Sugarcane (Saccharum spp. hybrids) is an economically important crop widely cultivated in the south of China, such as Guangxi, Yunnan, and Guangdong for use as the main raw material of the sugar and alcohol industry (Li and Yang et al. 2015). In July 2021, the sugarcane cultivar GT94-119 planted in Guangzhou (113° 22' E, 23° 09' N), Guangdong province, China showed red to brown ring lesions on the older leaves (Fig.1A). Multiple disease spots gradually merged, eventually leading to leaf wilting and necrosis was observed. Symptoms were present on 11% and 18% of plants in the two observation areas, respectively; however, since symptoms were primarily noted on older leaves, the yield effect was limited. Symptomatic leaf pieces (0.5 × 0.5 cm) were collected and surface-sterilized for 10s in 75% ethanol, followed by 10% NaClO for 30s, washed 3 times with distilled sterile water, blotted dry with sterile tissue, and plated on potato dextrose agar (PDA). The dishes were placed in an incubator at 28 ℃ for 72 h, and the resulting mycelia were transferred to new PDA to obtain pure cultures. The fungal colonies were brownish green, with concentric rings and radial edges (Fig.1B). The hyphae were transparent, separated, and apical hypertrophy (Fig.1C). Conidia were produced within 14 days, ranging in size from 20.0 to 25.5 × 2.5 to 4.5 µm (n=50), upright or curved spindle shaped, clustered or isolated at the end of the conidia stem, with a diaphragm (Fig.1D and E). Eleven isolates purified on PDA were obtained. Morphological identification showed that six of the 11 isolates were similar in morphology and preliminarily identified as Curvularia ischaemi (Mckenzie et al., 1981). One of the above six isolates, named GZ01, was selected for molecular identification. Following the CTAB method for extracting total DNA, the internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) region were amplified and sequenced by using ITS4/ITS5 primer (White et al. 1990) and GDF/GDR primer (Damm et al. 2012), respectively. The amplified sequence was compared to nucleotide sequence reported in GenBank using BLAST search, with 98.49% similarity to Curvularia ischaemi strain CBS 630.82 (GenBank MH861533.1) and 99.81% similarity to the GAPDH sequence of Curvularia ischaemi (GenBank LT715790.1). The phylogenetic tree based on sequence data for the two genes mentioned above and other reference sequences indicated that our isolate (GZ01) was closely identified as Curvularia ischaemi (Fig.2). To obtain a spore suspension of GZ01 for pathogenicity test, spores were cultured (28℃) in PDA for 14 days, washed with sterilized distilled water, and filtered with cheese cloth. The pathogenicity test was carried out in a greenhouse at 28℃ using a spore suspension (1×104 mL-1) and distilled water as inoculation sources. Healthy seedlings of the susceptible sugarcane cultivar LC05-136 were inoculated at the 5 to 6 leaf stage. The spore suspension was evenly sprayed on nine seedlings until the leaves were fully wet, additional nine seedlings were evenly sprayed with the same volume of sterile water to serve as the control. At 14 days after inoculation, all inoculated plants with suspension showed the same symptoms as observed in the greenhouse (Fig.1F), while all plants inoculated with sterile water showed no symptoms. Curvularia ischaemi was again isolated from the infected leaves with symptoms. The results confirm Koch's postulates. Curvularia ischaemi has been previously reported to cause disease on Batiki blusgrass (Ischaemum indicum) (Mckenzie et al. 1981). To our knowledge, this is the first report of C. ischaemi causing ring spot disease on sugarcane in China. For different ecological types of sugarcane areas, whether this disease will occur in the early stage of sugarcane growth and have an impact on sugarcane yield is worth further investigation.
Keywords: Curvularia ischaemi; pathogen; ring spot; sugarcane.