First Report of Puccinia engleriana Causing Leaf Rust of Tabernaemontana bufalina in Hainan, China

Abstract

Tabernaemontana bufalina Lour. is extensively cultivated as an ornamental plant in Hainan, Guangdong, and other regions of southern China. In January 2020, we observed a rust disease on T. bufalina leaves in Sanya (18.15。N and 109.30。E) Hainan, China, and the rust occurred all year-round. In the early stage of rust, yellow chlorotic spots appeared, and then uredinia on the abaxial leaf surface became visible. Uredinia (approximately 200-700 µm in diameter) were mostly yellowish-brown in color, solitary, and irregularly scattered. In the late stage of the disease, spots were connected into lesions, and eventually, the whole leaf became severely chlorotic. Urediniospores were light brown, subglobose, measured 25-30 µm × 20-25 µm. They had two pores and were echinulate with spines spaced 2-5 µm. The teliospores were naked, scattered, or aggregated on severely infected leaves. They were two-celled, measured 33-40 µm × 25-30 µm, elliptic, dark brown, and covered with tiny spines. The teliospores had a colorless pedicel at one end which was approximately 28-34 µm long and enlarged at the lower part. The morphological characteristics of the spores were consistent with the descriptions of Puccinia engleriana Henn. (Hennings 1905). In China, P. engleriana was first identified on the leaves of Tabernaemontana divaricata (L.) in Yunnan province, and recorded as new to China in 2012 (Zhuang 2012). Untill now, no leaf rust caused by P. engleriana has been reported in Hainan. Urediniospores were collected and DNA was extracted using a Quick-DNA extraction Kit (TIANGEN Biotech, Beijing, China). The nuclear large subunit (28S) region of the ribosomal DNA repeat was amplified with primers Rust28SF (Aime et al. 2018) and LR5 (Vilgalys and Hester 1990) following the protocol of Aime and McTaggart (2021). The length of the large subunit sequence was 1,010 bp. When searched the GenBank database, the sequence showed 97.07% homology to the large subunit ribosomal RNA gene (Sequence ID: MW147048.1) of P. engleriana, and 92.5% similarity with 18S ribosomal RNA gene (Sequence ID: KM249855.1) of P. hemerocallidis. This result was consistent with the morphological identification. As for the 3% difference in large subunit ribosomal RNA gene, it was speculated that it may be related to the differences of geographical distribution and host plants, as the reference P. engleriana was obtained from Tabernaemontana orientalis in Australia (Aime and McTaggart 2021). The large subunit sequence was submitted into the GenBank database, with accession No. MZ314895. T. bufalina cutting seedlings with 4 available leaves were used in the Koch's postulate test. These seedlings were planted in a greenhouse with a 14 h/10 h light/dark photoperiod at 28°C and 65% humidity. The urediniospores suspension (5107/ml in 0.05% Tween 20 solution) was sprayed on 6 healthy seedlings and other 6 seedlings were sprayed with 0.05% Tween 20 solution as a negative control. Two weeks after inoculation, leaf chlorosis and yellowish uredinia were observed on the inoculated seedlings, whereas the non-inoculated seedlings stayed healthy. To our knowledge, this is the first report of P. engleriana causing leaf rust on T. bufalina in Hainan province. This report will provide the reference for future investigation of T. bufalina leaf rust, and for further improvement on the knowledge of the geographical distribution of P. engleriana in China.