First report of maize rayado fino virus in corn fields in Santa Catarina, Brazil.

Maize ( L.) is the main cereal food of humans and animals in Brazil. In 2020 and 2021, a severe infestation of corn leafhoppers (; Hemiptera: Cicadellidae) was observed in Santa Catarina State (South of Brazil). Subsequently, symptoms of chlorotic stripes limited in leaf veins started to appear in maize plants. Given the similarity of symptoms and the presence of high populations of corn leafhoppers in corn production areas, 30 plants in reproductive stage showing systemic symptoms were collected in summer and autumn from commercial fields of five municipalities in Santa Catarina: Campos Novos (27°23'18.0"S, 51°12'52.7"W), Lages (27°47'17.8"S, 50°18'16.9"W), Mafra (26°06'42"S, 49°48'25"W), Fraiburgo (27°01'36"S, 50°55'19"W), and Abelardo Luz (26°34'02"S, 52°20'02"W). The young leaves of these samples were used for molecular analyses targeting the maize rayado fino virus (MRFV; : Marafivirus). Total nucleic acids were extracted using TRIzol® (Invitrogen, USA), following the manufacturer's instructions. These were used as a template for cDNA synthesis with the enzyme MMLV-RT (Promega, USA), following the manufacturer's instructions. The polymerase chain reaction (PCR) was performed using Gotaq® DNA polymerase (Promega, USA) and MRFV-09/MRFV-10 primers (Hammond et al. 1997). All PCR products were subjected to electrophoresis in 1% agarose gel and were visualized under ultraviolet light. Twenty-eight of the 30 tested plants were MRFV-positive, showing a fragment with an expected size of ~633 bp. To confirm our results, all MRFV-positive samples were sent for sequencing (GenBank accession numbers OM763708 - OM763710 and ON730784 - ON730806) and submitted to BLASTn search (https://blast.ncbi.nlm.nih.gov/Blast.cgi), resulting in identities ranging from 96.21% to 99.21% with the isolate "Brazil 26" of MRFV, which was detected in 2005 in São Paulo, Brazil (GenBank accession nº: AF186178) (Hammond and Bedendo 2005). A second set of primers was used to validate the first PCR, confirming MRFV infection (data not shown).Moreover, whitish streaks and leaf reddening were observed on the leaves of some plants; therefore, the identification for phytoplasmas ( Phytoplasma asteris) and spiroplasmas () from the corn stunt complex was performed. For this, previously extracted nucleic acids from each sample were used as templates for a multiplex PCR using the primers CSSR6/CSSF2 and R16F2n/R16R2 (Gundersen and Lee 1996; Barros et al. 2001). Two plants were infected with only spiroplasma, 17 samples were infected with Spiroplasma and MRFV, and three samples were infected by these three pathogens. An increasing incidence of corn stunt has been observed in commercial fields in Santa Catarina in recent years. Mollicutes are commonly found and mostly studied as causal agents of corn stunt disease. On the contrary, despite being present in Brazil since the 1970s, the virus is less studied because its contribution to the corn stunt complex is still unknown (Hammond and Bedendo 2001). In this report, indications that the virus is expanding to different regions in southern Brazil were observed, which raises an opportunity for further evaluation and its consideration in monitoring programs. Moreover, to the best of our knowledge, this is the first report of MRFV in Santa Catarina, Brazil.