Plum pox virus (PPV), is genetically diverse with nine different strains identified. Mutations, indel events, and inter-strain recombination events are known to contribute to the genetic diversity of PPV. This is the first report of intra-strain recombination events that contribute to PPV’s genetic diversity. Fourteen (14) isolates of the PPV strain Winona (W) were analysed including 9 new strain W isolates sequenced completely in this study. Isolates of other strains of PPV with more than 1 isolate with the complete genome sequence available in GenBank were included also in this study, for comparison and analysis. Five intra-strain recombination events were detected among the PPV W isolates, 1 among PPV C strain isolates, and 1 among PPV M strain isolates. Four (29%) of the PPV W isolates analysed are recombinants; one of which (P2-1) is a ‘mosaic’, with three recombination events identified. A new inter-strain recombinant event was identified between a strain M isolate and a strain Rec isolate, a known ...

Rubus stunt, caused by ‘Candidatus Phytoplasma rubi’ (Rubus stunt phytoplasma; RSP), is an economically important disease of Rubus spp. . This disease occurs in wild and cultivated Rubus in Europe but has not been reported from North America; however, its major leafhopper vector is well established in Western Canada and the United States. RSP has the potential to impact the cane-fruit industry by significantly compromising yields and impacting export potential for Rubus propagation material. To mitigate the risk of this disease entering or establishing, import and export testing of propagation material is a phytosanitary requirement in Canada, the U.S and other countries regulating RSP. In the absence of a specific test for RSP, the current testing scheme involves the use of a generic test to screen for phytoplasmas followed by additional time-consuming procedures to confirm the phytoplasma species. In this study, a real-time PCR assay, targeting 154 bp region of tuf gene, was developed for sensitive and specific detection of RSP in Rubus spp. The developed assay detected a minimum of 5 target copies, and no cross-reactivity was observed even with the ‘Ca. P. rubi’-related strain associated with blackberry witches’ broom which differs from RSP only by a single nucleotide polymorphism in the target region. Repeatability of the developed assay was checked on two real-time PCR platforms with acceptable results. In conclusion, this real-time PCR assay provides a sensitive and specific detection of RSP for mitigating the introduction and spread of Rubus stunt disease in Rubus spp..

Blackcurrant leaf chlorosis associated virus (BCLCaV) was detected recently by next-generation sequencing (NGS) and a new and distinct species in the genus Idaeovirus was proposed. Analysis of NGS-derived paired-end reads revealed the existence of bridge reads encompassing the 3′-terminus and 5′-terminus of RNA-2 or RNA-3 of BCLCaV. The full RNA-2 or RNA-3 could be amplified using outward facing or abutting primers; also, RNA-2/RNA-3 could be detected even after three consecutive RNase R enzyme treatments, with denaturation at 95 °C preceding each digestion. Evidence was obtained indicating that there are circular forms of BCLCaV RNA-2 and RNA-3.

GroE is a chaperonin folding system consisting of GroEL (Cpn60, a 60 kDa chaperonin), and the smaller co-chaperonin GroES (Cpn10). Many ‘client’ proteins require GroE to fold properly, including several that are essential for cell viability. GroE is found in nearly all bacteria and eukaryotes. Mollicutes are the only micro-organisms that lack GroE in almost all cases. Only two clades of Mollicutes have retained the ancestral GroE system, or perhaps reacquired one; these exceptions include the family Acholeplasmataceae , consisting of the genera Acholeplasma and ‘ Candidatus Phytoplasma’. The role of GroEL in these unique Mollicutes is a source of speculation, given how many non-canonical ‘moonlighting’ roles have been ascribed to this protein. GroEL has been suggested to play a role in pathogenesis in plant and animal pathogenic Mollicutes by binding to host cells and facilitating invasion. However, in one further layer of exception, the phytopathogenic taxon ‘ Candidatus Phytoplasma pruni’ (ribosomal group 16SrIII) was reported in 2012 to lack a GroE system. This study confirms the lack of a functional GroE system in 16SrIII by providing two new, high-quality, non-fragmented genome assemblies, as well as a thorough survey of other 16SrIII genomes for genes encoding GroEL/GroES, including those that may not resemble phytoplasma GroEL (i.e. acquired by horizontal gene transfer, HGT). We discuss the implications of a clearly phytopathogenic, invasive group of Mollicutes that nevertheless lacks GroE, in light of the presumed role of GroEL for this species. We determined that multiple genomes of 16SrIII contain short, non-functional groEL pseudogenes, while most of the reported genomes lack any semblance of a GroE system. Examination of the new assemblies allowed us to rule out HGT as a means of GroE acquisition.

Blackcurrant leaf chlorosis associated virus (BCLCaV) was detected recently by next-generation sequencing (NGS) and proposed as a new and distinct species in the genus Idaeovirus. Genomic components of BCLCaV that were detected and confirmed include: 1) RNA-1 that is monocistronic and encodes the replicase complex; 2) a bicistronic RNA-2 that encodes a movement protein (MP) and the coat protein (CP) of the virus, with open reading frames (ORF) that overlap by a single adenine (A) nucleotide (nt) representing the third position of an opal stop codon of the MP ORF2a and the first position of the start codon of the CP ORF2b; 3) a subgenomic form of RNA-2 (RNA-3) that contains ORF2b; and 4) a concatenated form of RNA-2 that consists of a complementary and inverted RNA-3 conjoined to the full-length RNA-2. Analysis of NGS-derived paired-end reads revealed the existence of bridge reads encompassing the 3’-terminus and 5’-terminus of RNA-2 or RNA-3 of BCLCaV. The full RNA-2 or RNA-3 could be amplified using outward facing or abutting primers; also RNA-2/RNA-3 could be detected even after three consecutive RNase R enzyme treatments with denaturation at 95 oC preceding each digestion. Evidence was obtained indicating that there are circular forms of BCLCaV RNA-2 and RNA-3.

GroE is a chaperonin folding system consisting of GroEL (Cpn60, a 60 kDa chaperonin), and the smaller co-chaperonin GroES (Cpn10). Many “client” proteins require GroE to fold properly, including several that are essential for cell viability. Unsurprisingly then, GroE is found in nearly all bacteria and eukaryotes. Mollicutes are the only microorganisms that lack GroE in almost all cases. Only two clades of Mollicutes have retained the ancestral GroE system, or perhaps reacquired one; these exceptions include the family Acholeplasmataceae (consisting of the genera Acholeplasma and Phytoplasma). The role of GroEL in these “exceptional” Mollicutes is a source of speculation, given how many non-canonical “moonlighting” roles have been ascribed to this protein. GroEL has been suggested to play a role in pathogenesis in plant and animal pathogenic Mollicutes, by binding to host cells and facilitating invasion. However, in one further layer of exception, the phytopathogenic taxon ‘Candidatus Phytoplasma pruni’ (ribosomal group 16SrIII), was reported to lack a GroE system. This study confirms the lack of a functional GroE system in 16SrIII by providing two new, high quality, non-fragmented genome assemblies, as well as a thorough survey of other 16SrIII genomes for genes encoding GroEL/GroES, including those that may not resemble phytoplasma groEL (ie. acquired by horizontal gene transfer, HGT). We discuss the implications of a clearly phytopathogenic, invasive group of Mollicutes that nevertheless lacks GroE, in light of the presumed role of GroEL for these microorganisms. We determined that three groups of genomes of 16SrIII contain short, non-functional groEL pseudogenes, while most of the reported genomes lack any semblance of a GroE system. Examination of the new assemblies allowed us to rule out HGT as a means of GroE acquisition.