Amorphophallus muelleri, known as konjac, is widely used in the biomedicine and food processing due to its richness in glucomannan. Between the years of 2019 to 2022, severe outbreaks of southern blight on Am. muelleri were observed during August and September in the main planting region of Mile city. The average disease incidence was 20%, resulted in 15.3% of economic losses in approximately 10,000 m2. Infected plants showed wilting and rotting and were covered with white dense mats of mycelia and sclerotia on both petiole base and tubers. Am. muelleri petiole base covered with mycelial mats were collected for pathogen isolation. The infected tissues (n=20) were washed with sterile water and surface disinfected with 75% alcohol for 60 seconds, rinsed three times with sterile water, cultured on rose bengal agar (RBA) and incubated at 27 ℃ for two days (Adre et al. 2022). Individual hyphae were transferred to new RBA plates and incubated at 27 ℃ for 15 days to obtain purified cultures. Five representative isolates were subsequently obtained and exhibited identical morphological characteristics. All isolates produced dense, cotton-white aerial mycelia and had a daily growth rate of 1.6 ± 0.2 mm (n=5). After 10 days, all isolates formed sclerotia in spherical (diameter range 1.1 to 3.5 mm, aver. 2.0 ± 0.5 mm; n=30) and irregular shapes. The number of sclerotia per plate ranged from 58 to 113 (aver=82; n=5). These sclerotia were initially white and gradually turned brown as they matured. A representative isolate (17B-1) was selected for molecular identification and the translation elongation factor (TEF, 480 nt.), internal transcribed spacer (ITS, 629 nt.), large subunit (LSU, 922 nt.), and small subunit (SSU, 1016 nt.) regions were amplified with the primers EF595F/EF1160R (Wendland and Kothe 1997), ITS1/ITS4 (Utama et al. 2022), NS1/NS4, and LROR/LR5 (Moncalvo et al. 2000), respectively. The ITS (GenBank accession no. OP658949), LSU (OP658955), SSU (OP658952), and TEF (OP679794) sequences were 99.19%, 99.78%, 99.31%, and 99.58% similar to those of At. rolfsii isolates (MT634388, MT225781, MT103059, and MN106270, respectively). Thus, the fungus represented by isolate 17B-1 was identified as At. rolfsii, corroborating the identification of Sclerotium rolfsii Sacc., the anamorph, based on cultural and morphological features. Pathogenicity tests were performed on six-month-old asymptomatic Am. muelleri plants (n=30) grown in pots with sterile soil in a greenhouse at 27 °C and 80% relative humidity. The petiole base was scratched with a sterile blade and 20 plants were inoculated by placing a 5 mm2 mycelial plug of five-day-old isolate 17B-1 on the wound. Sterile RBA plugs were used on 10 wounded control plants. After 12 days, all inoculated plants exhibited symptoms similar to those observed in the field, while the control plants showed no symptoms. The morphological and molecular identification of the fungus reisolated from inoculated petioles confirmed its identity as At. Rolfsii, fulfilling Koch's postulates. S. rolfsii was first reported on Am. campanulatus in India (Sarma et al. 2002). As At. rolfsii causes konjac diseases in all Amorphophallus growing areas (Pravi et al. 2014), the importance of At. rolfsii as an endemic pathogen of Am. muelleri in China needs to be recognized, and its prevalence should be determined as a first step to managing this disease.
Keywords: Athelia rolfsii; Konjac; Ribosomal RNA; TEF; polymerase chain reaction amplification.