First Report of Brown Apical Necrosis of Walnut Fruit Caused by Fusarium avenaceum in Hubei, China

Abstract

Brown apical necrosis (BAN) is a serious disease complex of walnut (Juglans regia L.) in France, Italy, Spain, and Turkey (Moragrega and Ozaktan 2010). Since 2014, BAN has been observed in 50 to 70% of trees in over 40,000 ha of walnut orchards in Baokang County and other counties of Hubei Province, China. In the early fruit development stages, external apical necrosis was observed exclusively at the fruit stigmatic or blossom end with small lesions and brown color. The lesion enlarged up to the fruit surface, invaded and decayed the inner tissues, and premature fruit drop occurred for many trees. Sections of the fruit revealed that inner tissues contained a brown to black rot. A total of 165 symptomatic fruits (cultivar Qingxiang) were collected periodically from Baokang County from May 2016 to 2018, and 165 fungal isolates and 104 bacterial isolates were obtained from infected fruits. Among the bacterial isolates, Pantoea agglomerans was the dominant species and accounted for 45.2% (2-year average), in agreement with Yang et al. (2011). Among the fungal isolates, Fusarium avenaceum (Fr.) Sacc. was the dominant species and accounted for 28.7% (3-year avg.), and Alternaria spp. for 25.5% (3-year avg.) and Colletotrichum spp. for 24.1% (3-year avg.). The frequency of F. avenaceum isolation between diseased and healthy areas was higher than that from rotted areas, but the opposite was observed for Alternaria spp. and Colletotrichum spp. Two Fusarium isolates, JRBK-5 and JRBK-10, were characterized and identified as F. avenaceum (Leslie and Summerell 2006).The translation elongation factor 1α (TEF-1α) gene and partial β-tubulin (TUB2) gene of isolate JRBK-5 were respectively amplified and sequenced (Zhu et al. 2014). The TEF-1α sequence (GenBank accession no. KY475585) showed 99.8% identity with F. avenaceum (KP170732) and the TUB2 sequence (KY475586) showed 100% identity with F. avenaceum (EU357852). Phylogenetic analyses showed that JRBK-5 was in the same clade as other isolates of F. avenaceum with high bootstrap support over 93%. To confirm pathogenicity, F. avenaceum JRBK-5 and JRBK-10, Alternaria spp., Colletotrichum spp., and P. agglomerans (10⁸ CFU ml⁻¹) were used to inoculate the stylar end of the detached premature fruits using 5-mm-diameter mycelial plugs with a non-wound inoculation method. Controls were treated with water and a PDA plug without the fungi. All fruits were incubated in a growth chamber at 25°C and 80 to 90% RH. Six days after inoculation, brown necrotic spots appeared on the fruits inoculated with all individual species. F. avenaceum showed higher virulence than the other fungi. The lesion diameter of F. avenaceum was 25.0 ± 2.0 mm, and those of the other fungi were <9.3 ± 1.4 mm. Combinations of F. avenaceum with the other fungi resulted in brown necrotic spots and virulence was the same as F. avenaceum alone. The inner tissues of inoculated fruits turned dark brown to black with decay. However, no symptoms developed on the control fruits. Reisolated F. avenaceum had the same cultural characteristics as the originally isolated F. avenaceum. The pathogenicity tests were repeated with similar results. Based on morphological characteristics, sequence analysis of the TEF-1α and TUB2, and Koch’s postulates, the isolates JRBK-5 and JRBK-10 were identified as F. avenaceum. To our knowledge, this is the first report of F. avenaceum causing BAN disease of walnut in Hubei, China.