Occurrence and identification of two Nothophoma species causing branch canker and leaf brown spot of walnut in China

In June 2020, branch canker and brown spot of leaves on walnut were observed in Qingyang, China. A total of five strains were isolated from symptomatic samples and verified for pathogenicity based on Koch's Postulates, which showed that strains Hz4-1, Hz5-2, and Hy3-1 were the causative agents, and preliminarily identified them as members of the genus Nothophoma based on morphological characteristics. Subsequently, multi-locus phylogenetic analysis of the partial sequences of internal transcribed spacer (ITS), nuclear large ribosomal subunit (LSU), partial beta-tubulin (TUB2), and RNA polymerase II second largest subunit (RPB2) showed that the three strains were clustered into two groups on the phylogenetic tree: strain Hz4-1 and Hz5-2 and Nothophoma quercina (CBS 6633.92) clustered into one branch with 84% support, while Hy3-1 and Nothophoma spiraeae (CFCC 53 929) clustered within a well-supported clade with a bootstrap value of 99%. As a result, combining morphological characteristics and multi-locus systematic analysis, the strains Hz4-1 and Hz5-2 were identified as N. quercina, and Hy3-1 was identified as N. spiraeae, which is the first report of N. spiraeae causing walnut disease in China.