How a chance finding and high-throughput sequencing helped unmask the probable causal agent of Brazilian curly top, a plant disease that disappeared over 70 years ago

Curly top disease (CTD) affects sugar beet, tomato and other crops, resulting in stunted plants with severely curled leaves and reduced yields. The disease occurs worldwide and is caused by geographically associated monopartite geminiviruses transmitted mostly by leafhoppers. However, the aetiology of CTD in South America remains unknown because the disease disappeared in the 1950s. Here, we describe how the chance finding of tomato plants with CTD-like symptoms in Brazil in 2016 and high-throughput sequencing helped identify a novel ~2.6 kb geminivirus DNA associated with curly top disease in Mato Grosso (GV-CTD-MT) that induced CTD symptoms in agroinoculated tomato and Nicotiana benthamiana Domin. plants and produced geminivirus-like virions in infected plants. Evidence GV-CTD-MT may be the genomic DNA of the historic Brazilian curly top virus (BraCTV) includes (i) occurring in the same geographic location (Brazil); (ii) inducing nearly identical CTD symptoms in tobacco ( Nicotiana tabacum L.) and tomato plants to those described in the 1940s for the BraCTV; (iii) inducing CTD in a similar broad host range of plant species as previously reported for BraCTV based on leafhopper transmission experiments; (iv) the co-phylogenetic analysis predicting the vector of GV-CTD-MT is Agallia sp. leafhoppers; and, importantly, (v) transmission experiments showing Agallia albidula , the known vector of BraCTV, is a competent vector of GV-CTD-MT. Sequence and phylogenetic analyses revealed that this novel CTD-inducing geminivirus has a chimeric genome, a long evolutionary history, and is closely related to monopartite geminiviruses recently identified in South America. These viruses were placed in the new genus Topilevirus , the fifth genus whose members induce CTD. Thus, our results suggest that BraCTD, which disappeared over 70 years ago, and possibly historic CTDs of sugar beet in South America were caused by topileviruses transmitted by indigenous leafhoppers, thereby solving the conundrum left by first-generation researchers.