Serendipitous identification of a new Iflavirus‐like virus infecting tomato and its subsequent characterization

The genomic sequence of a previously undescribed virus was identified from symptomless tomato plants (Solanum lycopersicum). The viral genome is a positive‐sense ssRNA molecule of 8506 nucleotides. It is predicted to encode a single polyprotein of 314·5 kDa that is subsequently processed into three coat protein components of 13·7, 17·9 and 13·5 kDa, and a viral replicase of approximately 207 kDa with conserved motifs for a helicase, a protease and RNA‐dependent RNA polymerase (RdRp). Pairwise analysis of the deduced amino acid sequence of the RdRp revealed that it shares closest identity with members of the family Iflaviridae, genus Iflavirus (19–47% identity). Evidence of replication in plants was detected by RT‐PCR of the viral replicative strand, and short interfering RNAs (siRNAs) matching the virus. The name Tomato matilda virus (TMaV) is proposed, and furthermore, that the genus Tomavirus (Tomato matilda virus) be created within the family Iflaviridae. This is the first report of a plant‐infecting virus resembling members of the Iflaviridae.     

Pathogenetic roles of beet necrotic yellow vein virus RNA5 in the exacerbation of symptoms and yield reduction,development of scab-like symptoms,and Rz1-resistance breaking in sugar beet

Beet necrotic yellow vein virus (BNYVV) generally has a four-segmented positive-sense RNA genome (RNAs 1–4), but some European and most Asian strains have an additional segment, RNA5. This study examined the effect of RNA5 and RNA3 on different sugar beet cultivars using a Polymyxa-mediated inoculation system under field and laboratory conditions. In field tests, the degree of sugar yield served as an index for assessing the virulence of BNYVV strains. Japanese A-II type isolates without RNA5 caused mostly 15%–90% sugar yield reductions, depending on the susceptibility of sugar beet cultivars, whereas the isolates with RNA5 induced more than 90% yield losses in the seven susceptible cultivars, but small yield losses in one Rz1-resistant and Rizor cultivars. However, a laboratory-produced isolate containing RNA5 but lacking RNA3 caused higher yield losses in Rizor than in susceptible plants, and induced scab-like symptoms on the root surface of both susceptible and resistant plants. In laboratory tests, A-II type isolates without RNA5 had low viral RNA accumulation levels in roots of Rizor and Rz1-resistant plants at early stages of infection, but in the presence of RNA5, viral RNA3 accumulation levels increased remarkably. This increased RNA3 accumulation was not observed in roots of the WB42 accession with the Rz2 gene. In contrast, the presence of RNA3 did not affect RNA5 accumulation levels. Collectively, this study demonstrated that RNA5 is involved in the development of scab-like symptoms and the enhancement of RNA3 accumulation, and suggests these characteristics of RNA5 are associated with Rz1-resistance breaking.     

Yellow tailflower mild mottle virus and Pelargonium zonate spot virus co‐infect a wild plant of red‐striped tailflower in Australia

Isolates of an Australian indigenous virus, Yellow tailflower mild mottle virus (YTMMV‐Kalbarri), and an exotic virus, Pelargonium zonate spot virus (PZSV‐SW13), are described from Anthocercis ilicifolia subsp. ilicifolia (red‐striped tailflower, family Solanaceae), a species endemic to Western Australia. This is the first report of either virus from this plant species. The complete genome sequences of YTMMV‐Kalbarri and of PZSV‐SW13 were obtained. YTMMV‐Kalbarri shared 97% nucleotide pairwise identity with the sequence of the type isolate YTMMV‐Cervantes. The sequence PZSV‐SW13 shared greatest sequence identity with the partial sequence of an Australian isolate of PZSV, also from a wild plant, and with a sunflower‐derived isolate of PZSV from Argentina. An experimental host range study was done of YTMMV‐Kalbarri using cultivated and wild solanaceous and non‐solanaceous plants. Most solanaceous plants became systemically infected, with symptoms of systemic infection ranging from symptomless to whole plant necrosis. Based on these studies, it is suggested that YTMMV has the potential to become a pathogen of commercial species of Solanaceae. This study provides further evidence that PZSV is present in wild plants in Australia, in this case an indigenous host species, and possible routes by which it invaded Australia are discussed.     

Resistance to Plum pox virus in plants expressing cytosolic and nuclear single‐chain antibodies against the viral RNA NIb replicase

The expression of engineered single‐chain variable fragments specific to the NIb RNA replicase of Plum pox virus (PPV) (scFv2A) in transgenic plants was successfully used as a strategy to interfere with viral infection. Different scFv2A fusion proteins were constructed to target those subcellular compartments, such as the cytosol, endoplasmic reticulum (ER) membrane structures and the nucleus, where NIb protein presumably accumulates. Several transgenic lines of Nicotiana benthamiana plants expressing the scFv2A targeted to the cytosol (2A lines), ER (6K2 lines) and nucleus (NLS lines) were obtained. The protective effect of scFv expression was determined by mechanical virus inoculation in five 2A, three 6K2 and four NLS transgenic lines. The strongest resistance was afforded with the 2A‐3 (six non‐infected plants out of 10), 6K2‐1 (17 out of 33) and NLS‐11 (16 out of 19) transgenic lines. The success of this interference with PPV infection opens new possibilities for the control of this RNA virus and could be exploited not only to confer resistance in transgenic plants, but also to elucidate the role of the non‐structural NIb protein in different cell compartments during viral infection.     

Impact of the potato inoculation date on potato virus Y load and viral distribution in daughter tubers at harvest

Aged plants are more difficult to infect than young plantlets. This modification of susceptibility is described as mature plant resistance (MPR). For potato virus Y (PVY), MPR is known to lead to low infection rates of plants inoculated at the postflowering stage and a decrease in the number of infected daughter tubers. However, the impact of inoculation date on the capacity of PVY to accumulate in daughter tubers has not been studied so far. Field and greenhouse experiments were carried out to better understand PVY epidemiology and to help potato growers to evaluate consequences of early/late infections on the quality of their crops. In field trials, potato plants (cv. Bintje) were covered by insectproof nets from planting to harvest except for a 14-day period to expose plants to natural PVY infections. Under controlled conditions, potato plants were mechanically inoculated with PVY at different dates from preflowering stages (early inoculations) to postflowering stage (late inoculations). At harvest, daughter tubers were individually collected and analysed to define proportions and viral load of infected tubers according to the time between virus inoculation and harvest. Our results showed that although the age of plants at the time of inoculation can modify their susceptibility to PVY infection, in return, early and late PVY inoculations lead to similar rates of infected tubers at the plant scale and equivalent viral accumulation in infected tubers. All together, these data revealed that both early/late infections are high risks for the sanitary quality of potato tubers.     

Occurrence of a new recombinant begomovirus species infecting tomato in the Al‐Batinah region of Oman

Whitefly‐transmitted begomoviruses are the most important limiting factor for tomato cultivation in Oman, particularly in the Al‐Batinah region, the major agricultural area of the country. Commercial farms in the Al‐Batinah region were surveyed during January–March 2013. Samples of tomato showing leaf curl disease symptoms typical of begomoviruses were collected and analysed. Full‐length sequences of five clones were shown to have relatively low percentage identity values to known begomoviruses, with the highest (88·6%) to isolates of Tomato leaf curl Oman virus (ToLCOMV), a begomovirus previously reported in Oman, indicating that these represent a newly identified species, for which the name Tomato leaf curl Barka virus (ToLCBrV) is proposed. Four isolates of ToLCBrV were found associated with Tomato leaf curl betasatellite (ToLCB). The five isolates of ToLCBrV characterized in this study were shown to be recombinants, with ToLCOMV as the major parent, and a fragment of Croton yellow vein virus (CrYVV) spanning the 3′ half of the replication‐associated protein. The significance of these findings is discussed.     

Viral infection‐induced endoplasmic reticulum stress and a membrane‐associated transcription factor NbNAC089 are involved in resistance to virus in Nicotiana benthamiana

Endoplasmic reticulum (ER) stress may induce two cell defence pathways, the unfolded protein response (UPR) or programmed cell death (PCD) upon unmitigated stress. This study confirmed that viral infection could induce ER stress through changing ER morphology and up‐regulating ER stress‐related genes, including NbNAC089. AtNAC089 serves as an ER stress sensor to regulate PCD in Arabidopsis. In this study, Nicotiana benthamiana NbNAC089 was identified. The gene encoded a 409 amino acid protein with a putative transmembrane domain near the C‐terminus and a NAC domain at the N‐terminus. NbNAC089 was localized to the ER membranes, and a truncated form of NbNAC089, lacking the transmembrane domain, was localized to the nucleus. Meanwhile, the full length of NbNAC089 was activated and cleaved in response to viral infection. The results suggest that the native protein may be translocated to the nucleus by release from the membrane during viral infection. Knock‐down of NbNAC089 in N. benthamiana increased susceptibility to Tobacco mosaic virus or Cucumber mosaic virus, and, in addition, promoted up‐regulation of UPR genes but impaired up‐regulation of PCD genes. These results show that NbNAC089 is a negative regulator of UPR and a positive regulator of PCD, and plays a role in the process of viral infection.     

Induction of systemic resistance against Cucumber mosaic virus by Penicillium simplicissimum GP17‐2 in Arabidopsis and tobacco

The plant growth‐promoting fungus, Penicillium simplicissimum GP17‐2, was evaluated for its ability to induce resistance against Cucumber mosaic virus (CMV) in Arabidopsis thaliana and tobacco plants. Treatment with barley grain inoculum (BGI) of GP17‐2 significantly enhanced fresh weight, dry weight and leaf number of A. thaliana and tobacco plants 6 weeks after planting. Two weeks after CMV inoculation, all plants treated with BGI of GP17‐2 or its culture filtrate (CF) showed a significant reduction in disease severity compared with non‐treated control plants, which exhibited severe mosaic symptoms by the end of the experiment. The enzyme‐linked immunosorbent assay (ELISA) demonstrated that CMV accumulation was significantly reduced in plants treated with GP17‐2 or its CF relative to control plants. Based on RT‐PCR, plants treated with GP17‐2 (BGI or CF) also exhibited increased expression of regulatory and defence genes involved in the SA and JA/ET signalling pathways. These results suggested that multiple defence pathways in A. thaliana and tobacco were involved in GP17‐2‐mediated resistance to CMV, although neither the transgenic NahG line, nor the npr1, jar1 or ein3 mutants disrupted the response in A. thaliana. This is the first report to demonstrate the induction of systemic resistance against CMV by GP17‐2 or its CF.     

Integrated management of Striga hermonthica in sorghum using a mycoherbicide and host plant resistance in the Nigerian Sudano‐Sahelian savanna

Striga hermonthica is a major biotic constraint to sorghum production in Nigeria, sometimes causing total yield loss. Recommendations for Striga management often include the use of cultural and agronomic practices, herbicides and host plant resistance when available. The use of biological control has not been commercialized. Fusarium oxysporum (isolate PSM 197)‐based mycoherbicide was used in combination with selected sorghums (the Striga‐resistant cultivar Samsorg 40, and the Striga tolerant landrace Yar'ruruka) as an Integrated Striga Management strategy (ISM) in on‐farm trials in the Sudano‐Sahelian savanna of Nigeria. Crop stands were significantly (P = 0.05) higher in ISM compared with non‐ISM plots on which the mycoherbicide was not applied. Similarly, ISM plots had significantly (P = 0.05) lower Striga counts than non‐ISM plots. Striga emergence was reduced by ISM by around 95%. Sorghum yields were 49.6% higher where integrated management was used. Cost benefit analysis of the ISM package shows that use of the mycoherbicide increased the profitability of sorghum production on Striga‐infested soils. Farmers’ preferences monitored during and after the trials highlighted the need for careful selection and integration of control components into an ISM package.     

Evidence for Lettuce big‐vein associated virus as the causal agent of a syndrome of necrotic rings and spots in lettuce

Lettuce big‐vein associated virus (LBVaV, genus Varicosavirus) was shown to be responsible for characteristic necrotic symptoms observed in combination with big‐vein symptoms in lettuce breeding lines when tested for their susceptibility to lettuce big‐vein disease (BVD) using viruliferous Olpidium virulentus spores in a nutrient film technique (NFT) system. Lettuce plants showing BVD are generally infected by two viruses: Mirafiori lettuce big‐vein virus (MiLBVV, genus Ophiovirus) and LBVaV. New mechanical inoculation methods were developed to separate the two viruses from each other and to transfer both viruses to indicator plants and lettuce. After mechanical inoculation onto lettuce plants MiLBVV induced vein‐band chlorosis, which is the characteristic symptom of BVD. LBVaV caused a syndrome of necrotic spots and rings which was also observed earlier in lettuce plants inoculated in the NFT system, resembling symptoms described for lettuce ring necrosis disease (RND). This observation is in contrast with the idea that LBVaV only causes latent infections in lettuce. De novo next‐generation sequencing demonstrated that LBVaV was the only pathogen present in a mechanically inoculated lettuce plant with symptoms, providing evidence that LBVaV was the causal agent of the observed necrotic syndrome and thus fulfilling Koch’s postulates for this virus. The necrotic syndrome caused by LBVaV in lettuce is referred to as LBVaV‐associated necrosis (LAN).