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First Report of Race 2 of Colletotrichum trifolii Causing Anthracnose on Alfalfa (Medicago sativa) in Wisconsin

Anthracnose of alfalfa (Medicago sativa), caused by Colletotrichum trifolii, is widespread in the United States. In addition to loss of forage due to death of stems, the pathogen causes crown rot, reducing stand life and winter survival (2), making it one of the most serious diseases of alfalfa. Three physiological races have been described (2). Race 1 is reported to be the dominant race that is present wherever alfalfa is grown, while race 2 was reported in a limited area in the Mid-Atlantic states, and race 4 was found in Ohio (1). Conspicuous, straw-colored dead stems with a “shepherd's crook” wilt and large, sunken, diamond-shaped lesions with a dark border were observed in experimental plots and breeding nurseries of experimental lines in Clinton and West Salem, WI, in August 2011 and in West Salem, WI, in mid-August 2012. Acervuli with black setae and orange spore masses were observed in lesions placed in moist chambers for 2 days at room temperature with ambient room lighting. Conidia were germinated on 1% water agar and then single hyphae were transferred to potato dextrose agar (PDA) plates. DNA was extracted from pure cultures of strains DA-1 (Clinton, WI) and FGI-3 (West Salem, WI), the rDNA ITS1-5.8S-ITS2 region was amplified with primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), the products sequenced directly, and the sequences compared to the ITS region of known race 1 and race 2 strains of C. trifolii. The sequences from DA-1 and FGI-3 were identical to the ITS sequence of C. trifolii 2sp2 (race 1; KF444778) and C. trifolii SB-2 (race 2; KF444779), but distinct from the ITS sequence of C. destructivum (JQ005764) and C. dematium (JX567507), which can cause anthracnose on alfalfa (1). Conidia from DA-1 and FGI-3 were harvested from 7-day-old cultures grown on PDA plates, diluted to 2 × 10⁶ conidia/ml, and sprayed to runoff on 10-day-old growth chamber grown plants of three differential cultivars: Saranac (susceptible to races 1 and 2), Arc (resistant to race 1, susceptible to race 2), and Saranac AR (resistant to races 1 and 2). Plants were maintained at 100% relative humidity for 48 h and then grown in a growth chamber at 24°C with a 16-h photoperiod. Symptoms were rated at 14 days after inoculation. In the three repetitions of the experiment using 75 plants of each cultivar in each experiment, less than 10% of the Saranac and Arc plants survived, while survival of Saranac AR was 31 to 44%. The approximate expected survival of differential cultivars inoculated with race 1 is 1% for Saranac, 65 to 70% for Arc, and 45% for Saranac AR (2). Aggressiveness of race 2 strains on Saranac AR is variable, ranging from 12 to 68% plant survival (3). The susceptibility of Arc when inoculated with DA-1 and FGI-3 is consistent with the reaction to race 2 strains, indicating that both strains are race 2. The isolation of race 2 strains in major alfalfa growing regions in Wisconsin indicates that this physiological race is currently more widespread than previously observed. Although most modern alfalfa cultivars have resistance to race 1, few cultivars with resistance to race 2 are available. The occurrence of C. trifolii race 2 in the Midwest United States should be considered in alfalfa breeding programs when developing multi-pest resistant alfalfa cultivars.

References: (1) J. J. Ariss and L. H. Rhodes. Plant Dis. 91:1362, 2007. (2) N. R. O'Neill. Plant Dis. 80:450, 1996. (3) N. R. O'Neill et al. Phytopathology 79:750, 1989.