore germination races in the world. The Mississippi isolates were collected from and penetration. The sexual stage of this fungus has been a seven-county area demonstrating diversity in pathogenicity reported only twice in North America and twice in England within a relatively narrow geographic range. Considerable variability in conidial size and colony color among single- conidial isolates has also been documented (Wenefrida and Received for publication 27 Nov. 2006. Accepted for publication 30 Mar. 2007. Spencer, 1993). This manuscript is Scientific Journal Series No. 061210167 of the University of Genetic diversity has been documented in D. rosae using Minnesota Department of Horticultural Science and has been supported in part by the Minnesota Agricultural Experiment Station. molecular marker analyses. Lee et al. (2000) examined 10 We thank Dr. Campbell Davidson, Dennis Eveleigh, Sam Kedem, Helen isolates using restriction fragment length polymorphism Nowell, Nick Howell, Ann Creamer, Donna Fuss, Paul Zimmerman, Jim (RFLP) analysis of the internal transcribed spacer region of Jacobi, Kathy Kilgore, Glenn Schroeter, Diane Brueckman, Julie Overom, rDNA. Distinct RFLP patterns were observed and isolates Kathy Ahlgren, and Charles Tubesing for collecting D. rosae isolates. We also acknowledge Marilena Aquino De Muro for contributing expertise in DNA clustered into three groups. Initial studies by British researchers extraction methodology. with simple sequence repeat and amplified fragment length 1Corresponding author. E-mail: [email protected]. polymorphism (AFLP) indicated that AFLP was the more 534 J. AMER.SOC.HORT.SCI. 132(4):534–540. 2007. reproducible and sensitive marker system for their isolates, Table 1. Fifty single-conidial isolates of Diplocarpon rosae, their although final results of their work have not yet been published geographic locations, and their host cultivars of origin.z (Drewes-Alvarez, 2003). Carlson-Nilsson (2002) conducted a Isolate Location Host cultivar Race randomly amplified polymorphic DNA analysis of 15 D. rosae CBB Birmingham, AL Cecile Brunner — y isolates collected from locations in Sweden, Canada, France, PEB Birmingham, AL Peace A and the United States, which were roughly separated by geo- LPB Birmingham, AL Love and Peace — graphic origin in a cluster analysis. The survey did not allow NDCH Jemison, AL New Dawn — in-depth analysis of geography, host of origin, pathogenic race, CBCH Jemison, AL Cecile Brunner — fungicide selection, or other factors on genetic similarity HECH Jemison, AL Heritage — among isolates, although diversity among isolates was demon- DDCH Jemison, AL Dortmund A strated. The current lack of knowledge about such factors and BACH Jemison, AL Ballerina — their influence on diversity complicates host resistance breed- SNC Bloomfield, CT Starry Night A ing efforts. PPC Bloomfield, CT Pink Panther — The objective of this study was a preliminary examination IGI Ames, IA Iceberg — of the genetic diversity present in 50 single-spore isolates of LPI Ames, IA Love and Peace A D. rosae collected from eastern North American using AFLP CMI Ames, IA Charles de Mills — analysis (Vos et al., 1995) and the manner in which diversity GMI Ames, IA Gold Medal — was distributed in relation to geographic origin, host of origin, PEL Shreveport, LA Peace A or pathogenic race structure of the isolates under study. Such UNM Morden, MB Unknown A information is useful for the screening and evaluation of host MSD Duluth, MN Morden Sunrise — resistance in rose germplasm targeted for North American FCD Duluth, MN Frontenac — markets. MSBD Duluth, MN Morden Snowbeauty — BAH Hastings, MN Ballerina — Materials and Methods HEH Hastings, MN Heritage — LPH Hastings, MN Love and Peace — COLLECTION. Leaves infected with D. rosae were collected JCH Hastings, MN John Cabot — from roses growing in 17 disparate locations across eastern GVH Hastings, MN George Vancouver C North America (Table 1; Fig. 1). Collection sites were chosen to WBN Chanhassen, MN William Baffin — represent a broad range of locations across the eastern United BAN Chanhassen, MN Ballerina — States and southern Canada. Collection sites contained multiple JCN Chanhassen, MN John Cabot A rose plants in small nurseries or botanical/display gardens; CMN Chanhassen, MN Charles de Mills — therefore, distances between individual plants in collection NWR Rochester, MN Nearly Wild — sites ranged from less than 3 m to no more than a few hundred LPM St. Louis, MO Love and Peace — meters apart. The only exception were isolates MSD and ANM St. Louis, MO Apricot Nectar — MSBD, which were collected from the same residential garden BAM St. Louis, MO Ballerina — in Duluth, MN, and isolate FCD, which was collected from a HEM St. Louis, MO Heritage — separate residential garden in the same city. A subset of leaf IGM St. Louis, MO Iceberg — samples was purposefully collected from the same host culti- LEM St. Louis, MO Love’s Promise — vars in different geographical locations to examine possible DDM St. Louis, MO Dortmund A effects of host of origin on the genetic diversity of D. rosae IGO Burlington, ON Iceberg — isolates. Infected leaves were stored at a temperature of –20 °C HEO Burlington, ON Heritage — for up to 4 months before pathogen isolation. MBO Burlington, ON Morden Blush A ISOLATION AND CULTURE. Fifty single-conidial isolates CBOD Newark, OH Cecile Brunner A were isolated from infected leaf pieces and increased from COH Kirtland, OH Carmenatta — single conidia (asexual spores) on potato dextrose agar (PDA) LFSC Landrum, SC La France — according to the methods described by Whitaker et al. (2007a). CBSC Landrum, SC Cecile Brunner — After 28 d of growth on PDA at room temperature, digital ANSC Landrum, SC Apricot Nectar A pictures were taken to record the colony color of all isolates. IGSC Landrum, SC Iceberg — Conidia from each single-conidial isolate were harvested