Report on Rice Yellowing Syndrome Caused by Co-infection of Rice Grassy Stunt Virus (RGSV) and Rice Ragged Stunt Virus (RRSV) in <i>Oryza sativa</i> in the Philippines

Rice yellowing syndrome (RYS) is a disease complex associated with co-infection by Rice grassy stunt virus (RGSV; Tenuivirus oryzabrevis) and Rice ragged stunt virus (RRSV; Oryzavirus oryzae), both transmitted by brown planthopper (BPH; Nilaparvata lugens) (Cabauatan et al. 2009). RYS was first reported in Vietnam’s Mekong Delta in 1989, with a major outbreak causing significant yield losses during 2005–2006. The disease affected 485,000 ha and caused US$120M losses (Du et al. 2007). Individual virus also caused major losses in Southeast Asia (Hibino et al., 1996). In the Philippines, RGSV and RRSV single infections were reported in the 1960s–1980s with a new RGSV strain (RGSV2) identified in 1985 (Hibino et al. 1985). However, the synergistic co-infection of RGSV and RRSV or RYS has not been documented so far in this country. In the 2025 dry season, ~90% incidence of RYS-like disease was observed in a 3-ha field of rice in Santa Cruz, Laguna, Philippines. According to a rice farmer from the RYS-infected field, yields dropped from 15 to 0.81 t ha⁻¹ (94.6% loss) due to panicle failure. Field symptoms include stunting, spreading growth and yellow-orange discoloration, resembling Rice tungro disease (Hibino 1996), RGSV2 (Hibino et al. 1985) or Rice orange leaf Phytoplasma (ROLP) (Jonson et al. 2025) infections (Figure 1). Leaf deformities (serration, twisting, curling) were also present, similar to RRSV or Southern black-streaked dwarf virus (SRBSDV) (Hibino 1996) symptoms. To determine the causal agent, DNA and RNA were extracted from 14 symptomatic plants and were tested against rice virus and phytoplasma diseases by PCR/RT-PCR. In this study new specific primers were designed to amplify approximately 500 bp amplicon in the coat protein region of RGSV, RRSV, Rice tungro bacilliform virus (RTBV), Rice tungro spherical virus (RTSV), and SRBSDV while 1.9 kb for ROLP primers designed by Lee et al. (1993) (Suppl. Table 1). The nucleic acid diagnostics showed all 14 samples were negative for RTBV, RTSV, ROLP, and SRBSDV, 10 were positive for both RGSV and RRSV and 4 for RGSV alone (Figure 2a, Suppl. Table 2). Enzyme Linked Immunosorbent Assay (ELISA) (Bajet et al. 1985) using leaf sap detected RGSV from 13 samples (Figure 2b). Two PCR fragments amplified using RGSV- and RRSV-specific primers were sequenced (Macrogen, Korea) (Suppl. Data 1). The resulting sequences were subjected to BLAST analysis against the NCBI database and showed the highest similarity to previously reported RGSV and RRSV strains from the Philippines (Suppl. Data 2). The sequence alignment showed that RGSV isolate shared 98% nucleotide identity with LG001 (KF438685.1), while the RRSV isolate shared 99% nucleotide identity with AY512585.1 (Suppl. Data 3). Collectively, our results indicated the co-presence of both RGSV and RRSV in the RYS-like diseased plants. Following Koch’s postulates, the pathogenicity of RGSV and RRSV in diseased plants was tested. Second–third instar N. lugens nymphs fed 4 days on RGSV-RRSV co-infected plants were transferred to 7-day-old Taichung Native 1 (TN1) seedlings (3 insects per seedling) for 1 day-inoculation. Inoculated seedlings (n = 196) were maintained in a greenhouse under natural light and temperature for 3 weeks. Initial yellowing symptoms were observed at 10 days post inoculation. After 3 weeks, the number of plants showing RGSV, RRSV, and RYS-like symptoms (Figure 2c) out of total inoculated plants was recorded. Out of 196 total inoculated seedlings, 89 were infected with various symptoms: 52 of those showed RYS-, 22 RGS- and 15 RRS- like symptoms (Suppl. Table 3). Co-presence of RRSV and RGSV in RYS-like plants was confirmed by RT-PCR/PCR, and RGSV was verified by ELISA (Suppl. Table 3). This insect transmission’s data demonstrated that co-infection by both RRSV and RGSV produced the RYS-like symptoms observed in the field. Based on the combined results from molecular diagnostic, field associated symptoms and transmission experiments, we conclude that the disease observed in Santa Cruz, Laguna is RYS caused by the RGSV-RRSV coinfection. This is the first documented report of RYS caused by RGSV–RRSV co-infection in the Philippines. The emergence of this RYS disease poses a new threat to Philippine rice production and underscores the need for regular disease monitoring and integrated virus and vector management to prevent potential outbreak in the future. Consequently, this finding was reported in a press release platform for urgent awareness to our stakeholders (Rice News Today, 2025). Also, a pest advisory was released by the Philippines Department of Agriculture particularly for the farmers to help mitigate the further spread of the disease in the next planting season. These include key practices include monitoring and controlling BPH populations with approved agents such as pymetrozine, triflumezopyrim, and buprofezin (Prashant et al. 2015). Also, cultural practices including plowing and fallowing fields after harvest to break their life cycle, and coordinating planting schedules with neighbors to reduce disease spread. Planting resistant varieties to BPH could be used to control RGSV and RRSV, for examples, some Philippine inbred rice varieties such as NSIC Rc 218, NSIC Rc 402, NSIC Rc 420, NSIC Rc 480, NSIC Rc 486, and PSB Rc 18, and NSIC Rc 216 (Philippines Rice Research Institute, 2026).