Fire Blight Susceptibility in Lilium spp. Correlates to Sensitivity to Botrytis elliptica Secreted Cell Death Inducing Compounds

Fire blight represents a widespread disease in Lilium spp. and is caused by the necrotrophic Ascomycete Botrytis elliptica. There are >100 Lilium species that fall into distinct phylogenetic groups and these have been used to generate the contemporary commercial genotypes. It is known among lily breeders and growers that different groups of lilies differ in susceptibility to fire blight, but the genetic basis and mechanisms of susceptibility to fire blight are unresolved. The aim of this study was to quantify differences in fire blight susceptibility between plant genotypes and differences in virulence between fungal isolates. To this end we inoculated, in four biological replicates over 2 years, a set of 12 B. elliptica isolates on a panel of 18 lily genotypes representing seven Lilium hybrid groups. A wide spectrum of variation in symptom severity was observed in different isolate-genotype combinations. There was a good correlation between the lesion diameters on leaves and flowers of the Lilium genotypes, although the flowers generally showed faster expanding lesions. It was earlier postulated that B. elliptica pathogenicity on lily is conferred by secreted proteins that induce programmed cell death in lily cells. We selected two aggressive isolates and one mild isolate and collected culture filtrate (CF) samples to compare the cell death inducing activity of their secreted compounds in lily. After leaf infiltration of the CFs, variation was observed in cell death responses between the diverse lilies. The severity of cell death responses upon infiltration of the fungal CF observed among the diverse Lilium hybrid groups correlated well to their fire blight susceptibility. These results support the hypothesis that susceptibility to fire blight in lily is mediated by their sensitivity to B. elliptica effector proteins in a quantitative manner. Cell death-inducing proteins may provide an attractive tool to predict fire blight susceptibility in lily breeding programs.

A Virus-Induced Gene-Silencing Vector Based on a Mild Isolate of Watermelon Mosaic Virus for Reverse Genetic Analyses in Cucurbits

As a response to viral infections, host plants trigger an RNA-mediated gene silencing defense, to which viruses respond with the expression of viral-encoded RNA silencing suppressors. If virus clones are manipulated to include sequences homologous to host endogenous genes, these are also targeted by the plant RNA silencing machinery. This so-called virus-induced gene silencing (VIGS) has become a powerful technique for reverse genetic analyses in plants, as an alternative to labor-intensive genome transformation. We show that a mild isolate of Watermelon mosaic virus (WMV, genus Potyvirus) can be used as a VIGS vector for reverse genetic analyses in melon. Recombinant WMV clones —in which fragments of the melon Phytoene desaturase (PDS) mRNA were inserted in sense, antisense, and hairpin modalities— induced a distinctive phenotype and significant silencing of the endogenous gene. While the foreign fragments in sense and antisense orientations were stable in the viral progeny, the hairpin was quickly lost. Nevertheless, the hairpin construct triggered a maintained silencing effect comparable to those of the sense and antisense constructs. The suitability of WMV as a VIGS vector was further confirmed targeting melon Magnesium chelatase subunit I (CHLI). These results also support that, although potyviruses express a strong silencing suppressor that usually precludes VIGS, mild isolates of this kind of viruses can be used as VIGS vectors. Finally, to facilitate the use of this new tool by cucurbit geneticists, we describe plasmid pGWMV-VIGS that allows easy cloning fragments of the genes of interest in a single Gibson assembly reaction.

Genetic characterization of a mild isolate of papaya ringspot virus type-P (PRSV-P) and assessment of its cross-protection potential under greenhouse and field conditions

A mild isolate of Papaya ringspot virus type-P, abbreviated as PRSV-mild, from Ecuador was sequenced and characterized. The most distinguishing symptom induced by PRSV-mild was gray powder-like leaf patches radiating from secondary veins. In greenhouse experiments, PRSV-mild did not confer durable protection against a severe isolate of the virus (PRSV-sev), obtained from the same field. Furthermore, isolate specific detection in mixed-infected plants showed that PRSV-sev becomes dominant in infections, rendering PRSV-mild undetectable at 90–120 days post superinfection. Virus testing using isolate-specific primers detected PRSV-mild in two out of five surveyed provinces, with 10% and 48% of incidence in Santo Domingo and Los Ríos, respectively. Comparative genomics showed that PRSV-mild lacks two amino acids from the coat protein region, whereas amino acid determinants for asymptomatic phenotypes were not identified. Recombination events were not predicted in the genomes of the Ecuadorean isolates. Phylogenetic analyses placed both PRSV-mild and PRSV-sev in a clade that includes an additional PRSV isolate from Ecuador and others from South America.

Genetic characterization of a mild isolate of papaya ringspot virus type-P (PRSV-P) and assessment of its cross-protection potential under greenhouse and field conditions

A mild isolate of Papaya ringspot virus type-P, abbreviated as PRSV-mild, from Ecuador was sequenced and characterized. The most distinguishing symptom induced by PRSV-mild was gray powder-like leaf spots radiating from secondary veins. In greenhouse experiments, PRSV-mild did not confer durable protection against a severe isolate of the virus (PRSV-sev), obtained from the same field. Furthermore, isolate specific detection in cross-protected plants showed that PRSV-sev becomes dominant in infections, rendering PRSV-mild undetectable at 90 - 120 days post superinfection. Virus testing using isolate-specific primers detected PRSV-mild in two out of five surveyed provinces, with 10% and 48% of incidence in Santo Domingo and Los Ríos, respectively. Comparative genomics showed that PRSV-mild lacks two amino acids from the coat protein region, whereas amino acid determinants for asymptomatic phenotypes were not identified. Recombination events were not predicted in the genomes of the Ecuadorean isolates. Phylogenetic analyses placed both PRSV-mild and PRSV-sev in a clade that includes an additional PRSV isolate from Ecuador and others from South America.

Control of Russet Crack Disease in Sweetpotato Plants Using a Protective Mild Strain of Sweet potato feathery mottle virus

A mild isolate (designated as 10-O) of Sweet potato feathery mottle virus (SPFMV) was obtained from Ipomoea batatas. This isolate rarely caused skin discoloration and did not cause russet crack disease on the storage roots of I. batatas that are typical of infection with the severe strain of SPFMV (SPFMV-S). The yield of the 10-O–infected I. batatas ranged from 92 to 105% of the yield of healthy I. batatas. The coat protein gene of 10-O encodes 315 amino acids and has sequence identities of 91.1% at the nucleotide level and 95.6% at the amino acid level with the corresponding region of SPFMV-S. When I. batatas cuttings were inoculated with 10-O and later challenged with SPFMV-S, russet crack symptoms were much reduced and SPFMV-S was not detected in the challenged plants. These results indicate that 10-O can be an effective biological control agent against russet crack disease.