Distribution of Phoma sclerotioides on Alfalfa and Winter Wheat Crops in the North Central United States J. E. Larsen, Department of Plant Pathology, University of Minnesota, St. Paul 55108; C. R. Hollingsworth, Northwest Research and Outreach Center and Department of Plant Pathology, University of Minnesota, Crookston 56716; J. Flor, Department of Plant Pathology, University of Minnesota, St. Paul 55108; M. R. Dornbusch, USDA- ARS-Plant Science Research, Department of Plant Pathology, University of Minnesota, St. Paul 55108; N. L. Simpson, Fort Valley State University, Fort Valley, GA 31030; and D. A. Samac, USDA-ARS-Plant Science Research, De- partment of Plant Pathology, University of Minnesota, St. Paul 55108 lower crown interferes with carbohydrate ABSTRACT and protein storage and increases suscepti- Larsen, J. E., Hollingsworth, C. R., Flor, J., Dornbusch, M. R., Simpson, N. L., and Samac, bility of alfalfa plants to winterkill (10). D. A. 2007. Distribution of Phoma sclerotioides on alfalfa and winter wheat crops in the North Hard red winter wheat (Triticum aesti- Central United States. Plant Dis. 91:551-558. vum L.) commonly exhibits widespread winterkill in Minnesota (21). Stand losses Brown root rot of alfalfa (Medicago sativa), caused by Phoma sclerotioides, has been reported in from adapted cultivars have been attributed several states in the northern United States and in western Canada. A survey was conducted to exclusively to weather-related plant determine the distribution of the fungus in Minnesota and Wisconsin. Isolates of the pathogen stresses. While the potential of P. sc l e - were recovered from roots of alfalfa, winter wheat, and perennial ryegrass plants. The internal transcribed spacer (ITS) 1, 5.8S, and ITS2 of the rDNA of the isolates from alfalfa and wheat rotioides to cause disease on winter wheat were identical and matched the sequences of a P. sclerotioides isolate from Wyoming. The fun- is currently unknown, it is a recognized gus was found to be widespread in both states and was detected in roots of alfalfa plants from 17 pathogen on over-wintering turfgrass spe- counties in Minnesota and 14 counties in Wisconsin using polymerase chain reaction (PCR)- cies (18). Although winter wheat acreage based assays. A real-time PCR assay was developed that increased sensitivity of detecting the continues to increase in Minnesota, many pathogen from plant tissues and soil. The isolates from alfalfa caused disease on inoculated win- growers avoid planting the crop because of ter wheat plants. Although the fungus was previously found associated with roots of diseased its risk for winterkill. Winter wheat may be cereal and turfgrass plants, this is the first demonstration of pathogenicity of P. sclerotioides on grown in rotation with alfalfa, a known wheat. host of P. sclerotioides, so it is critical to determine if it is a host for the fungus so Additional keywords: Lolium perenne, Triticum aestivum, winterkill that effective disease management strate- gies can be implemented. Polymerase chain reaction (PCR)-based techniques have become widely used for Brown root rot (BRR) of alfalfa (Medi- southwestern Wyoming (3), and was de- the detection of plant pathogens (2,13,14). cago sativa L.) is caused by Phoma scle- tected on a diseased alfalfa sample by the Because PCR is specific, sensitive, and rotioides G. Preuss ex Sacc. (formerly Montana Plant Disease Clinic (15). The rapid, it is ideal for identifying a slow- Plenodomus meliloti Dearness & Sanford). pathogen was later isolated from diseased growing fungus such as P. sclerotioides The fungus is a slow-growing plant patho- roots of alfalfa located in eight Wyoming (12). Real-time PCR is even more sensi- gen and saprophyte endemic to the prairie counties (4,5,9) and one Idaho county (8). tive, more efficient, and more effectively soils of Canada (1,17,18). The fungus was In the spring of 2003, P. sclerotioides was quantitative than conventional endpoint first studied on forage legumes during the first identified from diseased alfalfa plants PCR (2,14). Recent development of af- mid-1920s (1). Later, it was identified in Minnesota and Wisconsin (11,16). The fordable, real-time PCR techniques makes from diseased plants in the Northwest prevalence of the disease and distribution real-time PCR particularly suitable for Territories, Nova Scotia, Alaska, the of P. sclerotioides throughout the remain- high throughput detection and quantifica- Yukon, as well as British Columbia, Al- der of the United States, particularly the tion of pathogens in plant tissues and soil. berta, Saskatchewan, and Manitoba (1,19). upper-tier states, is largely unknown. Be- The objectives of this work were to (i) In the 1980s, the fungus was found causing cause optimal growth of the fungus occurs determine the distribution of P. sc l e - widespread root rot of alfalfa in Alberta at 10 to 15°C (5,8), the disease is sus- rotioides in the Upper Midwestern United (1). Only recently, P. sclerotioides was pected to contribute to winterkill in regions States by surveying for the pathogen in identified from diseased alfalfa in the con- where winters are extended. Brown root stands of alfalfa and winter wheat; (ii) tiguous United States. During the spring of rot is responsible for winter injury and determine if P. sclerotioides causes root rot 1996, P. sclerotioides was found to be the stand decline of a number of forage leg- of hard red winter wheat; and (iii) develop cause of widespread winterkill of alfalfa in umes including alfalfa, red clover (Tri- a sensitive and quantitative real-time PCR- folium pratense L.), bird’s foot-trefoil based assay to detect and quantify the (Lotus corniculatus L.), alsike clover (Tri- density of P. sclerotioides in roots of al- Corresponding author: D. A. Samac folium hybridum L.), sweet clover (Melilo- falfa, cereal crops, and soil. E-mail: [email protected] tus spp.), and common sainfoin (Ono- Accepted for publication 15 November 2006. brychis viciifolia Scop.) (1). MATERIALS AND METHODS Symptoms of BRR on alfalfa include Isolation of P. sclerotioides from al- brown, circular-shaped lesions on taproots falfa and winter wheat roots. Between doi:10.1094/ PDIS-91-5-0551 and lesions girdling, or banding, smaller 2003 and 2005, 12 alfalfa plants were col- This article is in the public domain and not copy- lateral or feeder roots. Over time, lesions rightable. It may be freely reprinted with custom- lected at random from a total of seven ary crediting of the source. The American Phyto- can coalesce and eventually cause plant commercial production fields located in pathological Society, 2007. death (1). Rot of the upper taproot and Marshall, Pennington, Red Lake, and Polk Plant Disease / May 2007 551 counties in northwest Minnesota and in Plants were collected from healthy and Samples were sequenced by the Advanced Otter Tail County in west central Minne- declining stands. From five to 20 plants Genetic Analysis Center (University of sota (84 total plant samples). Stand health were removed randomly across fields. At Minnesota) with primers ITS1 or ITS4. and age ranged from excellent (approxi- least five plants were assayed by PCR Sequences were aligned using ClustalW mately 1-year-old) to severely declining from each location. (1.83) Multiple Sequence Alignment. (3-years-old). Collected plants were placed In preparation for the PCR assay, DNA Inoculation of winter wheat with P. into self-sealing plastic bags and stored at was isolated from alfalfa, winter wheat, sclerotioides. On 4 October 2004, cultivar 4°C until processed. Roots were rinsed and perennial ryegrass roots using the Fast Jerry hard red winter wheat seed were with tap water to remove soil, and tissue DNA kit and Fast Prep instrument (Q- planted into Deepot Cells (Stuewe and isolations were made from discolored roots BIOgene Solon, OH). Approximately 200 Sons, Inc., Corvallis, OR) containing a of plants with symptoms and from ran- mg of root tissue was homogenized in 500 pasteurized native soil:sand mixture (1:1) domly selected roots of symptomless µl of buffer CLS-Y, 400 µl of buffer CLS- at a depth of 2.54 cm. Plants were main- plants. Root pieces were disinfected for 3 VF, and 100 µl of buffer PPS. Samples tained in the greenhouse at 10°C (±5°C) min in a 10% sodium hypochlorite solu- were agitated for 30 s on setting 5. DNA of under natural lighting for 7 days. A total of tion, rinsed for 3 min in sterile distilled P. sclerotioides was detected using a modi- 125 potted test plants were situated in water, and air-dried for 3 min on filter fied version of a previously established Deepot support trays (Stuewe and Sons). paper. The root pieces were then placed on endpoint PCR-based assay specific for P. Inoculum was prepared using a modified water agar and maintained in a dark incu- sclerotioides (12). PCR reactions were protocol (7) from four P. sclerotioides bator at 5°C for 4 months. If beaked performed in 25-µl volumes containing 50 isolates obtained from diseased alfalfa pycnidia characteristic of P. sclerotioides ng template DNA, 2.5 µl 10× reaction roots from four locations in Minnesota were observed in at least one petri dish, the buffer (Promega Corp., Madison, WI), 2.5 during 2004 (Thief River Falls [TRF], Red collection site was considered positive for µl dNTP mixture (2.5 mM each dNTP; Lake Falls [RLF], Holt, and Fergus Falls P. sclerotioides. Pure cultures of P. sc l e - TaKaRa Bio Inc., Otsu, Shiga, Japan), 1 [FF]). Sterile barley grains were placed rotioides were obtained by transferring µM SCAR primers, and 0.625 units Taq into five self-sealing plastic bags and in- disinfected pycnidia to potato dextrose DNA polymerase. Reaction conditions oculated with one each P. sclerotioides agar (PDA) plates.