The first report of Phytophthora multivesiculata causing black rot of Cymbidium and Ansellia africana from South Africa.

Abstract

Globally, various species and hybrids of Cymbidium are of horticultural importance. In January 2022, we visited a private orchidarium near Pretoria (25° 54' 30" S, 28° 24' 34" E). During this visit, the owner reported mortality among various pure and hybrid Cymbidium and Ansellia africana, an indigenous South African ornamental orchid. Phytophthora was identified as a possible causative agent based on an initial examination of the affected orchids. The infected orchids exhibited vascular wilting. Brown, water-soaked lesions covered the roots. The pseudobulbs showed black rot symptoms. Necrotic lesions were also visible towards the basal part of the leaves. For isolation of the causal agent, pieces of infected tissues from roots, pseudobulbs and leaves were surface sterilised using 70% ethanol and plated into Phytophthora selective medium, NARPH. All the plates were incubated at 21°C in darkness. After three days, Phytophthora-like mycelia emerged from all three tissue types. These colonies were transferred onto the PDA medium. For molecular identification, genomic DNA was extracted from four representative isolates (CMW58027-30) using a Zymo Research Fungal/Bacterial DNA MiniPrep kit. The complete ITS, and partial beta-tubulin (BT) and cytochrome oxidase 1 (COX1) gene regions were amplified using the primers DC6/ITS4 (Cooke et al. 2000; White et al. 1990), Btub_F1A/Btub_R1 (Blair et al. 2008; Kroon et al. 2004) and FM84/FM83 (Martin and Tooley 2003), respectively. BLAST searches in NCBI showed that the four isolates were from Phytophthora ITS Clades 2. ITS, BT, and COX1 datasets from Bose et al. (2021) were used for the phylogenetic identification of our isolates. Single gene and concatenated datasets were analysed using both maximum likelihood and Bayesian approaches, which confirmed the identity of the isolates as Phytophthora multivesiculata. All the sequences were submitted to the GenBank: ITS (OM967212-15), BT (OM966588-91), and COX1 (OM966592-95). Measurements of sporangia and gametangia overlapped with those from Ilieva et al. (1998): sporangia (28.3 - 56.3) 41.6 x 31.3 (21.5 -3 9.6) µm; L:B 1.42 (1.08 - 1.69); exit pore 11.2 (7.1 - 14.6) µm; oogonia 44.2 (24.5 - 56.3) µm; oospore 34.2 (21.6 - 53.2) µm; antheridia (5.8 - 14.6) 11.8 x 15.3 (6.2-14.9) µm. The pathogenicity trial was conducted following the protocol suggested by Ilieva et al. (1998). Five A. africana roots and the cut ends of seven Cymbidium leaves were immersed in separate beakers containing 100 ml of sterile distilled water and ten 5 mm agar discs excised from a 7-day-old culture of P. multivesiculata (CMW58027) grown on PDA. Sterile distilled water was used as the control, with an equal number of plants and leaves. All of the sets were incubated at 21°C. After seven days, the plants and leaves developed lesions similar to those observed on the symptomatic plants at the orchidarium. Trials were repeated once and the pathogen was reisolated from both trials and the identity was confirmed by amplifying the complete ITS gene region. Phytophthora multivesiculata has been previously reported from the Netherlands (Ilieva et al. 1998), Taiwan (Chern et al. 2011), Australia (Cunnington et al. 2009), New Zealand (Hill 2004) and elsewhere causing black rot of Cymbidium. However, this is the first report of P. multivesiculata causing black rot of Cymbidium and A. africana from Africa. We are now conducting follow-up surveys to determine the distribution range of this pathogen in South Africa.