First Report of Fusarium oxysporum on Birdsfoot Trefoil in Alabama

Lotus corniculatus (birdsfoot trefoil) is a common flowering plant native to Eurasia and North Africa. It is used as a forage plant and also grown for pasture, hay, and silage due to its nonbloating properties. A foliar blight and a crown rot disease of birdsfoot trefoil cultivars and breeding lines was observed at the Plant Breeding Unit of the E.V. Smith Research Center in Tallassee, Alabama, in 2015 and every season since then. Dying plants began to appear late in the season in hot weather (31°C) in fields with a sandy soil. Foliage became chlorotic, withered, and the plants died. The root system became brown to black with internal vascular discoloration of the hypocotyl. Entire symptomatic plants (47% incidence or 398 plants with 100% disease severity) were collected weekly from September 1 to October 27, 2015, from half-sib individuals that included Empire, Norcen, and Pardee cultivars. One stem and one root section per 187 plants were cut into 1-cm pieces, surface disinfected, and placed onto water agar and acid PDA (APDA), with 10 stems or roots per Petri dish. Dishes were incubated at 22 to 24°C for 5 to 14 days. Fusarium oxysporum was identified by observing morphological characteristics similar to those of Synder and Hansen (1940). White mycelium grew rapidly, covering the plate in 5 days. Phialides were short, singular growing laterally on the mycelium. Macroconidia were fusiform with a pointed apical cell and pedicel basal cell, typically 3 to 4 septa. Macroconidia were 24.9 to 50.0 (av. 38.6) µm long and 2.2 to 3.9 (av. 2.9) µm wide when 100 were measured. Microconidia were abundant and single celled and 4.0 to 8.0 (av. 5.8) µm long and 2.2 to 4.7 (av. 3.0) µm wide when 100 were measured. DNA sequencing was used to confirm morphological identity. DNA was isolated using the DNeasy Plant Mini Kit (Qiagen, U.S.A.). Two representative isolates were identified using partial sequences of translational elongation factor 1-α (primers EF-1 and Ef-2). Sequencing was conducted by Eurofins MWG Operon (U.S.A.). Sequence results MN481427 to MN481432 were cross-referenced with the Fusarium-ID database and showed 100% identity with F. oxysporum species complex. Koch’s postulates were performed using the cultures to test for pathogenicity on three commercially available cultivars (Empire, Norcen, and Pardee). All three demonstrated similar susceptibility results. The two F. oxysporum cultures were increased by placing 3-mm-diameter mycelial plugs from APDA plates into flasks containing 200 ml of potato dextrose broth. After 5 days of growth, the mycelium was blended into a slurry. Cultivars were planted in 50% potting mix/50% sand with F. oxysporum and without as the control. The 0.4% v/v F. oxysporum slurry (20 ml) was pipetted onto the soil mixture (500 cm³) before planting. Two complete experiments were conducted in a randomized complete block design with five replications per cultivar for a total of 60 experimental units (each pot containing 1 to 10 plants). Data were analyzed using SAS 9.4 PROC NPAR1WAY, and groups were compared using a Wilcoxon rank sum test. Reduced plant dried biomass was observed over all varieties: 0.7 versus 2.7 g (P = 0.004) between the F. oxysporum-inoculated pots and untreated control pots at 29 days after planting. Stems and hypocotyls collected from symptomatic plants confirmed the presence of F. oxysporum by reisolation and morphological identification. Koch’s postulates confirmed F. oxysporum is a foliar blight and root rot disease of birdsfoot trefoil. Fusarium spp. have been previously identified to infect birdsfoot trefoil in Maryland, North Carolina, and Virginia (Drake 1958) and also in New York (Wunsch et al. 2009). Because the Fusarium species complex is endemic to Alabama soils (Palmateer et al. 2004), potential growers of birdsfoot trefoil for livestock grazing and hay production should consider soil borne-fungus management strategies to minimize potential economic losses due to lower plant biomass production.