First Report of Ethiopia Maize-Associated Virus Infecting Sugarcane Variety R570 in Canal Point, Florida

Sugarcane (Saccharum interspecific hybrids) is a major crop contributing to both sugar production and economic stability, but various pathogens threaten its production. Unusual chlorosis was observed in several plants of variety R570 in a field trial established on 5,000 m 2 to investigate the impact of sugarcane mosaic virus (SCMV) and the leaf scald pathogen (Xanthomonas albilineans) on the microbiome of sugarcane varieties at Canal Point, FL. Disease (chlorosis) incidence in R570 rose from 6% in August 2023 (plant cane) to 28% by April 2025 (second ratoon). A total of five samples were collected from five plants showing chlorosis. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). For high-throughput sequencing (HTS), five single-indexed, ribosomal RNA-depleted cDNA libraries were prepared using the TruSeq® Stranded Total RNA Library Prep Plant Kit (Illumina, San Diego, CA). Sequencing was done on the Illumina NextSeq 2000 platform using a P2 100-cycle kit, yielding 11.12-21.49 million single-end reads. HTS reads were analyzed using PhytoPipe (Hu et al. 2023). BLASTn analysis of five assembled viral contigs (ranging from 2091 to 3149 nucleotides (nt); GenBank accessions PX394828-PX394832) resulted in the identification of Ethiopia maize-associate virus (EMaV; MF415880) as the top hit, with over 95% genome coverage and 82% nucleotide identity. Phylogenetic analysis using published umbra-like viruses (ULVs) confirmed that the sugarcane virus clusters with EMaV originally identified in maize in Ethiopia (Adams et al. 2017). Open reading frame (ORF) analysis of the derived viral sequence identified four ORFs, consistent with those of other Class 2 ULVs infecting monocots (Ying et al. 2024). The inferred amino acid sequences exhibited 84% similarity with the RNA-dependent RNA polymerase (RdRp) of EMaV. Additionally, a novel small ORF (180 nt) was identified in the 3′ UTR, absent from all known ULV sequences (Simon et al. 2024). Two pairs of specific RT-PCR primers were designed targeting RdRp regions of the EMaV genome: EMaV1: F-5′-TCCTCAAGTGCTACTCCGGA-3′; R-5′-GCATAAGTACGCAGTTGCCG-3′ and EMaV2: F-5′-GGCCCGGTGGGTAAAATACA-3′; R-5′-CCAGTGCTATCGCTCCAGAG-3′. These primers, along with those for the housekeeping gene NADH dehydrogenase subunit 5, were used to screen 21 samples from weed grasses growing near infected R570 plants, three symptomatic R570 leaf samples, and two samples from healthy controls. Detection of the expected 589-bp (EaMV1) and 733-bp (EaMV2) fragments confirmed the virus in R570. Sanger sequencing of the RT-PCR products showed 100% identity with the HTS-derived EMaV sequence. All weed samples and healthy controls tested negative, suggesting EMaV was confined to R570 plants. Similar to maize lethal necrosis disease, where EMaV co-infects the host with other viruses (Adams et al. 2017), coinfection by EaMV and widespread sugarcane viruses (sugarcane bacilliform virus, sugarcane mild mosaic virus, and sugarcane yellow leaf virus, representing the families Caulimoviridae, Closteroviridae, and Solemoviridae) in R570 caused severe stunting, resulting in yield losses up to 80% in Florida. This study provides the first evidence of EMaV infecting sugarcane, expanding the known host range of this ULV. The severe stunting and yield losses in cultivar R570 demonstrate its potential threat to sugarcane production and underscore the need for effective surveillance and management.