First Report of Aphanomyces euteiches Causing Aphanomyces Root Rot on Lentil in North Dakota

Aphanomyces euteiches (Drechs) is the causal agent of Aphanomyces root rot (ARR) on field pea (Pisum sativum L.), alfalfa (Medicago sativa), and lentil (Lens culinaris Medik.). ARR can cause severe yield loss in lentil. This disease is widespread in the Canadian prairies and was first reported in the U.S. Pacific Northwest in 2010 (Chatterton et al. 2019; Vandemark and Porter 2010). North Dakota is responsible for much of U.S. lentil production with >74,000 ha planted, primarily in the northwest corner of the state (USDA-NASS 2018). In 2018, lentil plants from a commercial field in North Dakota displayed symptoms consistent with ARR. Under microscopic examination, roots contained oospores consistent with A. euteiches. To confirm the pathogen, five cuttings were taken from symptomatic roots, placed onto metalaxyl-benomyl-vancomycin selective media for A. euteiches using the under the block technique, and incubated at 22 ± 2°C for 48 h (Pfender et al. 1984; Schmitthenner and Bhat 1994). Coenocytic mycelium, characteristic of A. euteiches, was transferred to PDA using hyphal-tip micromanipulation. The putative A. euteiches isolate was grown for 5 days on PDA at room temperature under ambient light. The isolate had coenocytic mycelia 5 to 13 µm in diameter (n = 12, average = 9.5 µm) and free-flowing cytoplasm (Scott 1961). The antheridia were monoclinous and diclinous, and they formed one to two antheridial attachments to the oogonia. Oogonia formed terminally, were 20 to 31 µm in diameter (n = 12, average = 24 µm), and were aplerotic. Oogonia developed characteristic smooth, thick walls. Four 5-mm-diameter PDA cores were excised from 5-day-old cultures and aseptically transferred to a sterile 250-ml Erlenmeyer flask containing eight corn kernels and 60 ml of deionized water. Flasks were incubated at 22 ± 2°C for 9 days and washed twice with sterilized tap water with a 3-hour resting period between washings. Sporangia formed 70 to 100 grape-like clusters of smooth primary zoospores (average diameter = 8 µm) at the terminal end of the hyphal stalk that yielded secondary biflagellate zoospores. Morphological identification was confirmed by sequencing using the universal primers ITS1 and ITS4 (White et al. 1990). BLASTn analysis indicated that the 620-bp sequence of isolate LenND18 was 99.52% identical to A. euteiches (GenBank KY593270.1). The sequence was deposited into GenBank as MN175977. To confirm pathogenicity of the isolate, lentils were inoculated and grown on a light shelf under laboratory conditions. Three sterile 640-ml pots were filled two-thirds full with pasteurized potting mix (Miracle Grow). Corn meal agar amended with streptomycin and neomycin from a single 90-mm Petri dish containing a 14-day-old A. euteiches culture was cut into 4-mm² sections and placed on top of the potting mix. Five surface-sterilized Richlea lentil seeds were placed on top of the cut agar pieces containing A. euteiches oospores in each pot and covered with potting mix. Seeds planted into three pots containing only pasteurized potting mix served as negative controls. Plants were grown at room temperature with a 16-h photoperiod. After 14 days, roots were washed and examined for symptoms. This experiment was repeated. Inoculated seedlings displayed symptoms characteristic of ARR. Golden-brown lesions were observed on the tap root, and symptomatic roots were soft with the outer portion (cortical tissue) easily stripped away. Control plants were asymptomatic. A. euteiches was reisolated from symptomatic plants, as confirmed by culture morphology and identification of oospores and mycelia using microscopy. This is the first report of A. euteiches on lentil in North Dakota. The recommended rotation for lentil is 3 to 4 years, but in the presence of long-lived oospores produced by A. euteiches, rotations may need to be lengthened to 6 to 8 years (Wu et al. 2018). Growers will need to monitor fields closely for symptoms and adopt longer rotations if ARR is observed.