Pythium ultimum Causing Black Stem Rot of Brassica oleracea var. alboglabra in China

Abstract

Brassica oleracea var. alboglabra, locally known as gailan, is an important leafy vegetable crop of China, widely cultivated in southern provinces. During 2016 to 2018, stem rot disease was seen in some agricultural fields around Guangzhou city of Guangdong province, China. Plants were affected by the disease at all growth stages. Black necrotic lesions developed and extended upward from the base of the stem on diseased plants. Symptoms first appeared as black lesions covering the stem surface. Under severe infestation, the infected stems got cracked and the whole plant collapsed. Mycelial masses were seen on the infected stem surface of some of the infected plants. Onset of rain increased disease incidence each year, and it reached up to 30% in severely infested fields. A large number of oospores were seen in infected plant tissues microscopically. Infected tissues were taken from the margins of the diseased stem and cut into small pieces (5 × 5 mm). This plant material was surface sterilized by 2% sodium hypochlorite solution, rinsed in sterile water several times, and placed on to V8 medium plates. Fungi were purified by successive subculturing on fresh media plates using the hyphal tipping technique. The fungi were rapidly growing with coenocytic mycelia. Light microscopy showed terminal and intercalary sporangia, thick-walled aplerotic oospores, and globose intercalary and terminal hyphal swellings. The diameters of 20 sporangia and oospores were 19.7 to 27.3 μm (mean 22.18 μm; SD 2.4) and 16.5 to 23.9 μm (mean 19.02 μm; SD 1.8), respectively. Morphological characters were consistent with Pythium ultimum Trow (Van der Plaats-Niterink 1981; Watanabe 2002). Genomic DNA was extracted from mycelial masses using a Dzup fungal DNA extraction kit (Sangon Biotechnology, Shanghai, China). Molecular identifications were performed by amplifying the internal transcribed spacer region (ITS) using primers ITS1/ITS4 (White et al. 1990) of five pathogen isolates. BLAST analysis showed that all the isolates shared 99% nucleotide identity with the previously submitted P. ultimum sequences (GenBank accessions KU210389, KU210177, and AY243070). The nucleotide sequence has been assigned GenBank accession number MK615615. Pathogenicity tests were performed on plants of B. oleracea of variety Dasun using five different pathogen isolates (VRI-1 to VRI-5). Pathogen isolates were considered as the treatments, and each treatment consisted of 12 individual plants. Uninoculated potato dextrose agar (PDA) was included as the control. The experiment was performed as a randomized complete block design and repeated twice. Plants were raised in plastic pots filled with sterilized commercial potting mix. Two weeks after emergence, plants were inoculated with quarter-strength PDA medium pieces (5 × 5 mm) colonized with different isolates of P. ultimum separately. The inoculum was placed 5 to 10 mm away from the plants. Plants were incubated in a growth room at 25 ± 2°C and 16-h/8-h photoperiod. Symptoms developed within 10 days after inoculation. Infected plants showed reduced growth and dark brown lesions on stem that coalesced and turned into a rotted mass. All five pathogen isolates caused disease, and none of the pathogen isolates showed disease incidence less than 80%. Stems of diseased plants showed numerous oospores when observed under a microscope. Control plants remained healthy and uninfected. The same pathogen was consistently reisolated from infected tissues of plants. Pathogen identity was reconfirmed by both molecular and morphological methods. To our knowledge, this is the first report of Pythium stem rot of B. oleracea var. alboglabra caused by P. ultimum in China. This disease can be of great economic loss because the whole aboveground portion of this plant is of economic importance and is consumed as a salad or cooked as a vegetable. Timely measures must be taken to manage this disease. This disease may increase in incidence in rainy seasons.