First Report of Globisporangium spinosum Causing Root Rot of Panax notoginseng in China.

Abstract

Panax notoginseng (Burkill) F.H. Chen, known as ''sanqi'', is widely used for its medicinal and tonic effects in China, such as treatment of cardiovascular diseases and antioxidant effects. However, root rot disease is the most destructive disease that hampers industrial development (Mi et al. 2017). In August 2018 and 2019, samples displaying symptoms of root rot, such as unnormal, stunted growth and chlorotic leaves, were dug out with a shovel and put in clean sample-bags from four production bases in Wenshan (around 23.5° N; 104° E), Yunnan province, the biggest "sanqi" production area in China, with over 20 thousand ha. Among production bases, root rot incidence typically ranged from 10% to 20%, but in a few number of severe cases, exceeded 70%. Typical symptomatic root tubers were washed with tap water, surface-sterilized in 5% NaClO for 1 min followed by 70% alcohol for 30 s, and rinsed three times with sterile water. The diseased tissues were excised and placed on rye sucrose agar medium (RSA) supplemented with 25 mg/L rifampin and 50 mg/L kanamycin, then incubated at 25°C in the dark for five days. Colonies with different features grew from the diseased pieces and twenty oomycete-like colonies, (white, dense, aerial mycelia with rapid radiate growth) were chosen for further investigation. Microscopic observations showed terminal and intercalary hyphal swellings, which were either globose or limoniform, thin-walled, mostly smooth, and 13-28 µm wide (average 20.3 µm). Oogonia were globose or rarely fusiform, mostly intercalary, 11-19 µm wide (average 13 µm), provided with a varying number of blunt, digitate projections. Oospores were plerotic, rarely aplerotic, thin-walled, and 16-19 μm wide (average 18.6 μm). Antheridia usually originated at various distances from the oogonium and appeared- clavate and crook-necked, making apical contact with the oogonium. Based on typical morphological features described, the isolates were putatively identified as Pythium spinosum (van der Plaats-Niterink 1981). The internal transcribed spacer region (ITS) and cytochrome c oxidase subunit II (CoxII) of isolates SQ00803 and SQ00903-1 were sequenced using universal primers ITS1/ITS4 and FM58/FM66 (Villa et al. 2006). The nucleotide sequences were deposited to GenBank with the accession numbers MN369530, MN370548, MN561687 and MN561688, respectively. Blastn analysis of ITS sequences showed 99.58% similarity to those of Globisporangium spinosum isolates CBS275.67 and CBS276.67 (accession number AY598701 and HQ643792). CoxII sequences showed 99.82% to 100% similarity to G. spinosum isolates (accession number GU071755 and AF196616). For pathogenicity tests, nine two-year-old healthy sanqi plants grown in sterilized substrate in plastic pots under greenhouse conditions were inoculated with 5×104 CFU/ml zoospore suspension of G. spinosum by root-drenching method (Dixon et al. 1984). Three non-inoculated plants drenched with sterile water were used as controls. All plants were incubated in a growth chamber at 25°C with a 16/8-h photoperiod. After two weeks, inoculated sanqi plants showed discoloration and chlorosis of leaves with water-soaked root rot. Control plants were symptomless and healthy. Colonies resembling G. spinosum were re-isolated from infected root tissues and showed the same morphological features as G. spinosum, thus fulfilling Koch's postulates. G. spinosum originally isolated from seedlings of Anthirrhinum majus has been reported to cause root rot disease on many plant species worldwide (Lévesque and De Cock 2004). Recently, based on whole genome sequencing and phylogenomic analysis, P. spinosum was transferred to Globisporangium genus. Therefore, P. spinosum is now classified as G. spinosum (Hai et al. 2022). To our knowledge, this is the first report of G. spinosum causing root rot of P. notoginseng in China. This research will contribute to the development of integrated management strategies for P. notoginseng root rot.