First Report of <i>Lasiodiplodia pseudotheobromae</i> Causing Maize Ear Rot in Guangxi Province in Southern China

Maize (Zea mays L.) is an important food and feed crop worldwide. Maize ear rot caused by more than 70 fungi usually leads to significant yield loss (Duan et al. 2022). In July 2024, about 2 percent of maize ears rot was observed in experimental field at Jiangnan District of Nanning City, Guangxi Province, China (22°64'N, 108°22'E).The symptoms observed were blackened and shrivelled maize kernels and blackened bracts along with abundant, black pycnidia (Fig. S1). Possible fungal causal agent was isolated from symptomatic samples surface-disinfected with 75% ethanol and 2% sodium hypochlorite, rinsed three times with sterile water, transferred to potato dextrose agar (PDA). After 2 days of incubation at 25°C in the dark, mycelia growing from kernels were transferred to fresh PDA plates. The mycelium exhibited rapid growth which covered a 9 cm Petri dish about 2 days. Initially colonies were gray white, rotund or subcircular, and mycelium showed sprawled growth; gradually colonies turned gray brown to gray black, villiform; cultures showed a dark underside. After 29 days, colonies produced black brown globose pycnidia. Conidia ellipsoidal, apex and base rounded, widest at the middle, thick-walled, initially hyaline and aseptate, becoming dark brown to black, one septate, and longitudinally striate with age, (21.1-32.6) × (9.6-15.0) µm, averaged 25.4 × 12.9 μm. The morphology of the fungus was similar to Lasiodiplodia spp (Alves et al. 2008). The isolate ZBSF008 was used for molecular identification. The pathogen identity to species level was verified by amplification of the internal transcribed spacer (ITS) region of rDNA, beta-tubulin (tub2) gene and calmodulin (cmdA) gene using ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass et al. 1995) and CAL-228F/CAL-737R primer sets, respectively. Accession numbers of gene sequences submitted to GenBank were PQ444004 for ITS; PQ456442 for tub2; and PQ456443 for cmdA. For all genes used, sequences were 99.60 to 100.00% identical to reference strain CBS 116459 of L. pseudotheobromae reported in GenBank (KF766193, EU673111, and KU886784). Maximum likelihood phylogenetic tree based on ITS-tub2-cmdA sequence showed that ZBSF008 located on the same clade with strains of L. pseudotheobromae (Fig. S2). Koch’s postulates were completed by using Zhengdan958 as experimental materials growing in the experimental field of Hebei Province. Each of the six maize ears was injected with 2 ml conidial suspension of the isolate through the silk channel. Control groups were inoculated with sterile distilled water. About 9 days post inoculation, the seed coat of all inoculated kernels was grayish white and covered with white mycelium, the bract showed gray. After 35 days, the seed coat of all inoculated kernels was white or grey spot when the infection was mild. The kernel became black and mildewed, the seed was shriveled, and the endosperm was black. When infected maize were split open there was grayish-white and black mold developing among kernels. The cob was also infected, becoming gray black when the infection was severe. When air humidity was high, inoculated kernels and bracts were grayish-black that were covered grayish-white and black mold. (Fig. S3). Meanwhile, the control ears did not exhibit any disease symptoms. The same fungus was re-isolated from the inoculated kernels and its identity was re-confirmed by cultural, morphological characteristics and sequence analysis. The L. pseudothebromae was previously reported to have affect several hosts such as acacia mangium, mango, persimmon and gossypium hirsutum (Alves et al. 2008; Castro et al. 2014; Nogueira et al. 2017; Alam et al. 2021; Sumera et al. 2021). In India, L. pseudotheobromae is known to cause maize stalk rot, with the incidence ranged from 10 – 18% and yield loss of up to 30% (Swamy et al. 2020). To our knowledge, this is the first report of L. pseudotheobromae causing ear rot on maize in China.