First Report of Wilt on Alfalfa in China Caused by Verticillium nigrescens.

Abstract

Alfalfa (Medicago sativa Linn.), widely grown throughout the world, is an important perennial forage crop. It is high in protein and digestible fiber and is an excellent source of several vitamins (A, D, E, and K) and minerals for beef cattle, horses, sheep, goats, and even humans (2). Wilt symptoms on alfalfa were observed during a disease survey in Yangling, Shaanxi, China in 2009. Symptoms included discoloration, shortened internode, and plant death. However, the vascular tissue of diseased alfalfa plants did not exhibit discoloration and typical "V" symptoms of Verticillium albo-atrum infection. Eleven fungal isolates were obtained from diseased alfalfa plants in Yangling by a tissue isolation method (1). Isolates were cultured on Czapek Dox Agar (CDA; pH 7.2) slants at 22 ± 1°C in darkness. Colonies on CDA plates were whitish and cream-white when viewed from the underside, later becoming dark gray due to the formation of gray or dark brown chlamydospores in single or in short chains. DNA was extracted from each isolate and the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA) was amplified and sequenced using primers ITS-1F and ITS4. The 11 isolates were divided into five groups based on their in vitro morphological characters. A single isolate from each of the five groups was chosen for ITS sequencing. All five isolates had the same ITS sequence (GenBank Accession No. AB551216). On the basis of the ITS sequence and morphology (4), these isolates were identified as V. nigrescens Pethyhr. (recently renamed as Gibellulopsis nigrescens). Five representative isolates were used to fulfill Koch's postulates. Alfalfa seeds (cv. Cossack) were surface sterilized with 75% ethanol for 5 min, allowed to dry, and planted into cow dung compost that had been autoclaved at 160°C for 2 h. Plants were cultivated under controlled greenhouse conditions at 23 to 25°C with a photoperiod of 14 h. Inoculum was prepared by comminuting 15-day-old cultures and sterile deionized water into a suspension of mycelial fragments and conidia (105 to 106 CFU/ml) in a blender. Seedlings (four-leaf stage) were inoculated by immersing roots in the inoculum suspension for 60 min (3). Each isolate was inoculated onto 30 seedlings, six in each pot; another 30 seedlings were soaked with sterile deionized water for 60 min as a control. After 20 days in the greenhouse, all inoculated plants exhibited wilt symptoms similar to the original wilt symptoms observed on diseased alfalfa plants. In contrast, none of the control plants showed wilt symptoms. The pathogen was reisolated from all diseased plants and confirmed to the original ones. To our knowledge, this is the first report of V. nigrescens infecting M. sativa in China, indicating V. nigrescens as one possible important pathogen of alfalfa.