Ramie is an important natural fibre crop and a perennial herbaceous plant of the nettle family Urticaceae. In 2013, brown root rot disease was observed on ramie plants in Yuangjiang, Hunan Province, China. Symptoms included stunted plants, reduced number of ramets per plant, and brown to purple-black discolouration of the root. The pathogen was identified as Pythium vexans according to morphological features and sequence identity (99–100%) to the published data for internal transcribed spacer (ITS), 18S and 28S rDNA sequences. The pathogenicity was confirmed on healthy fibrous roots of seedlings and on detached storage roots under greenhouse conditions. To our knowledge, this is the first report of brown root rot disease caused by Pythium vexans on ramie.

Phytocystatins play multiple roles in plant growth, development and resistance to pests and other environmental stresses. A ramie (Boehmeria nivea L.) phytocystatin gene, designated as BnCPI, was isolated from a ramie cDNA library and its full-length cDNA was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA sequence (691 bp) consisted of a 303 bp open reading frame (ORF) encoding a protein of 100 amino acids with deduced molecular mass of 11.06 kDa and a theoretical isoelectric point (pI) of 6.0. The alignment of genome DNA (accession no. MF153097) and cDNA sequences of BnCPI showed that an intron (~104 bp) exists in the coding region. The BnCPI protein contains most of the highly conserved blocks including Gly⁵-Gly⁶ at the N-terminal, the reactive site motif QxVxG (Q⁴⁹V⁵⁰V⁵¹S⁵²G⁵³), the L⁷⁹-W⁸⁰ block and the [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N (L²²G²³R²⁴ F²⁵A²⁶V²⁷ D²⁸D²⁹H³⁰ N³¹) block that is common among plant cystatins. BLAST analysis indicated that BnCPI is similar to cystatins from Glycine max (77%), Glycine soja (76%), Hevea brasiliensis (75%) and Ricinus communis (75%). The BnCPI was subcloned into expression vector pSmart-I and then overexpressed in Escherichia coli BL21 (DE3) as a His-tagged recombinant protein. The purified reBnCPI has a molecular mass of 11.4 kDa determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Purified reBnCPI can efficiently inhibit the protease activity of papain and ficin toward BANA (Nα-benzoyl-L-arginine-2-naphthyamide), as well as the mycelium growth of some important plant pathogenic fungi. The data further contribute to our understanding of the molecular functions of BnCPI.