番茄静脉间萎黄病病毒(ToICV;Begomovirussolanumintervae,Begomovirus属,双生病毒科)已被描述为在巴西东北部(NE)感染番茄(Solanumlycopersicum)和甲脂甲苷类(Albuquerque等人。,2012;席尔瓦等人。,2012).在2020年的一项调查中,在Alagoas州观察到了表现出病毒样症状的豆科杂草Rhynchosiaminima植物,东北巴西。随机收集R.minima的症状叶样品(n=15;补充图1)。来自每个样品的总DNA用作模板,用于使用简并引物对PAL1v1978和PAR1c496对双生病毒通用的部分双生病毒DNA-A序列进行PCR扩增(Rojas等人。,1993).从12个样品中观察到~1.2kbp的扩增子,尽管这不应该被认为是发病率,因为只收集了有症状的植物。为了鉴定与小R.minima相关的双生病毒,使用滚环扩增(RCA)从PCR阳性样品中扩增病毒基因组(Inoue-Nagata等人。,2004).用HindIII消化RCA产物,克隆到pBluescriptIIKS质粒载体中,并进行双向Sanger测序(MacrogenInc.,首尔)。BLASTn搜索表明,此处报告的克隆(n=4)对应于BegomovirusDNA-A成分,两两比较表明,他们与ToICV有着最高的身份,在92.4-94.7%的核苷酸序列同一性。基于Begomovirus属≥91%核苷酸同一性的物种划分标准(Brown等人。,2015),从R.minima获得的Begomovirus是ToICV的新分离株。长度为2,619-2,623nt的新DNA-A序列以登录号PP639092至PP639095保存在GenBank中。使用在MEGAv.11中实施的MUSCLE算法(Kumar等人。,2018),并在RaxML-NG中重建了最大似然(ML)树(Kozlov等人。,2019),假设一般的时间可逆(GTR)核苷酸取代模型具有γ(G)模型的速率异质性和1,000个自举重复。基于DNA-A的树显示ToICV序列聚集成单系群,另外支持这些分离株作为茄科病毒物种的成员。在ToICV分离株中预测了至少两个独立的种间重组事件,断点位于Rep编码区域和ToICV(GenBank加入JF803253),番茄斑驳卷叶病毒(JF803248)和大豆起泡花叶病毒(MN486865)被检测为推定亲本。据我们所知,这是ToICV在全球范围内感染R.minima的第一份报告,扩大了这种初生病毒的宿主范围。非栽培植物,例如R.minima作为菜鸟病毒的储库和接种物来源起着至关重要的作用(Paz-Carrasco等人。,2014),加强它们与社会经济重要作物的相关性。
Tomato interveinal chlorosis virus (ToICV;
Begomovirus solanumintervenae, genus
Begomovirus, family Geminiviridae) has been described infecting tomato (Solanum lycopersicum) and Macroptilium lathyroides in Northeastern (NE) Brazil for more than a decade (Albuquerque et al., 2012; Silva et al., 2012). During a survey in 2020, plants of the leguminous weed Rhynchosia minima exhibiting virus-like symptoms such as mosaic and interveinal chlorosis were observed in the state of Alagoas, NE Brazil. Symptomatic leaf samples of R. minima were randomly collected (n=15; supplementary figure 1). Total DNA from each sample was used as a template for PCR amplification of partial begomoviral DNA-A sequences using the degenerate primer pair PAL1v1978 and PAR1c496, universal for geminiviruses (Rojas et al., 1993). Amplicons of ~1.2 kbp were observed from 12 samples, although this should not be considered as incidence since only symptomatic plants were collected. To identify the
begomovirus associated with R. minima, viral genomes were amplified from PCR-positive samples using rolling circle amplification (RCA) (Inoue-Nagata et al., 2004). The RCA products were digested with HindIII, cloned into the pBluescript II KS+ plasmid vector and bidirectionally Sanger-sequenced (Macrogen Inc., Seoul). BLASTn searches indicated that the clones (n=4) reported here corresponded to a
begomovirus DNA-A component, and pairwise comparisons showed that they shared the highest identity with ToICV, at 92.4-94.7% nucleotide sequence identity. Based on the species demarcation criteria of ≥91% nucleotide identity for the genus
Begomovirus (Brown et al., 2015), the begomoviruses obtained from R. minima are new isolates of ToICV. The new DNA-A sequences of 2,619-2,623 nt in length were deposited in GenBank under accession numbers PP639092 to PP639095. Multiple nucleotide sequence alignments were prepared using the MUSCLE algorithm implemented in MEGA v.11 (Kumar et al., 2018), and a maximum likelihood (ML) tree was reconstructed in RaxML-NG (Kozlov et al., 2019), assuming a general time reversible (GTR) nucleotide substitution model with a gamma (G) model of rate heterogeneity and 1,000 bootstrap replicates. The DNA-A-based tree showed that the ToICV sequences clustered into a monophyletic group, additionally supporting these isolates as members of the species Begomovirus solanumintervenae. At least two independent interspecies recombination events were predicted among the ToICV isolates, with breakpoints located in the Rep-encoding region and ToICV (GenBank Accession JF803253), tomato mottle leaf curl virus (JF803248) and soybean blistering mosaic virus (MN486865) detected as putative parents. To the best of our knowledge, this is the first report of ToICV infecting R. minima worldwide, expanding the host range of this
begomovirus. Non-cultivated plants such as R. minima play a crucial role as reservoirs and sources of inoculum for begomoviruses (Paz-Carrasco et al., 2014), reinforcing their relevance to socioeconomically important crops.