Resistance Breeding Through RNA Silencing of Host Factors Involved in Virus Replication.

RNA silencing is a sequence-specific suppression of gene expression conserved in eukaryotes including fungi, plants, and animals. Based on this mechanism, crop improvements have been made to confer pathogen resistance and abiotic stress tolerance. Here we have applied this technique to produce virus resistant tomato plants using host genes involved in viral replication. Tomato homologs of Arabidopsis TOM1 involved in tobamovirus replication has been isolated and used to construct the plasmids that carried inverted repeats of the genes for induction of RNA silencing. Tomato plants were transformed by the plasmids via Agrobacterium, and tested for virus resistance. Actually, the T2 and T3 plants showed resistance to tomato mosaic virus. Here we describe the method to construct RNA silencing-inducing plasmids, to transform tomato plants and to check the introduction of transgenes and virus resistance.

Selection Pressure Pathways and Mechanisms of Resistance to the Demethylation Inhibitor-Difenoconazole in Penicillium expansum.

Penicillium expansum causes blue mold, the most economically important postharvest disease of pome fruit worldwide. Beside sanitation practices, the disease is managed through fungicide applications at harvest. Difenoconazole (DIF) is a new demethylation inhibitor (DMI) fungicide registered recently to manage postharvest diseases of pome fruit. Herein, we evaluated the sensitivity of 130 P. expansum baseline isolates never exposed to DIF and determined the effective concentration (EC50) necessary to inhibit 50% germination, germ tube length, and mycelial growth. The respective mean EC50 values of 0.32, 0.26, and 0.18 µg/ml indicate a high sensitivity of P. expansum baseline isolates to DIF. We also found full and extended control efficacy in vivo after 6 months of storage at 1°C. We conducted a risk assessment for DIF-resistance development using ultraviolet excitation combined with or without DIF-selection pressure to generate and characterize lab mutants. Fifteen DIF-resistant mutants were selected and showed EC50 values of 0.92 to 1.4 µg/ml and 1.7 to 3.8 µg/ml without and with a DIF selection pressure, respectively. Resistance to DIF was stable in vitro over a 10-week period without selection pressure. Alignment of the full CYP51 gene sequences from the three wild-type and 15 mutant isolates revealed a tyrosine to phenylalanine mutation at codon 126 (Y126F) in all of the 15 mutants but not in the wild-type parental isolates. Resistance factors increased 5 to 15-fold in the mutants compared to the wild-type-isolates. DIF-resistant mutants also displayed enhanced CYP51 expression by 2 to 14-fold and was positively correlated with the EC50 values (R 2 = 0.8264). Cross resistance between DIF and fludioxonil, the mixing-partner in the commercial product, was not observed. Our findings suggest P. expansum resistance to DIF is likely to emerge in commercial packinghouse when used frequently. Future studies will determine whether resistance to DIF is qualitative or quantitative which will be determinant in the speed at which resistance will develop and spread in commercial packinghouses and to develop appropriate strategies to extend the lifespan of this new fungicide.

Sensitivity of Phacidiopycnis spp. Isolates from Pome Fruit to Six Pre- and Postharvest Fungicides.

Phacidiopycnis washingtonensis and P. pyri cause speck rot and Phacidiopycnis rot on apple and pear, respectively. Infection occurs in the orchard and remains latent, and symptoms appear after months of storage. Decay management relies on orchard sanitation and pre- and postharvest fungicides. In a 2017 survey, speck rot accounted for 6.4% of apple decay in central Washington, whereas Phacidiopycnis rot accounted for 3.9 and 6.7% of total pear decay in Washington and Oregon, respectively. Sensitivities of baseline populations of 110 P. washingtonensis and 76 P. pyri isolates collected between 2003 and 2005 to preharvest fungicides pyraclostrobin (PYRA) and boscalid (BOSC) and to postharvest fungicides thiabendazole (TBZ), fludioxonil (FDL), pyrimethanil (PYRI), and difenoconazole (DFC) were evaluated using a mycelial growth inhibition assay. Mean effective concentrations necessary to inhibit 50% growth (EC50) of P. washingtonensis were 0.1, 0.3, 0.8, 1.8, 2.1, and 4.8 µg/ml for FDL, PYRI, TBZ, DFC, PYRA, and BOSC, respectively. Respective mean EC50 values for P. pyri were 0.2, 0.6, 1.6, 1.1, 0.4, and 1.8 µg/ml. The sensitivity of exposed P. washingtonensis and P. pyri populations collected in 2017 revealed potential shifts toward BOSC and PYRA resistance. The efficacy of the six fungicides to control isolates of each pathogen with different in vitro sensitivity levels was evaluated on apple and pear fruit. FDL, DFC, and PYRI controlled both Phacidiopycnis spp. regardless of their EC50 values after 5 months of storage at 0°C in a regular atmosphere. The consistent occurrence of Phacidiopycnis spp. will require continuous monitoring and development of disease management strategies based on fungicide phenotypes and efficacy of existing fungicides assessed herein.

Molecular analysis of transgenic melon plants showing virus resistance conferred by direct repeat of movement gene of Cucumber green mottle mosaic virus.

Cucumber green mottle mosaic virus (CGMMV) is a major limiting factor in the production of melon plants worldwide. For effective control of this virus using the transgenic approach, the direct repeat of the movement protein gene of CGMMV was used for transforming melon plants by Agrobacterium tumefaciens. PCR and Southern blot analyses of T3 confirmed that they carried the transgene. Northern blot analysis with total RNA showed that transgene transcript RNA as well as siRNA was observed in all plants tested. Separate leaves or individual plants were inoculated with CGMMV and subjected to ELISA and RNA blot analysis using the coat protein gene probe of the virus. Compared to nontransgenic control, these plants were shown to have high virus resistance. Furthermore, cytosine of the transgene DNA in the plants was methylated. Thus, these results reveal that the transgenic lines were highly resistant to the virus through RNA silencing. Key message High virus resistance was obtained in transgenic melon plants with direct repeat of movement protein gene of Cucumber green mottle mosaic tobamovirus through RNA silencing.