Table_1_Construction of an Infectious Clone of the Badnavirus Cacao Swollen Shoot Ghana M Virus and Infectivity by Gene Gun- and Agrobacterium-Mediated Inoculation.DOCX

Cacao swollen shoot disease (CSSD) is a damaging disease of Theobroma cacao L. associated with infection by a group of poorly characterized badnaviral species. To establish causality and characterize the symptomatology associated with infection by the badnavirus cacao swollen shoot Ghana M virus (CSSGMV), an infectious clone (1.3-mer) was constructed and used to inoculated cacao “Amelonado” seedlings by biolistic inoculation (BI; n = 18) and agroinoculation (AI; n = 15). Newly expanded leaves of BI (10/18) and AI (12/15) plants developed foliar mosaic and curling symptoms 30-days post inoculation (dpi), with chlorotic mottling and necrotic crinkling being evident by 90 dpi. By 120 dpi, three of 15 AI plants exhibited characteristic stem-swelling. Viral infection was verified by PCR-amplification and sequencing of a 1068 bp fragment of the CSSGMV ORF3 from newly expanding leaves 60 dpi. The PCR results indicated that 14 of 18 and 15 of 15 BI and AI plants, respectively, were systemically infected. The complete CSSGMV genome sequence was determined, by Illumina sequencing, from representative AI and BI plants and shared >99.5% pairwise nucleotide identity with CSSGMV-Nig9 (GenBank Accession No. MH785299). Based on the development of characteristic CSSD symptoms and recovery of partial and complete genome sequences of CSSGMV-Nig9 from systemically infected cacao plants, Koch's postulates have been fulfilled.

可可肿枝病（CSSD）是可可植物（Theobroma cacao L.）的一种破坏性病害，与一组难以表征的Badnaviridae科病毒种类感染相关。为了确立因果联系并描述可可肿枝病（Ghana M病毒，CSSGMV）感染所伴随的症状学特征，构建了一个感染克隆（1.3-mer）并用于通过生物粒子介导接种（BI；n = 18）和农业接种（AI；n = 15）对可可‘Ameleonado’幼苗进行接种。BI组（10/18）和AI组（12/15）的植物新展开的叶子在接种后30天（dpi）出现叶片花叶和卷曲症状，到90 dpi时，可见叶片出现黄斑和坏死皱缩。到120 dpi时，AI组中的3株植物表现出典型的茎部肿胀。通过PCR扩增和测序新展开叶子中CSSGMV ORF3的1068 bp片段（60 dpi）来验证病毒感染。PCR结果指出，18株BI植物中的14株和15株AI植物中均有系统性感染。通过Illumina测序从代表性AI和BI植物中确定了完整的CSSGMV基因组序列，并与CSSGMV-Nig9（GenBank登录号：MH785299）的序列共享超过99.5%的成对核苷酸同源性。基于CSSD典型症状的发展，以及从系统性感染的可可植物中恢复CSSGMV-Nig9的部分和完整基因组序列，Koch法则得以满足。