Biology and chemistry of an Umbravirus like 2989 bp single stranded RNA as a possible causal agent for Opuntia stunting disease (engrosamiento de cladodios) - A Review

Abstract

Perhaps the most economically important disease of Opuntia ficus indica fruit cacti in Mexicois the “engrosamiento de cladodios” or macho disease. The symptoms of this disease, whichhas been suggested to be caused by a phytoplasma, are severe stunting of cladodes, flowersand fruits. In the mid-1980s this disease appeared in commercial cactus fruit orchards ofD’Arrigo Bros near Gonzalez, California. It was performed more than 30 PCR-based tests forviruses as well as various extraction methods and polymerase chain reaction (PCR) tests forphytoplasmas but were unable to find any of the known viruses or mycoplasmas in thestrongly symptomatic cactus with this disease. As almost all plant viruses go through areplication phase involving double stranded RNA (dsRNA), a dsRNA extraction wasperformed and a dsRNA species of about 600 bp identified. Then, reverse-transcribed thedsRNA, amplified the resultant cDNA by PCR, and cloned and sequenced the 600 bpfragment that were identified in symptomatic tissue. When this sequence was compared totranslated DNA in the National Center for Biotechnology Information (NCBI) nucleotide database (BLAST analysis) it was most similar to the Tobacco bushy top virus (E score of 2e-39),which is a single stranded RNA virus with no DNA intermediate. Primers made from this 630bp fragment were used to extend this sequence to 2989 sequence. This sequence appears tobe a full-length sequence with three open reading frames (ORF) and is shorter than theclosest class of viruses, the Umbraviruses that can be spread by mechanical transmissionand by aphids. It was not possible to transmit the virus or symptoms mechanically. Over asix-year period using traditional PCR, this virus was found in hundreds of symptomatic cactibut not in non-symptomatic pads. RT-PCR has found low levels of this virus on nonsymptomaticcladodes (3.7 fg) on a symptomatic plant and much higher concentrations(1x102 to 1x105 fg) on symptomatic cladodes from the same plant. Black bean aphids (Aphis fabae), that are the vector for a closely related Umbravirus known as groundnut rosetta virus,have been routinely found on the unopened flowers of cactus. This Umbravirus was found inaphids feeding on symptomatic cladodes. As Umbraviruses cannot infect plants without acompanion Luteovirus, that provides the protein coat for the Umbravirus, degenerateLuteovirus primers were used and a probable incomplete Luteovirus-like 4797 bp sequencewas found on aphids feeding on symptomatic cactus. This Luteovirus was not found inOpuntia cladodes using PCR. A micro RNA assembly of six pooled symptomatic Opuntiasdid not find a contig that spanned the 4797 putative Luteovirus sequence, but somefragments as large as 44 bp were exact matches to the Luteovirus. As Umbraviruses occurthroughout the plant but Luteoviruses only occur in the phloem, lower Luteovirusconcentrations would be expected. Two successive one hour 60°C heat treatmentseliminated these symptoms on new growth that was also PCR negative. A 5839 bpPotexvirus was found in some of these cladodes but its presence was not correlated with anysymptoms. Similar symptomatic cacti in Italy, South Africa and Mexico should be examinedwith these primers and dsRNA to see if similar correlations between presence/absence of thisfragment and symptomatic plants can be obtained. It is suggested that this disease be knownas OSD (Opuntia Stunting Disease).

Umbravirus-like RNA viruses are capable of independent systemic plant infection in the absence of encoded movement proteins

“…Similarly, it was expected that ULVs would require a helper virus to support infection as is observed for umbraviruses, which lack a CP and silencing suppressor. Curiously, several reports using next-generation sequencing did not identify a helper virus in plants infected with Group 2 ULVs, despite specific attempts to do so [ 40 , 44 , 60 ]. We have found that agroinfiltration of CY1 in N .…”

“…General time reversible model with gamma distribution rates and invariable frequency (GTR+G+I) was determined to be the best-fit substitution model in MEGA X and was implemented to construct the tree. The following ULV sequences were used: PMeV2 (KT921785), PpVQ (MT113180), BabVQ (MT113182) [ 42 , 93 , 94 ], OULV (MH579715, MT909563, MT909562) [ 44 ], FULV (MW480492, MW480492) [ 60 ], CY1 (JX101610) [ 40 ], CY2 (MT893741) [ 77 ], WULV (OK573479) [ 95 ], GULV(OP886321) [ 96 ], PIULV (OL330774) [ 97 ], SbaVa (MK211274) [ 98 ], TULV (OK018180) [ 99 ], MULV (OM937759) [ 41 ], JgULV (OM937760) [ 41 ], SULV (MN868593) [ 100 ], EMaV (MF415880), AgULV (OP660856), GPpTV1 (OM514399), ArULV (OQ102001) [ 101 ]. To analyze the phylogenetic relationship of ORF5 proteins with selected CPs and MPs, protein sequences were aligned using PROMALS3D [ 53 ] and trimmed in MEGA X.…”

“…Surprisingly, several Group 2 ULVs were reported to exist in plants in the apparent absence of a helper virus [ 40 – 42 , 44 ]. These include members of Class 1 that infect papaya and babaco trees throughout Ecuador, and Class 2 opuntia umbra-like virus (OULV) found in opuntia plants.…”

A Guide to Cannabis Virology: From the Virome Investigation to the Development of Viral Biotechnological Tools

“…While it is known that Luteoviridae provides the coat protein for trans-encapsidation of the Umbravirus RNA and allows its persistent transmission through an aphid vector [ 47 , 48 ], no helper virus has currently been associated with ulaRNAs. Indications that ulaRNAs must also possess a helper virus come from the finding of a Polerovirus-like virus along with OULV in aphids feeding on opuntia [ 49 ], even though evidence on actual transmissibility has never been provided. Thus, it can be hypothesized that, in cannabis as well as in other hosts, the occurrence of ulaRNAs dissociated from helper viruses comes from their loss after aphid transmission or during plant vegetative propagation.…”