First Report of Leaf Spot Disease on Pineapple Caused by Fusarium solani in China

Pineapple (Ananas comosus) is an economically important tropical fruit in Guangdong Province, China. The plants were seriously infected with a year-round leaf spot disease. During September to November 2022, the leaf spot disease of pineapple was found in Xuwen city and Zhanjiang city of Guangdong Province. A disease survey of 1 ha revealed that pineapple was affected at an incidence ranging from 30% to 50%. The disease caused economic loss to control plant diseases with chemicals. The initial symptoms were observed after 1 month of planting in October and included yellow spot and developed brown necrotic lesions. The leaves of pineapples showed symptoms of Large brown necrotic lesions appear on leaves especially on the tip of basal leaves. The diseased leaves were collected and surface-disinfested in 1% NaClO for 2-3 m, rinsed with sterilized water, air dried, placed on potato dextrose agar (PDA) medium, and incubated for 3 to 5 days at 28°C. Two isolates (PLF1 and PLF2) were collected and purified using the single-spore method. Colonies developing on PDA were in a circle with abundant white, densely fluffy aerial mycelium, pale to colorless after 3 d and pale orange 5-15 d later. Cultures of the isolates produced macroconidia which were falcate, 1-3-septate, hyaline, 18.2-43.4×4.8-6.8 µm(n=50). Cultures of the isolate also produced large amount of conidia which were hyaline, oblong, no septate, 5.2-10.6×2.7-5.2 µm (n=50). These characteristics were consistent with the description of Fusarium sp. (Chitrampalam et al. 2018). For molecular identification, the genomic DNA of the 2 isolates was extracted. The fragments of internal transcribed spacer (ITS), translation elongation factor 1α (EF1α) and β-tubulin were amplified and sequened using the primer pairs of ITS4/ITS5, EF1/EF2 (O'Donnell et al. 2008) and T1/T2 (O'Donnell et al. 1997). These sequences were deposited in GenBank (OR501466, OR501467 for the ITS; OR499874, OR499875 for elongation factor; OR499876, OR499877 for β-tubulin). Phylogenetic trees were constructed in MAGA 11 using the Maximum likelihood (ML) method based on the concatenated sequences of ITS, EF1α, and Tublin (Figure 1). The 2 isolates were grouped with F. solani A01-1 (GCA_027945525.1) with a bootstrap value of 100 in the phylogenetic tree. The morphology and multi-gene phylogenetic analysis indicated that the new isolates are F. solani. The 2 isolates were selected for pathogenicity tests to fulfill Koch's postulates. Six plants at seven- to ten-leaf stage were inoculated with each isolate separately. Three sites of each leaf were wounded with a sterile needle and covered with a piece of cotton drenched with 200 µl spore suspension (107 spores/ml) from each isolate cultured in PD medium. Leaves inoculated with PD medium served as negative controls. Inoculated plants were placed in an incubator at 28°C, and 80% humidity under a 12-h light/dark cycle for 7 days. After 7 days of incubation, necrotic spots were observed in all the inoculated plants except the negative control. The pathogenicity tests were conducted three times with similar results. The strains were then reisolated from the lesions and found to be Fusarium solani as those of the inoculum. F．ananatum， F. guttiforme and F. subglutinans have been reported to infect all parts of pineapplefusariosis disease (Jacobs et al. 2010; Stępień et al. 2013; Ventura et al. 1993). To our knowledge, this is the first report of fusariosis on Pineapple caused by F. solani. Identification of F. solani as a disease agent on pineapple will assist in disease management for this important fruit tree.