First Report of Verticillium Wilt of Goji ( <i>Lycium barbarum</i> ) Caused by <i>Verticillium dahliae</i> and <i>V. nonalfalfae</i> in China

Goji (Lycium barbarum) is a highly valued traditional medicinal herb and functional food. As the world's dominant producer and supplier, China yielded over 420,000 tons in recent years (Wang et al. 2024). In July 2023, symptoms including leaf yellowing, defoliation, vascular bundle browning, and plant death were observed in goji plants (approximately 20, 000 m2) at Nuomuhong farm in Qinghai province (36°25'39.6"N, 96°16'42.8"E), with an incidence of about 67%. To identify the causal agent, infected tissue pieces (5 × 5 mm) from five plants were surface sterilized with 75% ethanol for 5 s, followed by 3% NaClO for 30 s, and rinsed three times with sterile water. The tissues were then plated on PDA and incubated at 25°C in darkness. After single hyphal tip purification, twelve isolates were obtained. All colonies on PDA were initially white, later darkened in the center, and produced verticillate conidiophores with hyaline, ellipsoidal conidia (5.24 ± 0.79 × 2.38 ± 0.26 µm, n = 50). These morphological characteristics matched Verticillium spp. (Inderbitzin et al. 2011). For molecular identification, we compared DNA sequence data from five gene regions: the nuclear rDNA internal transcribed spacer region (ITS), actin (ACT), translation elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS) (Inderbitzin et al. 2011). All isolates represented two distinct haplotypes (GenBank accession numbers, haplotype Ⅰ: PQ663849, PQ683745, PQ683757, PQ683749, PQ683753; haplotype Ⅱ: PQ663851, PQ683747, PQ683759, PQ683751, PQ683755), with each haplotype derived from a different infected plant. A maximum likelihood phylogenetic tree was constructed based on the combined dataset of ITS, ACT, EF, GPD and TS sequences from four representative isolates (haplotype I: H165 and H167; haplotype II: H197 and H227). The tree showed that: isolates H165 and H167 grouped in the same clade with the ex-type isolate of V. dahliae (100% bootstrap value), and isolates H197 and H227 clustered within the clade representing V. nonalfalfae (100% bootstrap value). For pathogenicity testing, ten healthy 6-month-old goji cutting seedlings per isolate (V. dahliae: H167; V. nonalfalfae: H227) were inoculated by soil drenching around the root zone with 50 ml of a conidial suspension (1 × 107 conidia/ml), using sterile water as the negative control. All treatments were maintained in a greenhouse (25 ± 2°C, relative humidity ≥ 75%, 12-h photoperiod). At 10-15 days post-inoculation (dpi), all inoculated plants exhibited similar symptoms, including leaf wilting, desiccation, and vascular discoloration in the stems. Disease progression was more rapid in plants inoculated with V. nonalfalfae (H227), resulting in complete plant mortality at around 20 dpi, compared with approximately 30 dpi for V. dahliae (H167). Both pathogens caused 100% final disease incidence and mortality, whereas all control plants remained asymptomatic. Pathogenicity tests were repeated three times. In all trials, the pathogen was successfully re-isolated from symptomatic tissues and confirmed based on morphology, thereby fulfilling Koch's postulates. Verticillium dahliae has previously been reported causing Verticillium wilt on goji in Turkey (Özer and Bayraktar 2016) and Italy (Ruano-Rosa et al. 2017). To our knowledge, this is the first report of V. dahliae infecting goji in China, and the first global report of V. nonalfalfae as a pathogen on goji.