First report of Fusarium oxysporum causing wilt on Perilla frutescens in China.

Abstract

Perilla frutescens, commonly known as "ZI SU", is an herbaceous plant known for its culinary and medicinal properties and is distributed throughout China (Igarashi and Miyazaki, 2013). In October 2023, wilt symptoms were observed on P. frutescens plants in Shaowu (27.34°N, 117.49°E), Fujian province, China (incidence rate: 30%). The stems first showed discoloration, which transitioned from green to dark brown or black. The leaves drooped and often curled, eventually drying out. Fifteen typical symptomatic stems were randomly selected and samples were taken from the margins of the lesions and cut into 5 × 5-mm pieces. The samples were surface sterilized with 5% sodium hypochlorite solution for 1 min, rinsed with sterile water three times, and dried. Samples were incubated on potato dextrose agar (PDA) medium for 5 d at 25°C in the dark, then transferred using monospore isolation to new PDA plates and incubated for another 5 d at 25°C in the dark. Thirteen of the 20 isolates obtained had similar colony morphology (isolation rate 65%). The front of colonies exhibited a flat, cottony texture. The reverse side of the colonies was pale yellow. Two representative isolates, 1380 and 1383, were cultivated on carnation leaf agar (CLA) medium. Microconidia of the isolates were oval, cylindrical or ovate, and were 5.56 to 15.26 × 2.82 to 4.49 μm (n = 50). Macroconidia had 2 to 5 septa, usually 3 septa, and were straight or slightly curved, 2.6 to 5.47 × 20.8 to 42.4 μm (n = 50). Chlamydospores were roundish, terminal or intercalary. The morphological characteristics were consistent with the description of Fusarium (Leslie and Summerell 2006). DNA was extracted from the two isolates using the cetyltrimethylammonium bromide (CTAB) method. The partial internal transcribed spacer (ITS) region, the translation elongation factor (TEF-1α) and partial RNA polymerase second largest subunit (RPB2), were amplified (GenBank accession numbers for TEF-1α were PV057161 and PV057162, for RBP2 were PV057159 and PV057160, and for ITS were PV639174 and PV639175 for 1380 and 1383, respectively) (Crous et al 2009). When compared with other Fusarium species in GenBank, both isolates exhibited 99.85% (TEF-1α, MN417200), 99.90% (RBP2, AB986568), and 100% (ITS, OR753367) similarity with Fusarium oxysporum. A phylogenetic tree which combined ITS, TEF-1α and RPB2 was constructed using MEGA6 with the maximum likelihood method (Xu et al 2022), and the two isolates clustered with F. oxysporum with 99% bootstrap values. To test pathogenicity, a conidial suspension of 1383 with a concentration of 1 × 106 conidia/mL was sprayed on three stems of one-month-old P. frutescens seedlings. For the control, sterile water was sprayed on two control stems and there were 3 biological replicates. Seedlings were covered with plastic film for 24 h and kept in a culture room at 25°C, and inoculated stems exhibited similar symptoms to those in the field 4 days' post inoculation, while control stems had no symptoms. Koch's postulates were fulfilled by reisolating the same isolate from diseased stems based on morphological characteristics. The morphology and molecular biology results confirm that the isolates were F. oxysporum. This is the first report of F. oxysporum causing wilt on P. frutescens. China is the main cultivation and export country of P. frutescens, and this disease poses a threat and damage to production. Since F. oxysporum has a wide host range, strategies to manage it should be developed.