Identification of Leaf Spot and Stem Rot on Plectranthus verticillatus Caused by Rhizoctonia solani AG-4 HGI in China

Abstract

Plectranthus verticillatus (Swedish ivy) is a plant in the family Lamiaceae (Labiatae), native to South-east Africa, that is commonly grown as a houseplant in China. In June 2018, the initial symptoms water-soaked spots were found on the stem base of sixteen P. verticillatus plants (1-year-old plants) in a commercial greenhouse, situating at Daxing district, Beijing, China (116°22’E, 39°42’N), where there were 150 potted P. verticillatus plants obtained by cuttage propagation and grown in conventional nutrient soils. Almost a month later, the disease was more severe and leaves appeared symptoms. Infected leaves displayed water-soaked, dark brown lesions and fell off easily. Symptoms on the stems were dark brown, rotted and lodging. In the survey, the incidence of affected plants was 70%. To isolate the causal agent, the infected leaf and stem tissues were cut into 3 mm segments and surface disinfected in 1% NaClO for 1 min, then rinsed three times with sterile distilled water and cultured on potato dextrose agar (PDA) at 25±1 °C with a 12-h photoperiod. After 2 days, hyphal tips were transferred to new PDA plates to obtain pure cultures. Six morphologically similar isolates (CC1 to CC6) were obtained from the symptomatic samples. All the fungal colonies were white initially, turned brown gradually, and formed irregular shaped sclerotia after 9 days of incubation on PDA. Observation of microstructures indicated that all the isolates resembled Rhizoctonia sp.(Sneh et al. 1991), including septate hyphal with right-angled branching, a slight constriction at the base of hyphal branches and 3 to 8 nuclei per cell. Total genomic DNA was extracted from pure isolates using a Fungal DNA Kit (GBCBIO, Guangzhou, China) and the internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 and sequenced (White et al. 1990 and Garibaldi et al. 2019).The results showed that sequences of 6 isolates were identical and had a 100% identity with those of R. solani AG-4 HGI (GenBank accession no. MH172679 and MH172664). The phylogenetic analysis performed by the neighbour-joining method (MEGA 7 software) showed that the representative isolate (accession no. MZ723940) was assigned to group of R. solani AG-4 HG-I. The pathogenicity of these six isolates to the leaves and stems of P. verticillatus were conducted. Healthy P. verticillatus plants (1-year-old) were grown in 2-liter pots with sterile substrate in a greenhouse at 14–19°C (night)/23–28°C (day). After 7 days, five P. verticillatus plants for each isolate were inoculated with a 6-mm-diameter PDA plug taken from a 10-day-old PDA culture, which was placed onto leaf blade and the base of the stem. Five control pots were inoculated with sterile PDA plugs. All the inoculated plants were incubated in a glass cabinet under 90% humidity and 25°C for 2 days and were then moved back to the greenhouse with natural daylight conditions. The test was repeated three times. Ten days later, all inoculated leaves and stems appeared dark brown and water-soaked lesions similar to those observed on naturally infected plants, whereas control plants remained asymptomatic. The fungus that was reisolated from diseased tissues had the same morphological characters as the original isolates and confirmed to be R. solani AG-4 HG-I by molecular identification. R. solani AG-4 HGI has an extensive host range, which had previously been recorded to cause leaf, root and stem rot on Plectranthus sp. in Florida in 1984 (Alfieri Jr. et al. 1984). To our knowledge, this is the first report of R. solani AG-4 HGI infecting P. verticillatus in China. Its confirmation is a significant step toward management recommendations for growers.