First Report of Fusarium sacchari That Causes Sugarcane Wilt Disease in China

Abstract

China is the world’s third-largest sugar producer after India and Brazil, with sugarcane plants accounting for 90% of total sugar production in China. Susceptible sugarcane cultivars were vulnerable to severe damage by wilt, which in China has caused yield losses of about 10.0%. During a disease survey in a nationwide test of sugarcane varieties in different regions in August 2019, 5-month-old plants of the sugarcane hybrid variety Zhongzhe1 (ZZ1) grown in Beihai (109°06′52″E, 21°28′37″N) of Guangxi Zhuang Autonomous Region were found to exhibit dried internodal tissue, yellowed leaves, and hollow stalks consistent with wilt. A total of 18 sugarcane stalk samples affected by symptomatic wilt were collected from Beihai. Symptomatic internal stalk tissues were dissected into 0.5 × 0.5-cm pieces that were surface sterilized with 0.1% HgCl₂ for 45 s, followed by 70% ethanol for 60 s, and rinsed three times with sterile water. Around 5 to 10 pieces were placed on potato dextrose agar (PDA) and incubated at 28°C for 2 to 3 days. Growing hyphal tips were picked and transferred to fresh PDA plates to purify fungal isolates and maintained on PDA and carnation leaf-piece agar (CLA) for observing conidia and conidial fructification. A total of six fungal strains were isolated from the 18 symptomatic stalk samples (Ho and Ko 1997). These isolates produced abundant white mycelia on the PDA medium for 3 days. The macroconidia typically had three to five septa and were slightly falcate, relatively slender, and had thin walls. The microconidia were slim, oval, and primarily nonseptate. The microconidia grown on CLA had an oval shape that was similar to Fusarium sacchari (Leslie and Summerell 2006), which had no long chains and produced false heads from mono- and polyphialides. Genomic DNA from the six single-spore cultured isolates was extracted, amplified, and sequenced using two specific primer pairs of the translation elongation factor TEF1-α (EF1/EF2, 0.6 kb) and the second-largest subunit of RNA polymerase II, RPB2 (5f2/7cr and 7cf/11ar, 1.8 kb) (O’Donnell et al. 2013). All 12 sequences were submitted to GenBank (MN894584 to MN894595) and had over 99% homology to F. sacchari with 657/657 for TEF1-α (MK152503) and 1,700/1,706 for RPB2 (MK829741) through pairwise alignment against the Fusarium MLST database. A phylogenetic tree on the TEF1-α and RPB2 gene sequences was constructed by neighbor-joining methods with the reference sequences from other Fusarium species, including F. sacchari, F. mangiferae, F. fujikuroi, F. proliferatum, F. thapsinum, and F. verticillioides. Sequence alignment confirmed that the six isolates belonged to F. sacchari. A pathogenicity test was conducted on 5-month-old cultivar ZZ1. Stalk inoculation was achieved through a borehole made at the third internode from the top visible dewlap of the plant using a 5-mm-diameter hole punch (Viswanathan et al. 2011). A mycelial plug of the three strains isolated from wilt-affected plants (GX-BH01, GX-BH02, and GX-BH03) was placed in the borehole and sealed with Parafilm. A plug of the sterile medium was used as a control. All pathogenicity assays were repeated three times. The inoculated plants were placed in a growth chamber at 25 to 28°C with a 12-h day/12-h night photoperiod. After 30 days, the canes inoculated with the fungal isolates exhibited tissue discoloration and hollow stalks that were similar to those observed in field-grown sugarcane, whereas the control plants had no symptoms. The reisolated fungi were identified as F. sacchari based on morphological, cultural, and molecular characteristics. Our results confirmed that F. sacchari is the causative pathogen of sugarcane wilt, and this is the first report of this pathogen in China. This report has epidemiological significance for the planning of suitable management practices to prevent wilt disease in China.