Putative recombination signature and significance of insertion/deletion events in the RNA-dependent RNA polymerase coding region of sugarcane yellow leaf virus

The 5898 nucleotide single-strand RNA genome of Sugarcane yellow leaf virus (SCYLV) contains one long open reading frame, which is translated into a 120.6 kDa polyprotein. The sequences of SCYLV isolates from the two SCYLV-susceptible cultivars from Hawaii had a deletion of 48–51 nt in ORF1. SCYLV from 12 sugarcane hybrid cultivars from different origins were tested by RT-PCR using a specific set of primers, to investigate the genome segment for this deletion. Only three cultivars were found not to have the deletion (H87-4319, JA-605 and CP52-43), while SCYLV from nine cultivars (H73-6110, H87-4094, H78-7750, GT54-9, G84-47, H78-4153, H65-7052, C1051-73, Ph-8013) along with aphid (Melanaphis sacchari), which fed on SCYLV-infected H73-6110, contained a deletion of about 50 nt. The deleted sequence was located in the overlap frameshift of ORF1 and ORF2. Thus, ORFs 1 and 2 of SCYLV are translated via ribosomal frameshift and yield the 120.6 kDa viral replicase. ORF1 plays most likely a role in the replication and is a source of large variability among the virus population. To identify possible recombination events located in the RdRp domain of the Hawaiian isolates, two programs were used: RDP v.4.3 and RECCO. It is noteworthy that according both methods Haw73-6110 was found as a potential recombinant. On the other hand, opposed to the RDP package, RECCO revealed that Haw87-4094 isolate was also a recombinant whereas Haw87-4319 was not.     

The Torradovirus‐specific RNA2‐ORF1 protein is necessary for plant systemic infection

Tomato apex necrosis virus (ToANV, species Tomato marchitez virus, genus Torradovirus, family Secoviridae) causes a severe tomato disease in Mexico. One distinctive feature of torradoviruses compared with other members of the family Secoviridae is the presence of an additional open reading frame (ORF) in genomic RNA2 (denominated RNA2‐ORF1), located upstream of ORF2. RNA2‐ORF2 encodes a polyprotein that is processed into a putative movement protein and three capsid proteins (CPs). The RNA2‐ORF1 protein has homologues only amongst other torradoviruses and, so far, no function has been associated with it. We used recombinant and mutant ToANV clones to investigate the role of the RNA2‐ORF1 protein in various aspects of the virus infection cycle. The lack of a functional RNA2‐ORF1 resulted in an inability to systemically infect Nicotiana benthamiana and tomato plants, but both positive‐ and negative‐strand RNA1 and RNA2 accumulated locally in agroinfiltrated areas in N. benthamiana plants, indicating that the RNA2‐ORF1 mutants were replication competent. Furthermore, a mutant with a deletion in RNA2‐ORF1 was competent for virion formation and cell‐to‐cell movement in the cells immediately surrounding the initial infection site. However, immunological detection of the ToANV CPs in the agroinfiltrated areas showed that this mutant was not detected in the sieve elements even if the surrounding parenchymatic cells were ToANV positive, suggesting a role for the RNA2‐ORF1 protein in processes occurring prior to phloem uploading, including efficient spread in inoculated leaves.     

Complete Genome Sequence and Genome Analysis of Eggplant mottled dwarf virus‐Iranian Isolate

The full‐length nucleotide sequence of the Iranian isolate of Eggplant mottled dwarf virus (EMDV), a phytorhabdovirus, was determined using the random polymerase chain reaction method (rPCR) followed by PCR with specific primers to fill in the gaps. The negative‐sense RNA genome of the Iranian isolate of EMDV contains 13154 nucleotides and seven open‐reading frames (ORFs) in the order 3′‐leader‐N‐X‐P‐Y‐M‐G‐L‐trailer‐5′. These ORFs encode the nucleocapsid, X protein (of unknown function), phosphoprotein, Y protein (putative movement protein), matrix protein, glycoprotein and RNA‐dependent RNA polymerase, respectively. EMDV has a 199 nt 3′ leader RNA and a 151 nt 5′ trailer, and the ORFs are separated by conserved intergenic sequences. Phylogenetic analyses indicate that EMDV is most closely related to Potato yellow dwarf virus, which has a distinctly different geographical distribution.     

Molecular Detection and Complete Genome Sequences of Tomato chlorosis virus Isolates from Infectious Outbreaks in China

Tomato chlorosis virus (ToCV) is a whitefly‐transmitted, phloem‐limited, bipartite Crinivirus. In 2012, severe interveinal symptoms characteristic of ToCV infections were observed in greenhouse tomato plants in the Shandong province of China. High levels of infestation by whiteflies (Bemisia tabaci), which transmit ToCV, were also observed on tomato plants in all the greenhouses investigated. The presence of ToCV was confirmed by specific RT‐PCR either in the sampled plants or in the whiteflies collected from the ventral surface of the leaves of diseased plants. The complete genomic nucleotide sequences (RNA1 and RNA2) of the Shandong isolate of ToCV (ToCV‐SDSG) were determined and analysed. ToCV‐SDSG RNA1 consisted of 8594 nucleotides encompassing four open reading frames (ORFs). ToCV‐SDSG RNA2 consisted of 8242 nucleotides encompassing nine ORFs. Phylogenetic analysis suggests that the Chinese ToCV‐SDSG isolate is most similar to the ToCV‐Florida isolate.     

Experimental demonstration of accelerated extinction in source‐sink metapopulations

Population extinction is a fundamental ecological process which may be aggravated by the exchange of organisms between productive (source) and unproductive (sink) habitat patches. The extent to which such source‐sink exchange affects extinction rates is unknown. We conducted an experiment in which metapopulation effects could be distinguished from source‐sink effects in laboratory populations of Daphnia magna. Time‐to‐extinction in this experiment was maximized at intermediate levels of habitat fragmentation, which is consistent with a minority of theoretical models. These results provided a baseline for comparison with experimental treatments designed to detect effects of concentrating resources in source patches. These treatments showed that source‐sink configurations increased population variability (the coefficient of variation in abundance) and extinction hazard compared with homogeneous environments. These results suggest that where environments are spatially heterogeneous, accurate assessments of extinction risk will require understanding the exchange of organisms among population sources and sinks. Such heterogeneity may be the norm rather than the exception because of both the intrinsic heterogeneity naturally exhibited by ecosystems and increasing habitat fragmentation by human activity.     

Molecular characterisation of a calmodulin gene,VcCaM1, that is differentially expressed under aluminium stress in highbush blueberry

Calmodulin (CaM), a small acidic protein, is one of the best characterised Ca²⁺ sensors in eukaryotes. This Ca²⁺‐regulated protein plays a critical role in decoding and transducing environmental stress signals by activating specific targets. Many environmental stresses elicit changes in intracellular Ca²⁺ activity that could initiate adaptive responses under adverse conditions. We report the first molecular cloning and characterisation of a calmodulin gene, VcCaM1 (Vaccinium corymbosum Calmodulin 1), in the woody shrub, highbush blueberry. VcCaM1 was first identified as VCAL19, a gene induced by aluminium stress in V. corymbosum L. A full‐length cDNA of VcCaM1 containing a 766‐bp open reading frame (ORF) encoding 149 amino acids was cloned from root RNA. The sequence encodes four Ca²⁺‐binding motifs (EF‐hands) and shows high similarity (99%) with the isoform CaM 201 of Daucus carota. Expression analyses showed that following Al treatment, VcCaM1 message level decreased in roots of Brigitta, an Al‐resistant cultivar, and after 48 h, was lower than in Bluegold, an Al‐sensitive cultivar. VcCAM1 message also decreased in leaves of both cultivars within 2 h of treatment. Message levels in leaves then increased by 24 h to control levels in Brigitta, but not in Bluegold, but then decreased again by 48 h. In conclusion, VcCaM1 does not appear to be directly involved in Al resistance, but may be involved in improved plant performance under Al toxicity conditions through regulation of Ca²⁺ homeostasis and antioxidant systems in leaves.