New World Cactaceae Plants Harbor Diverse Geminiviruses

Abstract

The family Cactaceae comprises a diverse group of typically succulent plants that are native to the American continent but have been introduced to nearly all other continents, predominantly for ornamental purposes. Despite their economic, cultural, and ecological importance, very little research has been conducted on the viral community that infects them. We previously identified a highly divergent geminivirus that is the first known to infect cacti. Recent research efforts in non-cultivated and asymptomatic plants have shown that the diversity of this viral family has been under-sampled. As a consequence, little is known about the effects and interactions of geminiviruses in many plants, such as cacti. With the objective to expand knowledge on the diversity of geminiviruses infecting cacti, we used previously acquired high-throughput sequencing results to search for viral sequences using BLASTx against a viral RefSeq protein database. We identified two additional sequences with similarity to geminiviruses, for which we designed abutting primers and recovered full-length genomes. From 42 cacti and five scale insects, we derived 42 complete genome sequences of a novel geminivirus species that we have tentatively named Opuntia virus 2 (OpV2) and 32 genomes of an Opuntia-infecting becurtovirus (which is a new strain of the spinach curly top Arizona virus species). Interspecies recombination analysis of the OpV2 group revealed several recombinant regions, in some cases spanning half of the genome. Phylogenetic analysis demonstrated that OpV2 is a novel geminivirus more closely related to viruses of the genus Curtovirus, which was further supported by the detection of three recombination events between curtoviruses and OpV2. Both OpV2 and Opuntia becurtoviruses were identified in mixed infections, which also included the previously characterized Opuntia virus 1. Viral quantification of the co-infected cactus plants compared with single infections did not show any clear trend in viral dynamics that might be associated with the mixed infections. Using experimental Rhizobium-mediated inoculations, we found that the initial accumulation of OpV2 is facilitated by co-infection with OpV1. This study shows that the diversity of geminiviruses that infect cacti is under-sampled and that cacti harbor diverse geminiviruses. The detection of the Opuntia becurtoviruses suggests spill-over events between viruses of cultivated species and native vegetation. The threat this poses to cacti needs to be further investigated.

Keywords: Becurtovirus; Cactaceae; Geminiviridae; diversity; recombination.

Figure 1

(A) Neighbor-joining phylogenetic tree of the genome sequences of the eleven genotypes of OpV2 and the Opuntia becurtovirus, together with representative sequences from various genera in the family Geminiviridae. Branches with <60% bootstrap support have been collapsed and the phylogenetic tree is midpoint-rooted. The genomic organization of OpV2 and the Opuntia becurtovirus are illustrated on the right side of the phylogenetic tree near their respective groups. (B) Organization and orientation of the replication-associated interactive sequences “iterons” in the intergenic region of both eleven genotypes of OpV2 and the Opuntia becurtovirus. The arrows indicate the orientation of the iteron sequences relative to the nonanucleotide and lower-case letters indicate that the nucleotide is variable among sequences in that genotype. Some genotypes presented more than one type of “iteron” sequence.

Figure 2

Maximum-likelihood phylogenetic tree of the Rep and CP amino acid sequences of the eleven representative genotypes of OpV2 and Opuntia becurtovirus, and representative sequences from various genera in the family Geminiviridae. Branches with <0.8 aLRT support have been collapsed and the trees are rooted with sequences of genomoviruses [62].

Figure 3

(A) Neighbor-joining phylogenetic tree of the 42 OpV2 sequences with genotypes denoted by symbols. (B) Maximum-likelihood phylogenetic tree of the 42 OpV2 sequences with recombination regions removed. The eleven genotypes of OpV2 are represented by symbols. Graphical representation of each genome representing the recombination event with the breakpoint location within the genome.

Figure 4

(A) Neighbor-joining phylogenetic tree of the 32 Opuntia becurtovirus sequences. Branches with <60% bootstrap branch support have been collapsed and the tree is midpoint-rooted. (B) Maximum-likelihood phylogenetic tree of the 32 Opuntia becurtovirus sequences with recombination regions removed. Graphical representation of each genome representing the recombination event with the breakpoint location within the genome.

Figure 5

(A) Neighbor-joining phylogenetic tree of the OpV2 and becurtovirus representative sequences, together with representatives from the genera Becurtovirus and Curtovirus. Branches with <60% bootstrap branch support have been collapsed and the tree is midpoint-rooted. (B) Maximum-likelihood (ML) phylogenetic tree of the Opv2 and becurtovirus representative sequences, together with representatives from the genera Becurtovirus and Curtovirus with recombination regions removed. Branches with <60% bootstrap support have been collapsed and the tree is midpoint-rooted. Graphical representation of each genome, representing the recombination event with the breakpoint location within the OpV2 genome for reference, which is color coded according to the genera of the major and minor parent sequences of each event. In bold are the two sequences that represent OpV2 and Opuntia becurtovirus.

Figure 6

Bar graph of the viral loads (genomic units/ng of total DNA) determined by real-time quantitative PCR for the Opuntia virus 1 (OpV1), Opuntia virus 2 (OpV2), and Opuntia becurtovirus in a subset of cactus samples collected in this study. The graphs show (A) the group of single-infected cacti and (B) the cactus samples that presented mixed infection with standard deviation for the triplicate reactions. The Opuntia becurtovirus was the only virus that did not present detectable levels of genomic units.

Figure 7

Bar graph of the viral loads (genomic units/ng total DNA) observed through real-time quantitative PCR for the Opuntia virus 1 (OpV1) and Opuntia virus 2 (OpV2) in N. benthamiana plants at 17 and 45 days post-inoculation. (A) N. benthamiana inoculated with OpV1; (B) N. benthamiana inoculated with OpV2 and (C) N. benthamiana plants with mixed infection of OpV1 and OpV2 with standard deviation for the triplicate reactions.

Figure 8

Sequence similarity network analysis of the CP amino acid sequences of geminiviruses present in GenBank (dataset was created with an amino acid identity cut-off of 90%), together with representatives of the 11 genotypes from OpV2, 15 genotypes from OpV1, and one Opuntia becurtovirus. Each dot represents a sequence and is colored based on the genus or group. The genera that have known insect vectors are highlighted in a light grey box with the insect vector name displayed at the top. Clusters or singletons marked with a brown halo have no known insect vector associated with them. ApGV, apple geminivirus; ACSV, Axonopus compressus streak virus; CaCDaV, Camellia chlorotic dwarf-associated virus; CCDaV, citrus chlorotic dwarf-associated virus; DfasMV, dragonfly-associated mastrevirus; CBCSV, common bean curly stunt virus; ECSV, Eragrostis curvula streak virus; EMSV, Eragrostis minor streak virus; GraGV, grapevine geminivirus; JmaV, Juncus maritimus-associated virus; LaaV, Limeum africanum-associated virus; MCaV, mulberry crinkle- associated virus; MiSV, Miscanthus streak virus; MSMV, maize streak Reunion virus; PYLCV, parsley yellow leaf curl virus; PMLCV-1, paper mulberry leaf curl virus 1; PMLCV-1, paper mulberry leaf curl virus 2; PCMoV, passion fruit chlorotic mottle virus; PgaV, Polygala garcinii-associated virus; RLV1, rice latent virus 1; RLV2, rice latent virus 2; SMaV, switchgrass mosaic-associated virus; SpSMV1, sweet potato symptomless mastrevirus 1; SSMV1, Sporobolus striate mosaic virus 1; SSMV2, Sporobolus striate mosaic virus 2; SStV-A, sugarcane striate virus A; SStV-D, sugarcane striate virus D; SWSV, sugarcane white streak virus; TaGV, tomato-associated geminivirus; ToALCV, tomato apical leaf curl virus.