First Report of Sea buckthorn Stem Wilt Caused by Fusarium proliferatum in Liaoning, China

Sea buckthorn（Hippophae rhamnoides L.） is a flowering shrub native to cold-temperate regions of Eurasia, which is also valuable for its berries and leaves containing various vitamins and flavonoids (Pundir et al. 2021). In late June 2020, high mortality (more than 70%) was observed in sea buckthorn in a 1.6-ha seedling nursery in Chaoyang City, Liaoning province, China, where 16 Chinese and Russian cultivars (cv.) had been planted since 2014 (cv. Shenqiuhong, eshi01 through eshi15). The mortality of two introduced sea buckthorn varieties (eshi02, eshi04) was 100% (125 trees died in total). The symptoms include massive drooping leaves and dried-up stems on 6-year-old infected trees. Pieces of tree roots and stems with brown discoloration in the xylem vessels were selected. Small tissue fragments (0.2-0.5 cm) were surface disinfested (3 min in 75% ethanol, rinsed with sterile distilled water), air-dried, and placed on potato dextrose agar (PDA) medium for 5 days at 25°C in the dark. A fungus was consistently isolated from both diseased roots and stems tissues, and a representative isolate (LC-1) was harvested. Genomic DNA was extracted for amplification and sequencing of the partial translation elongation factor-1α (EF1 and EF2 primers, accession Nos. MZ669853) (O'Donnell et al. 1998) and RNA polymerase II second largest subunit (RPB2) (7cf/11aR primers, accession Nos. MZ669854) (O'Donnell et al. 2007). The sequences were further analyzed at the Fusarium MLST (https://fusarium.mycobank.org/) for identity confirmation, and showed 99.8% (over 95.2% query coverage) and 96.4% (over 88.4% query coverage) similarity to Fusarium proliferatum (NRRL 13584, 13591). Isolates on Spezieller Nahrstoffarmer agar (SNA) produced abundant aerial white mycelia and yellow pigmentation. The 30 macroconidia measured ranged from 28.5 - 62.5 × 3.2 - 5.4 μm, were thin, slender, with 3-5 septa. The aseptate microconidia ranged from 4.7 - 13.6 × 2.2 - 4.3 μm (n = 30). Pathogenicity tests were performed on healthy, potted 1-year-old sea buckthorn seedlings (cv. eshi05) using two isolates in a greenhouse at 25 °C, 80% relative humidity, and 12-hour light/dark photoperiod. Ten potted seedlings were inoculated on the stems by placing a 5-mm-diameter mycelial plug (5-day-old PDA cultures for each isolate) into the surface of a wound created with a needle, and the inoculation sites were covered with Parafilm to maintain moisture. Ten seedlings were inoculated with PDA plugs as controls. Six to ten days after inoculation, color of the leaves in the middle of the stems was variegated, and then dark necrotic lesions on leaf margins were observed. Three weeks after inoculation, 80% of inoculated stems were wilted, while control plants remained asymptomatic. The pathogen was consistently re-isolated and the recovered isolates were identified as F. proliferatum by amplifying the EF-1α gene. The typical symptoms on inoculated plants were dark to brown necrotic lesions on chlorotic leaves initially, and black withered stems in the terminal stage, similar to those observed on sea buckthorn trees infected with Fusarium sporotrichioides in Gansu and Heilongjiang provinces (Song et al. 2010; Xia et al. 2021). To our knowledge, this is the first report of sea buckthorn stem wilt caused by F. proliferatum in Liaoning province, China, which will be beneficial for expanding knowledge of Fusarium disease in sea buckthorn and provide more information for sustainable disease management in sea buckthorn.