Occurrence of Fusarium fujikuroi Causing Fusarium Wilt on Carthusian Pink in China.

Abstract

Carthusian pink (Dianthus carthusianorum) is native to Europe and is widely grown in China for landscaping. In September 2022, wilting symptoms of carthusian pink were found in Xixia City (33°18'31″ N, 111°29'45″ E), Henan Province, China, with a disease incidence of 65%. Approximately 100 plants were surveyed on the landscaping lawns of the park. Initial symptoms were yellow to brown lesions on the base of stems and leaves. Later, the lesions spread throughout the plants, turning leaves yellow, and leading to root and leaf rot. Eventually, the plants shriveled and died (Figure S1a). Thirty diseased tissues isolated from the roots and leaves were cut into 5×5 mm pieces, which were surface sterilized with 75% ethanol solution for 30 seconds and 1% NaClO solution for 1 minute, rinsed three times in sterilized water, placed on potato dextrose agar (PDA) plates supplemented with 50 μg ml-1 streptomycin, and incubated at 28°C for five days. A total of 25 purified fungal strains with similar phenotypic features were obtained. Three representative strains named OSZ-P1, OSZ-P2, and OSZ-P3 were selected for identification. Fungal colonies developed an abundant aerial mycelium, initially white, which subsequently developed red to purple pigments (Figure S1b). Macroconidia were slender, straight, and measured 12.74 to 49.39 × 2.07 to 4.39 μm (n=50), with two to five septa. Microconidia were clavate and measured 6.31 to 11.61 × 2.15 to 4.02 μm (n=50) (Figure S1c). These morphological characteristics were consistent with Fusarium spp.. The rDNA internal transcribed spacer (ITS), β-tubulin gene (tub2), translation elongation factor 1-alpha gene (tef1), calmodulin (cmdA), RNA polymerase largest subunit (rpb1), and RNA polymerase II second largest subunit (rpb2) were amplified with primers ITS1/ITS4, BT-2a/BT-2b, EF1/EF2, CL1/CL2A, Fa/G2R, and 5F2/7Cr, respectively, for further identification (Yilmaz et al. 2021, O'Donnell et al. 2022). ITS (OQ726389, OQ726390, OQ726391), tub2 (OQ730191, OQ789645, OQ789646), tef1 (OR088904, OR088905, OR088906), cmdA (OR133730, OR133731, OR133732), rpb1 (OR088907, OR088908, OR133729), and rpb2 (OR133733, OR133734, OR133735) nucleotide sequences of the strains OSZ-P1, OSZ-P2, and OSZ-P3 were submitted to GenBank. BLASTn analysis of OSZ-P1 sequences exhibited 99 to 100% similarity with Fusarium fujikuroi sequences (strains Augusto2, I1.3, and CSV1) CP023096, CP023108, CP023084 of cmdA, CP023089, CP023077 of rpb1, and CP023093, CP023105, CP023081 of rpb2. A Phylogenetic tree was constructed of combined genes (tub2, tef1, cmdA, rpb1, rpb2) of sequences, alongside the sequences of the type strains by the neighbor-joining method. The three strains formed a clade with the type strains CBS257.52 and Augusto2 of F. fujikuroi in phylogenetic trees, being clearly separated from other Fusarium spp. (Figure S2). The morphological features and molecular analyses supported the strains as members of F. fujikuroi. To verify the pathogenicity, aboveground parts of the plants of five healthy six-month-old potted plants were sprayed with 100 µl of conidial suspension per pot (106 conidia ml-1), and five similar plants were sprayed with sterilized water as a control. All plants were placed in a climate incubator at 28°C and 90% relative humidity. Seven days after inoculation, withered and yellowed lesions were observed, similar to the natural lesions (Figure S1e). No symptoms were observed on the control plants. The whole pathogenicity tests were performed thrice. Reisolation resulted in cultures that were morphologically and molecularly identical to the original isolates, fulfilling Koch's postulates. Fusarium wilt disease has been reported on other plants of the genus Dianthus. Vascular wilt on Dianthus caryophyllus (carnation) caused by Fusarium oxysporum is the most destructive disease of carnation crops worldwide (Ardila et al. 2014). Fusarium acuminatum causing Dianthus chinensis root rot and foliage blight has recently been reported in Nanjing, China (Xu et al. 2022). To our knowledge, this is the first report of F. fujikuroi causing Fusarium wilt on carthusian pink worldwide. The host range of F. fujikuroi still needs to be clarified for accurate disease management in the selection of plant species for landscape.