First Report of Charcoal Rot Caused by Macrophomina phaseolina on Lily in China

Lily (Lilium spp.) is widely cultivated in many regions of China because of its high ornamental and edible value. In May 2023, a new disease of lily, called charcoal rot, was detected in Wuhan, China, causing widespread wilting and mortality of the Oriental lily hybrid 'Roselily Esra'. The incidence was up to 90%. The charcoal rot disease mainly affected lily leaves, with reddish-brown spots appearing on the upper part of the leaves in the early stage, gradually expanding to form brown patches, losing green color and turning yellow at the disease junction, and creating a clear yellow halo around the lesion. The leaves became brown, necrotic, and invaginated in the later stages, eventually leading to extensive discoloration. To isolate the pathogen, symptomatic leaves were excised into small pieces (0.5 cm × 0.5 cm), surface sterilized with 75% ethanol for 30 s, and washed three times with sterile distilled water. These sterilized leaf pieces were then incubated on potato dextrose agar (PDA) plates for 3 days at 25℃ in the dark. When the mycelia grew around the tissues, a single hyphal tip isolation method was used to purify the isolates on new PDA plates. Three strains that exhibited similar colony morphology were obtained. The front of the colonies exhibited cottony texture. The reverse side of the colonies was grey to black. One representative isolate, MP02, was selected for further study. The colonies initially appeared white, then gradually changed from gray to dark gray, and ultimately turned black. The colonies produced black sclerotia at the later stage of growth. The aerial mycelia of the colony were cottony. Microscopically, the mycelia were septate, and the sclerotia were black, measuring 36.28 to 91.32 × 37.12 to 91.15 μm (average 55.33 to 59.90 μm; n=30). The morphological characteristics matched those of the genus Macrophomina (Sarr et al. 2014). To identify the species of the fungus at the molecular level, ITS, TEF, and 28S rRNA genes were amplified and sequenced using primers ITS1/ITS4 (Gardes et al. 1993), 758F/986R (Carbone and Kohn 1999), and LROR/LR7 (Vilgalys and Hester 1990). The BLASTn results in GenBank showed that the ITS (PQ357630), TEF (PQ464126), and 28S rRNA (PQ472084) sequences of the fungus shared 99.80% (506/507), 99.22% (252/257) and 98.50% (1124/1145) identity with the distinct M. phaseolina strains (GenBank accession MZ726450.1, ON461806.1 and EU754168.1, respectively). The fungus was identified as M. phaseolina through phylogenetic analysis of concatenated ITS, TEF, and 28S rRNA sequences using MEGA 7.0 with the neighbor-joining method. In the pathogenicity test, three healthy leaves were detached and inoculated in vitro with 0.5-cm-diameter mycelium discs and then incubated in an incubator (22℃, 90% humidity, 16 h light/8 h darkness). For the control, three detached healthy leaves were treated exactly the same as the inoculated leaves, but with sterile PDA discs instead. Six healthy potted plants maintained in a greenhouse (25℃-28℃) were also used for the pathogenicity tests. Nine leaves from three potted plants were randomly inoculated in vivo using 0.5-cm-diameter mycelium discs. Another three potted plants were used as controls and inoculated with sterile PDA discs. The inoculated leaves all showed dark brown spots similar to those observed under natural conditions, while the control leaves were asymptomatic. The pathogen was re-isolated from the inoculated tissues, and the morphological identification and the DNA sequencing results confirmed that the isolate was M. phaseolina. To our knowledge, this is the first report of lily charcoal rot caused by M. Phaseolina. Our study provides important reference information for disease identification and the development of control methods.