The Cucumber mosaic virus (CMV) 2b protein is a counter-defense factor and symptom determinant. Conserved domains in the 2b protein sequence were mutated in the 2b gene of strain Fny-CMV. The effects of these mutations were assessed by infection of Nicotiana tabacum, N. benthamiana, and Arabidopsis thaliana (ecotype Col-0) with mutant viruses and by expression of mutant 2b transgenes in A. thaliana. We confirmed that two nuclear localization signals were required for symptom induction and found that the N-terminal domain was essential for symptom induction. The C-terminal domain and two serine residues within a putative phosphorylation domain modulated symptom severity. Further infection studies were conducted using Fny-CMVΔ2b, a mutant that cannot express the 2b protein and that induces no symptoms in N. tabacum, N. benthamiana, or A. thaliana ecotype Col-0. Surprisingly, in plants of A. thaliana ecotype C24, Fny-CMVΔ2b induced severe symptoms similar to those induced by the wild-type virus. However, C24 plants infected with the mutant virus recovered from disease while those infected with the wild-type virus did not. Expression of 2b transgenes from either Fny-CMV or from LS-CMV (a mild strain) in Col-0 plants enhanced systemic movement of Fny-CMVΔ2b and permitted symptom induction by Fny-CMVΔ2b. Taken together, the results indicate that the 2b protein itself is an important symptom determinant in certain hosts. However, they also suggest that the protein may somehow synergize symptom induction by other CMV-encoded factors.

Several plant virus mutants, in which genes encoding silencing suppressor proteins have been deleted, are known to induce systemic or localized RNA silencing against themselves and other RNA molecules containing homologous sequences. Thus, it is thought that many cases of cross-protection, in which infection with a mild or asymptomatic virus mutant protects plants against challenge infection with closely related virulent viruses, can be explained by RNA silencing. We found that a cucumber mosaic virus (CMV) mutant of the subgroup IA strain Fny (Fny-CMVD2b), which cannot express the 2b silencing suppressor protein, cross-protects tobacco (Nicotiana tabacum) and Nicotiana benthamiana plants against disease induction by wild-type Fny-CMV. However, protection is most effective only if inoculation with Fny-CMVD2b and challenge inoculation with wild-type CMV occurs on the same leaf. Unexpectedly, FnyCMVD2b also protected plants against infection with TC-CMV, a subgroup II strain that is not closely related to Fny-CMV. Additionally, in situ hybridization revealed that Fny-CMVD2b and Fny-CMV can co-exist in the same tissues but these tissues contain zones of Fny-CMVD2b-infected host cells from which Fny-CMV appears to be excluded. Taken together, it appears unlikely that cross-protection by Fny-CMVD2b occurs by induction of systemic RNA silencing against itself and homologous RNA sequences in wild-type CMV. It is more likely that protection occurs through either induction of very highly localized RNA silencing, or by competition between strains for host cells or resources. INTRODUCTIONCucumber mosaic virus (CMV) is the type species of the genus Cucumovirus of the family Bromoviridae (Van Regenmortel et al., 2000). Based on serological relationships and sequence criteria, the species is divided into three distinct subgroups: IA, IB and II (Roossinck et al., 1999). CMV has the largest host range of any known plant virus and is transmitted in a non-persistent manner by aphids belonging to over 80 species. Taken together, these factors probably contribute to the worldwide distribution of the virus and its economic importance (Palukaitis et al., 1992;Palukaitis & García-Arenal, 2003). The CMV genome consists of three positive-sense RNA molecules. These are RNAs 1, 2 and 3, which also function as mRNAs for the synthesis of the 1a and 2a replicase proteins, and the 3a movement protein, respectively. During replication, subgenomic mRNAs encoding additional proteins are synthesized. The subgenomic RNA 4, derived from RNA 3, is the mRNA for CMV coat protein (CP) and RNA 4A, which is derived from RNA 2, is the mRNA for the multifunctional 2b protein (Ding et al., 1994). Direct control of CMV or prevention of its transmission by aphids by using insecticides is difficult to achieve. One promising approach for controlling CMV is the use of pathogen-derived transgenes in genetically modified plants (Palukaitis & Zaitlin, 1997;Gaba et al., 2004). Pathogenderived resistance to CMV and other viruses works either through the triggering of...

A range of fungal isolates was tested in a three-stage screening system for their ability to degrade sclerotia of Sclerotium cepivorum on agar and in soil, and to reduce white rot disease on onion seedlings. Biological control agents (BCAs) were identified that could degrade up to 60% of sclerotia in soil and significantly reduce white rot disease on onion seedlings. The efficacy of the BCAs was enhanced when applied as wheat bran cultures compared with spore suspensions, and two of the best BCAs from the screening procedures were both identified as Trichoderma viride (L4, S17A). When L4 and S17A were fluid-drilled in guar gum with bulb onion seed in the field white rot symptoms were significantly reduced, but stem base applications applied mid-season had little effect. The strategy of selecting and using BCAs that degrade sclerotia of S. cepivorum and integration with other control methods is discussed.

Laboratory assays demonstrated that two isolates of Trichoderma viride and one isolate of Trichoderma pseudokoningii degraded up to 80% of sclerotia of four isolates of Sclerotium cepivorum in a silty clay soil, and also degraded up to 60% of sclerotia in three other soil types. Relationships were defined between the degree of sclerotial degradation by the two T. viride isolates in the silty clay soil and both temperature and soil water potential. Sclerotia were degraded between 10 and 25°C at −0·00012 MPa, but there was little activity of T. viride at 5°C or at −4 MPa. Degradation of S. cepivorum sclerotia also occurred in the absence of Trichoderma at soil water potentials approaching saturation. Experiments using onion seedling bioassays showed that the efficacy of Trichoderma isolates for the control of white rot using the same selection of soils and S. cepivorum isolates was variable, but that there was significant disease control overall. The importance of environmental factors and pathogen isolate in relation to effective biological control of white rot is discussed.

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