First Report of Black Root Rot Disease on Morinda Officinalis caused by Lasiodiplodia pseudotheobromae in China

Morinda officinalis is a wildly cultivated medicinal plant in Zhaoqing, Guangdong Province of China. The annual production of M. officinalis in Deqin county of Zhaoqing accounts for 90% of China’s total production. In 2018, a disease on M. officinalis caused significant economic losses by affecting medicinal plant quality. Symptomatic plants exhibited blackened necrotic discoloration of roots. A diseased plant was collected in June 2018 from Zhaoqing, Guangdong, surface sterilized in 75% ethanol for 1 min and then in 2% NaClO for 3 min, and rinsed three times in sterile distilled water; internal necrotic tissue was then transferred to potato dextrose agar (PDA) and incubated at 28°C for 3 days. The fungal colonies were initially white and then became green to dark green with sporulation. Some small black pellets were found on the edge of the colony cultured on PDA medium for 25 days. The morphology of black fungal pellets was examined by light microscopy. The conidia were ellipsoidal, initially hyaline, unicellular, becoming dark brown, and developing a thick wall and a central septum. Conidia measured 16.01 to 21.74 μm long and 8.83 to 11.43 μm wide (n = 60). The conidial morphology matched that of Lasiodiplodia, a member of the Botryosphaeriaceae family (Alves et al. 2008). Moreover, the internal transcribed spacer region of the ribosomal RNA was amplified by using primers ITS1 and ITS4 (White et al. 1990) and sequenced (GenBank accession no. MK090538). The 542-bp sequence was compared with the GenBank database using nucleotide BLAST, and the isolate sequence was 100% similar to the sequence of Lasiodiplodia pseudotheobromae (GenBank accession no. MH663527). The β-tubulin gene region of the ribosomal DNA was amplified by using primers T1 (O’Donnell and Cigelnik 1997) and CYLTUB1R (Crous et al. 2004) and sequenced (GenBank accession no. MK328528). The 459-bp sequence was compared with the GenBank database using nucleotide BLAST, and the isolate sequence was 100% similar to the sequence of L. pseudotheobromae (GenBank accession no. KY583260.1). On the basis of morphological characteristics and nucleotide homology, the isolate was identified as L. pseudotheobromae, a member of the Botryosphaeriaceae family. To satisfy Koch’s postulates, both roots and leaves of M. officinalis were inoculated by placing a mycelium plug from the growing margin of 7-day-old colonies upside down directly into a fresh wound. After 3 days, the root xylem of inoculated plants turned brown and gradually became dark, similar to symptoms observed in the field. The leaves also turned brown and gradually became dark brown after 4 days. The disease spots were round or nearly circular. No symptoms were observed on the control plants. The pathogen was reisolated from root lesions, and its identity was confirmed by morphological characteristics. To our knowledge, this is first report of L. pseudotheobromae causing black root rot of M. officinalis in China.