Table_3_A Temporal Diversity Analysis of Brazilian Begomoviruses in Tomato Reveals a Decrease in Species Richness between 2003 and 2016.docx

Understanding the molecular evolution and diversity changes of begomoviruses is crucial for predicting future outbreaks of the begomovirus disease in tomato crops. Thus, a molecular diversity study using high-throughput sequencing (HTS) was carried out on samples of infected tomato leaves collected between 2003 and 2016 from Central Brazil. DNA samples were subjected to rolling circle amplification and pooled in three batches, G1 (2003–2005, N = 107), G2 (2009–2011, N = 118), and G3 (2014–2016, N = 129) prior to HTS. Nineteen genome-sized geminivirus sequences were assembled, but only 17 were confirmed by PCR. In the G1 library, five begomoviruses and one capula-like virus were detected, but the number of identified viruses decreased to three begomoviruses in the G2 and G3 libraries. The bipartite begomovirus tomato severe rugose virus (ToSRV) and the monopartite tomato mottle leaf curl virus (ToMoLCV) were found to be the most prevalent begomoviruses in this survey. Our analyses revealed a significant increase in both relative abundance and genetic diversity of ToMoLCV from G1 to G3, and ToSRV from G1 to G2; however, both abundance and diversity decreased from G2 to G3. This suggests that ToMoLCV and ToSRV outcompeted other begomoviruses from G1 to G2 and that ToSRV was being outcompeted by ToMoLCV from G2 to G3. The possible evolutionary history of begomoviruses that were likely transferred from wild native plants and weeds to tomato crops after the introduction of the polyphagous vector Bemisia tabaci MEAM1 and the wide use of cultivars carrying the Ty-1 resistance gene are discussed, as well as the strengths and limitations of the use of HTS in identification and diversity analysis of begomoviruses.

解析双生病毒（begomoviruses）的分子演化与多样性变化，对预测番茄作物双生病毒病害的未来暴发至关重要。为此，本研究以2003年至2016年间从巴西中部采集的感病番茄叶片样本为材料，利用高通量测序（high-throughput sequencing, HTS）开展分子多样性分析。在进行HTS前，我们对DNA样本实施滚环扩增（rolling circle amplification），并将其分为三个混合池：G1组（2003–2005年，样本量N=107）、G2组（2009–2011年，N=118）与G3组（2014–2016年，N=129）。本研究共组装获得19条基因组级别的双生病毒序列，但经聚合酶链式反应（polymerase chain reaction, PCR）验证后，仅17条序列得到确认。在G1文库中，共检测到5种双生病毒与1种类卡普拉病毒（capula-like virus）；而G2与G3文库中鉴定到的病毒种类缩减至3种双生病毒。本调查中检出的优势双生病毒为二分体双生病毒番茄严重皱缩病毒（tomato severe rugose virus, ToSRV）与单分体双生病毒番茄斑驳曲叶病毒（tomato mottle leaf curl virus, ToMoLCV）。分析结果显示，从G1到G3阶段，番茄斑驳曲叶病毒（ToMoLCV）的相对丰度与遗传多样性均呈显著上升趋势；番茄严重皱缩病毒（ToSRV）则在G1至G2阶段同步上升，但二者的丰度与多样性均在G2至G3阶段出现下降。这表明在G1至G2阶段，番茄斑驳曲叶病毒与番茄严重皱缩病毒相较于其他双生病毒具备竞争优势；而在G2至G3阶段，番茄严重皱缩病毒则被番茄斑驳曲叶病毒反超。本研究还探讨了双生病毒的潜在演化历程：在多食性媒介烟粉虱MEAM1隐种（Bemisia tabaci MEAM1）入侵以及携带Ty-1抗性基因的栽培品种广泛推广后，双生病毒从野生本土植物与杂草向番茄作物转移的潜在路径；同时也讨论了高通量测序在双生病毒鉴定与多样性分析中的应用优势与局限性。