First report of leaf spot disease caused by Fusarium oxysporum on Litsea cubeba in China.

Abstract

Litsea cubeba, an important industrial plant species that originated in China, produces fruit essential oil extensively applied in the chemical industry (Xiang et al. 2020). In July 2020, a large-scale outbreak of leaf spot disease on Litsea cubeba was first observed and then monitored over time in Yueyang (29°37'N; 113°13'E) and Changsha (28°06'N; 113°02'E), Hunan province, China. Symptoms of this disease consisted of round-shaped lesions that initially appeared as small light-brown spots. With the increase in number, these small spots coalesced into larger, dark-brown lesions leading to yellowing and abscission of the leaves. To identify the causal agent this disease, the pathogen was isolated with a tissue separation method (Gao et al. 2020). The infected leaf tissues surface-disinfected with 75% ethanol and 0.1% HgCl were aseptically cut into small pieces (11 cm) and then placed onto potato dextrose agar (PDA) medium with cephalothin (0.2 mg/ml) and incubated at 28°C for 3-5 days. The purified colonies on PDA exhibited fluffy white hyphae, secreted a dark red pigment that had been observed in previous studies (Xiao et al. 2015) and produced microconidia and macroconidia. The microconidia were single-celled, non-septate, ovoid, and ranged from 3.08 to 13.89 μm long and 2.17 to 3.62 μm wide (n=50). Macroconidia were three to five-septate, slightly curved, and ranged from 11.77 to 26.85 μm long and 3.31 to 4.50 μm wide (n=50). These morphological features suggested that theisolates were most likely Fusarium oxysporum (Savian et al. 2021). To further confirm the identity of this pathogen (designated as Fox-1), the TEF-1a gene (Genbank accession No. OM281065) and rDNA ITS region (Genbank accession No. OM250084) were cloned and then sequenced (Cui et al, 2021). Sequence alignments indicated that the ITS and TEF-1a sequences shared 99.8% (504/505) and 99.7% (665/667) similarities with that of F. oxysporum (Genbank accession No. MF667966, KT230848), respectively. Both of the morphological characteristics and molecular data were used to identify this pathogen as F. oxysporum Schltdl.: Fr. 1824. To further verify whether these isolates of F. oxysporum can cause leaf spot disease, Koch's postulates were tested (Gradmann 2014). The purified pathogens were inoculated on artificial wounds of detached Litsea cubeba leaves and the leaves on the field plants of Litsea cubeba, respectively. The wounds of leaves were inoculated with sterile distilled water as negative controls. The experiment was performed independently three times, each with three leaves and three inoculated wounds on each leaf. All pathogen-inoculated wounds developed dark brown or black lesions on detached leaves within 3 days and on leaves on plants within 9 days, whereas the controls showed no symptoms. Re-isolations from infected leaves confirmed that the re-isolated pathogens possessed identical morphological characteristics to those of the original pathogens. To our knowledge, this is the first report of leaf spot infection of Litsea cubeba caused by F. oxysporum in China. This disease severely delays plant development and significantly decreases the yield of essential oil of Litsea cubeba. Our results laid a foundation for the subsequent research into pathogenic mechanisms drug sensitivity tests, which will contribute to the prevention and cure of leaf spot disease of Litsea cubeba.