Genetic and Morphological Evidence that Phoma sclerotioides, Causal Agent of Brown Root Rot of Alfalfa, Is Composed of a Species Complex

Phoma sclerotioides, causal agent of brown root rot of alfalfa, causes severe root and crown lesions on alfalfa and other perennial forage legumes in regions with harsh winters. Isolates of P. sclerotioides exhibit diverse cultural morphologies on potato dextrose agar (PDA), suggesting that they may exhibit a high degree of genetic diversity. To investigate the genetic relatedness of P. sclerotioides isolates, 154 isolates from North America were sequenced at 10 loci. Maximum parsimony and maximum likelihood analyses of the complete 10-locus data set placed isolates into multiple strongly supported clades, and analyses of gene-jackknife and single-gene partitions of the data set indicated robust support for six major clades and three subclades. Genetic differences corresponded closely to differences in conidial size and septation, pycnidial neck length, mycelial pigmentation, and growth rate in axenic culture at 18 and 25°C. Isolates exhibited morphologies broadly consistent with the species description of P. sclerotioides, and new species were not designated. On the basis of genetic and morphological differences, we propose establishing seven infraspecific varieties within P. sclerotioides: P. sclerotioides var. sclerotioides, champlainii, viridis, obscurus, steubenii, macrospora, and saskatchewanii. All varieties of P. sclerotioides caused brown root rot of alfalfa and grew well at low temperatures.

First Report of Brown Root Rot of Alfalfa Caused by Phoma sclerotioides in Maine, Ontario, and Pennsylvania

Brown root rot (BRR), caused by the fungal pathogen Phoma sclerotioides G. Preuss ex Sacc. (synonym Plenodomus meliloti Dearn. & G.B. Sanford), is associated with yield loss of alfalfa (Medicago sativa L.) in regions with severe winters (1). In the spring of 2007, 9 to 69 alfalfa plants were collected from each of five production fields in Maine, 10 fields in Ontario, and nine fields in Pennsylvania. All alfalfa stands existed at least two winters. P. sclerotioides was isolated from alfalfa root and crown lesions from five fields in Maine (Penobscot, Somerset and Waldo counties), seven fields from southwestern (Woodstock and Niagara), south-central (Lindsay and Belleville), and southeastern Ontario (near Ottawa), and four fields in Pennsylvania (Columbia, Crawford, and Jefferson counties; 41.1 to 41.6°N). BRR incidence was 9 to 29% in Maine, 5 to 29% in Ontario, and 8 to 22% in Pennsylvania. In Ontario, some lesions girdled the crown; in three fields in Maine, large pycnidia characteristic of P. sclerotioides were present on alfalfa crowns and overwintered stems. On potato dextrose agar, conidia (5 to 8 × 2 to 3 μm, unicellular, hyaline, and ovoid) and pycnidia (0.33 to 1.15 mm in diameter with multiple beaks) of single-conidium isolates were characteristic of P. sclerotioides (2). Diagnostic PCR (3) of isolates resulted in a single amplicon of expected size (500 bp). The internal transcribed spacer (ITS) 1, 5.8S, and ITS2 of the rDNA were sequenced for 12 representative isolates, and sequences (GenBank Accession Nos. FJ179151 to FJ179162) were 95.5 to 100% identical to P. sclerotioides ATCC isolate 56515 over a 488-bp alignment. Eight months after seeding, potted ‘Vernal’ alfalfa was inoculated (4), kept at 4°C for 8 weeks, 0 to –2°C for 12 weeks, 4°C for 8 weeks, and 10 to 15°C for 7 weeks. Of 108 plants inoculated with the Maine isolates, 35 developed severe cortical lesions and 16 died. Of 18 plants inoculated with the Ontario isolates, 16 developed severe cortical lesions and eight died. Of 18 plants inoculated with a Pennsylvania isolate, 11 developed severe cortical lesions and five died. Lesions were typical of BRR: light to very dark brown, sometimes with a darker border, and often containing abundant pycnidia. Plant mortality was associated with lesions that girdled the root and crown. Of 18 plants in the control treatment, three developed severe cortical lesions and none died. BRR is common in Alberta, Saskatchewan, and Manitoba, but in eastern Canada it has been reported only in Nova Scotia. To our knowledge, this is the first report of BRR in Maine, Ontario, and Pennsylvania and the southernmost report of BRR in eastern North America. References: (1) B. Berkenkamp et al. Can. J. Plant Sci. 71:211, 1991. (2) G. H. Boerema et al. Persoonia 15:431, 1994. (3) R. C. Larsen et al. Plant Dis. 86:928, 2002. (4) M. J. Wunsch et al. Plant Dis. 91:1293, 2007.

First Report of Brown Root Rot of Alfalfa Caused by Phoma sclerotioides in Colorado and New Mexico

Brown root rot (BRR), caused by the fungal pathogen Phoma sclerotioides G. Preuss ex Sacc. (synonym Plenodomus meliloti Dearn. & G.B. Sanford), is associated with winterkill, slow emergence from winter dormancy, and yield loss of alfalfa (Medicago sativa L.) (1,2). BRR is a problem in regions with severe winters and is common in Alaska and Alberta, Saskatchewan and Manitoba, Canada. It was first observed in the continental United States in Wyoming during 1996 (2) and has subsequently been found in Idaho, Minnesota, Montana, New Hampshire, New York, Vermont, and Wisconsin. In the intermountain valleys of northern New Mexico and western Colorado, winters can be severe; alfalfa winterkill events occur periodically, but it is unknown if BRR is present. In May 2006, alfalfa plants were collected from production fields in Huerfano, Otero, and Rio Grande counties in Colorado and Rio Arriba and Taos counties in New Mexico and assessed for BRR. Two to three fields were sampled per county and 20 or 40 plants were collected per field. All fields existed for at least two winters. Fields sampled in Rio Grande County exhibited severe winterkill, with most plants completely girdled by crown lesions. Plants from other fields exhibited a range of root and crown rots. Isolation of P. sclerotioides was attempted from all plants with a previously described protocol (4). The pathogen was isolated from crown lesions of one alfalfa plant each from Rio Grande and Taos counties. Both lesions extended into the cortex. On potato dextrose agar and water agar with barley (4), single-conidium cultures of each isolate produced large pycnidia (0.35 to 0.80 mm in diameter) with multiple beaks, white cirri darkening to yellow with age, and unicellular, hyaline, ovoid conidia 5 to 7 μm long by 2 μm wide. Diagnostic PCR of the cultures using P. sclerotioides-specific primers (3) resulted in a single amplicon of expected size (500 bp). The internal transcribed spacer (ITS) 1, 5.8S, and ITS2 of the rDNA were amplified and sequenced using primers ITS1 and ITS4. The ITS sequences (GenBank Accession Nos. EU265669 and EU265670) were >98% identical to P. sclerotioides ATCC isolate 56515 over 503 bp of aligned sequence. Potted ‘Vernal’ alfalfa was inoculated 4 months after seeding, kept at 4°C for 5.5 weeks, 0 to –2°C for 12 weeks, and 4°C for 3 weeks. Of the 14 plants inoculated with the Colorado isolate, 11 developed cortical lesions and 8 winterkilled. Of the 23 plants inoculated with the New Mexico isolate, 22 developed cortical lesions and 16 winterkilled. Lesions were light to very dark brown, sometimes with a darker border and often containing abundant pycnidia. Winterkill was associated with lesions girdling the crown. P. sclerotioides was isolated from the lesions. To our knowledge, this is the southernmost report of BRR in North America and the first report of BRR in New Mexico and Colorado. The incidence and severity of BRR in the region surveyed appear to be considerably lower than in the more northern regions. References: (1) B. Berkenkamp et al. Can. J. Plant Sci. 71:211, 1991. (2) C. R. Hollingsworth et al. Can. J. Plant Pathol. 25:215, 2003. (3) R. C. Larsen et al. Plant Dis. 86:928, 2002. (4) M. J. Wunsch et al. Plant Dis. 91:1293, 2007.

Distribution, Impact, and Soil Environment of Phoma sclerotioides in Northeastern U.S. Alfalfa Fields

We report brown root rot (BRR) of alfalfa, caused by the fungal pathogen Phoma sclerotioides, for the first time in the eastern United States. Alfalfa production fields in New York, Vermont, and New Hampshire were sampled in spring 2005, and soil characteristics were related to variability in BRR incidence and severity in two New York fields sampled extensively. BRR was detected in 8 of 10 fields sampled in New York, 6 of 7 fields sampled in Vermont, and 5 of 6 fields sampled in New Hampshire. Lesions on both roots and crowns were common in all three states, and most BRR lesions extended into the cortical tissues. Diagnostic polymerase chain reaction (PCR) of P. sclerotioides isolates produced a single amplicon of the expected size. In vivo conidia and pycnidia morphology of northeastern isolates was consistent with published descriptions of P. sclerotioides, and P. sclerotioides was reisolated from symptomatic lesions after pathogenicity testing. In two New York fields sampled extensively, BRR severity varied with soil strength, soil texture, soil saturation, and alfalfa stand density. The spatial pattern of BRR within fields suggests the pathogen was not recently introduced. The results suggest BRR is widespread in alfalfa production fields in New York, Vermont, and New Hampshire.

Distribution of Phoma sclerotioides on Alfalfa and Winter Wheat Crops in the North Central United States

Brown root rot of alfalfa (Medicago sativa), caused by Phoma sclerotioides, has been reported in several states in the northern United States and in western Canada. A survey was conducted to determine the distribution of the fungus in Minnesota and Wisconsin. Isolates of the pathogen 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 were identical and matched the sequences of a P. sclerotioides isolate from Wyoming. The fungus was found to be widespread in both states and was detected in roots of alfalfa plants from 17 counties in Minnesota and 14 counties in Wisconsin using polymerase chain reaction (PCR)-based assays. A real-time PCR assay was developed that increased sensitivity of detecting the pathogen from plant tissues and soil. The isolates from alfalfa caused disease on inoculated winter wheat plants. Although the fungus was previously found associated with roots of diseased cereal and turfgrass plants, this is the first demonstration of pathogenicity of P. sclerotioides on wheat.

First Report of Brown Root Rot of Alfalfa Caused by Phoma sclerotioides in Wisconsin

Brown root rot (BRR) has been associated with winterkill of alfalfa (Medicago sativa L.) in the temperate regions of North America where winters are severe (1). Although suspected, BRR has not been associated with winterkill of alfalfa in the upper Midwestern United States. Alfalfa plants exhibiting symptoms resembling those induced by the causal agent Phoma sclerotioides G. Preuss ex Sacc. were collected from fields in Marinette, Pierce, and Marathon counties in Wisconsin during the spring and early summer of 2003. Symptoms included stunting and decline in 1- to 3-year-old plants that were slow to break dormancy in the early spring. Roots frequently exhibited dark brown lesions or were entirely decayed. Advanced lesions often formed dark bands around the circumference of tap and secondary roots. Beaked pycnidial structures typical of P. sclerotioides were also observed on many samples with advanced lesions. Plants with symptoms of BRR were also observed in Clark, Langlade, Lincoln, Oconto, Shawno, Taylor, and Wood counties. Several lesion areas of tissue on the tap and lateral roots of each sample were excised with a sterile scalpel. Total DNA was extracted using the Fast DNA kit (Bio 101, Carlsbad, CA). In addition, soil samples were collected in the root rhizosphere of symptomatic plants from four fields in two counties. Soil DNA was extracted with the Ultra-Clean DNA soil extraction kit (Mo Bio, Solana Beach, CA). DNA extractions were diluted 1:10 or 1:50, and samples were evaluated for the presence of P. sclerotioides using polymerase chain reaction (PCR)-based sequence-characterized amplified region (SCAR) markers according to the method described previously (4). Amplicons of the expected size (499 bp) were detected from alfalfa roots sampled from Marathon (4 of 4), Marinette (4 of 5), and Pierce (4 of 4) counties but not in roots from healthy controls produced in the greenhouse at Prosser, WA. PCR amplicons were also produced from all field soil samples in Marathon and Marinette counties. Proof of pathogenicity via Koch's postulates for this host-pathogen system was not attempted because of the extensive time period required (1). However, characteristic beaked pycnidia were present, and the pathogen was identified using PCR on DNA from roots symptomatic of BRR. Detection of BRR has been limited in the United States to Wyoming (2), but has been thought to occur in other states with severe winters (3). To our knowledge, this is the first report of P. sclerotioides in Wisconsin. References: (1) J. G. N. Davidson. Brown root rot. Pages 29–31 in: Compendium of Alfalfa Diseases. 2nd ed. D. L. Stuteville and D. C. Erwin, eds. The American Phytopathological Society, St. Paul, MN, 1990. (2) F. A. Gray et al. Pages 27–28 in: Proc. 10th Western Alfalfa Improv. Conf., 1997. (3) C. R. Hollingsworth et al. Can. J Plant Pathol. 25:215, 2003. (4) R. C. Larsen et al. Plant Dis. 86:928, 2002.

Assessing resistance to spring black stem and leaf spot of alfalfa caused by Phoma spp.

The disease reaction of alfalfa (Medicago sativa) cultivars to spring black stem was evaluated in field trials and greenhouse experiments. In field trials, differences in cultivar reaction to leaf spot (predominantly spring black stem) were observed in 9 of 16 station years. The reaction of certain cultivars was consistent across most trials, but other cultivars were quite variable. Under controlled conditions, one isolate each of Phoma sclerotioides and P. exigua produced symptoms on alfalfa leaves that were similar to those caused by P. medicaginis. These results indicate that P. medicaginis is not the only pathogen responsible for symptoms of spring black stem on alfalfa in the prairie region. In a detached-leaf study, one isolate each of P. medicaginis, P. sclerotioides and P. exigua produced leaf lesions on all 18 alfalfa cultivars assessed. Disease incidence in Absolute, Algonquin, Pickseed 3006 and Anik (M. sativa subsp. falcata) was lower than in 630 and AC Blue J. Inoculation of eight selected cultivars using a range of spore concentrations under controlled conditions showed a similar pattern; all three isolates produced leaf lesions on all eight cultivars. Ino culation with conidial suspensions of P. medicaginis resulted in a lower disease incidence on Absolute than on Beaver. Key words: Medicago sativa, Medicago sativa subsp. falcata, Phoma medicaginis, P. sclerotioides, P. exigua, detached leaves.

A Rapid Method Using PCR-Based SCAR Markers for the Detection and Identification of Phoma sclerotioides: The Cause of Brown Root Rot Disease of Alfalfa

A rapid technique for identification and detection of Phoma sclerotioides, the causal agent of brown root rot of alfalfa, has been developed using polymerase chain reaction (PCR). Amplification products obtained from random amplified polymorphic DNA (RAPD) reactions were cloned and sequenced, and two extended primer sets were designed from the resulting data that were used to detect sequence-characterized DNA markers. A single 499-bp DNA amplification product was consistently obtained from primers PSB12499 that was specific for 19 isolates of P.sclerotioides but was not produced from Phoma medicaginis or Phoma betae, or from other soilborne pathogens including Aphanomyces euteiches, Rhizoctonia solani, Fusarium oxysporum, Pythium ultimum, or Phytophthora infestans. A 499-bp amplification product was also produced from root tissue known to be infected with the fungus as verified by microscopic examination. A similar PCR product was obtained from soil samples collected from fields with an established infection of P. sclerotioides on alfalfa. This PCR-based assay enables detection of P. sclerotioides from alfalfa root tissue and in soil samples in a single day, including extraction of DNA, compared with standard methods that require up to 100 days for identification using agar media.

First Report of Brown Root Rot on Alfalfa Caused by Phoma sclerotioides in the Continental United States

Phoma sclerotioides G. Preuss ex Sacc. (previously named Plenodomus meliloti Dearn. & G.B. Sanford) is associated with root rot and extensive winterkill of leguminous forage crops, such as clover (Trifolium and Melilotus spp.), sainfoin (Onobrychis viciifolia), and alfalfa (Medicago sativa). Winterkill and root rot of irrigated alfalfa were observed for the first time in a field of cv. Multiplier in western Wyoming during the spring of 1996. Dark brown to black, sunken, rotting lesions were noted on upper secondary roots and taproots of dead and living diseased plants. Superficial and embedded beaked pycnidia and pycnosclerotia were observed near root lesions. A Phoma sp. isolated from a diseased plant in Farson, WY, was maintained on potato dextrose and half-strength V8-juice agars. Beaked pycnidia, typical of P. sclerotioides, were observed in culture when grown at 10°C for 2 months. A pathogenicity test was performed on cv. Multiplier. Two barley seeds colonized by a Phoma sp. derived from a Wyoming isolate were positioned on taproots of healthy, greenhouse-grown, 5-month-old plants ≈2.5 cm below the crown and were covered with a small piece of sterile cotton. Three replicate samples (24 plants inoculated and 24 plants uninoculated per replicate) were winter-hardened for 4 weeks (15.6°C/10°C, day/night, for 2 weeks, followed by 10°C/7.2°C, day/night, for 2 weeks) and placed outside during January 1998 in Laramie, WY, for a 4-month winter exposure period. Plants were rated for disease during June 1998. A disease severity rating of 1 to 5 was assigned to each experimental unit, where 1 = no disease and 5 = dead plant. The percentage of diseased plants at each severity rating for all inoculated plants was 1 = 19%, 2 = 33%, 3 = 31%, 4 = 13%, and 5 = 4%. Mycelium typical of P. sclerotioides was found on 99% of inoculated plant roots whether or not they had pycnidia. Pycnidia were found on the lower stems and petioles of some inoculated plants. Three percent of control plants also developed brown root rot (BRR) symptoms (taproot lesions or discoloration) by June 1998. The percentage of diseased plants at each severity rating for all uninoculated plants was 1 = 96%, 2 = 4%, and 3 through 5 = 0%. Aboveground propagule placement likely contributed to the spread of BRR by raindrop splash and wind-driven plant debris to adjacent alfalfa. Most inoculated plants had immature pycnidia or protopycnidia (94%), whereas 6.9% of the plants also had fully mature, beaked pycnidia. Pure fungal cultures were obtained from several diseased roots and compared with the original Wyoming Phoma sp. culture and a Canada isolate of P. sclerotioides (ATCC no. 56515) (2): colony, pycnidial, and conidial morphologies were identical, completing Koch's postulates. This is the first report of BRR on alfalfa in the continental United States. References: (1) J. G. N. Davidson. 1990. Brown root rot. Pages 29–31 in: Compendium of Alfalfa Disease. 2nd ed. The American Phytopathological Society, St. Paul, MN. (2) C. R. Hollingsworth et al. Phytopathology 88(suppl.):S39, 1999.

Resistance of alfalfa cultivars to brown root rot

Alfalfa cultivars were evaluated for resistance to the low-temperature disease, brown root rot, caused by Phoma sclerotioides, and for forage yield in field trials with naturally occurring disease. The cultivar Peace was the most resistant, followed by Algonquin, Beaver, Apica, Anchor and Pacer. Yields were closely related to root rot resistance. Key words: Brown root rot, alfalfa, Phoma sclerotioides, Plenodomus meliloti, resistance, losses