First report of Fusarium solani causing wilt disease on Gleditsia sinensis in Tianjin, China.

The Gleditsia sinensis Lam. widely grown in China is a perennial plant with medicinal properties (Zhang et al. 2016). Since 2019, the leaves of G. sinensis have exhibited yellowing and wilting, and the plants have gradually become stunted and dead in Taifeng park of Binhai New Area in Tianjin (39.02° N; 117.65° E). In this park, there are two types of G. sinensis, one is with round branch thorns, the other is with flat branch thorns. The G. sinensis with round branch thorns did not grow well and almost all plants had disease symptoms. The samples were collected on October, 2021 and deposited in Plant Disease Laboratory of Tianjin Agricultural University under accession no. PATAU211018. The disease symptoms consisted of foliage wilt (Figure 1A), plant drying and vascular tissue discoloration (Figure 1B). The stem sections from different plants were surface-disinfested in 0.6% NaClO, wiped with 75% ethanol and rinsed with sterile water. Thirty tissue samples were placed on Potato Dextrose Agar (PDA) medium and cultured at 28℃ for 7 days (Uppala et al. 2013). Thirty fungal isolates with the same morphological characteristics were obtained from the samples. Five representative isolates (PATAU211018-05, PATAU211018-07, PATAU211018-10, PATAU211018-12 and PATAU211018-21) were collected and purified using the single-spore method (Li et al. 2022). Colonies of the five isolates on PDA grew in a circular shape and showed abundant white densely fluffy aerial mycelium (Figure 1C). Morphological characteristics included septate and hyaline hyphae, long cylindrical monophialides (Figure 1D), macroconidia (Figure 1E) and microconidia (Figure 1F). Macroconidia were falcate, 2-5-septate, hyaline, 18-40 × 4-6 μm (n = 50). Microconidia were hyaline, oblong, 0-1-septate, 5-14 × 2-6 μm (n = 50). These morphological characteristics were consistent with the description of Fusarium solani. (Chitrampalam et al. 2018). PATAU211018-12 was randomly chosen for molecular analysis as the representative isolate given the similarity of these isolates. For further identification, the genomic DNA of isolate PATAU211018-12 was extracted. The fragments of internal transcribed spacer (ITS), translation elongation factor 1α (EF1α) gene and RNA polymerase II subunit (RPB2) were amplified and sequenced (O'Donnell et al. 2008; Carbone et al. 1999). The sequences of ITS, EF1α, and RPB2 of PATAU211018-12 were deposited in GenBank under the accession no. of OP735578, ON630412 and OP746032, respectively. Phylogenetic trees were constructed in MAGA X (Kumar et al. 2018) using the neighbor-joining (NJ) method based on the concatenated sequences of ITS, EF1α, and RPB2 (Figure 2). The isolate (PATAU211018-12) grouped with F. solani (JS-169) with a bootstrap value of 100 in the phylogenetic tree. The morphology and multi-gene phylogenetic analysis indicated that the new isolate is F. solani. Pathogenicity tests were carried out on one-year-old G. sinensis seedlings with round branch thorns (n=6). The F. solani isolate PATAU211018-12 was cultured in Potato Dextrose Broth (PDB) at 28°C on a shaker at 150 rpm for 5 days. Mycelia were filtered through four layers of sterilized lens paper and the conidia were obtained for pathogenicity tests. G. sinensis was infected by F. solani through root soaking method. The roots were inoculated by dipping in conidial suspension with the concentration of 107 conidia/mL for 30 minutes. Control plants (n=6) were treated with distilled water. Plants were in pots indoors at 25℃. At 20 days after inoculation, the leaves of inoculated plants were chlorotic and wilted (Figure 1G), symptoms similar to those observed in the park. In contrast, the leaves of control plants were symptomless (Figure 1H). The pathogenicity assay was repeated three times. The fungal isolate was re-isolated from the disease tissues and verified as F. solani based on morphology and molecular character (ITS, EF1α and RPB2). F. solani has been reported as pathogens on many plants, such as Eriobotrya japonica (Wu et al. 2021), Fragaria × ananassa (Pastrana et al. 2014), Gastrodia elata (Li et al. 2022) and Hedysarum boreale (Uppala et al. 2013). To our knowledge, this is the first report of F. solani causing disease on G. sinensis in China. Identification of F. solani as a disease agent in G. sinensis will assist in disease management for this important tree crop.