Monitoring spore dispersal and early infections of Diplocarpon coronariae causing apple blotch using selected spore traps and a new qPCR method

Apple blotch (AB) is a major disease of apples in Asia and recently also emerging in Europe and the USA. It is caused by the fungus Diplocarpon coronariae (Dc) (formerly: Marssonina coronaria; teleomorph: Diplocarpon mali) and leads to severe defoliation of apple trees in late summer and thus to reduced yield and fruit quality. To develop effective crop protection strategies, a sound knowledge of the pathogen's biology is crucial. However, especially data on the early phase of disease development is scarce, and no data on spore dispersal for Europe is available. In this study, we assessed different spore traps for their capacity to capture Dc spores, and we developed a highly sensitive TaqMan qPCR method to quantify Dc conidia in spore trap samples. With these tools, we monitored the temporal and spatial spore dispersal and disease progress in spring and early summer in an extensively managed apple orchard in Switzerland in 2019 and 2020. Our results show that Dc overwinters in leaf litter and that spore dispersal and primary infections occur already in late April and beginning of May. We provide the first results on early-season spore dispersal of Dc, which, combined with the observed disease progress, helps to understand the disease dynamics and improve disease forecast models. Using the new qPCR method, we finally detected Dc in buds, on bark and on fruit mummies, suggesting that these apple organs may serve as additional overwintering habitats for the fungus.

Deciphering the role of fungal calmodulin gene using Host-mediated gene silencing approach in apple

Premature leaf fall caused by Marssonina coronaria is one of the most destructive diseases of apple in India. In this study, host induced gene silencing approach was exploited to develop resistance to this disease in an apple cultivar ‘Red Chief’. Calmodulin gene (CaM) having its role in fungal differentiation, development and pathogenicity was selected as target. hpRNAi construct was prepared from the conserved off target free partial gene sequence of CaM and used for transformation trials. Upto 6% kanamycin resistant shoots were obtained on selective medium having 5–6 mg/l kan after 7 weeks of coculture. In PCR analysis of 13 RNAi putative transformants, 10 lines were found positive with CaM and nptII gene specific primers and six lines showed hybridization signal. Semi qRT-PCR revealed variable levels of transgene expression among RNAi lines which seems to be related to copy number of integrated gene. In vitro detached leaf assay revealed lesion development and disease progression in wild type after 5 dpi but not visible in five CaM RNAi lines. Microscopic examination of infected control leaves showed fully developed, septate mycelium, and conidia along with necrosis of whole tissue while three transformants showed reduced growth and differentiation of fungus and in rest three, hyphal development and necrosis were strongly restricted. We conclude that trafficking of dsRNA/ siRNA from apple plants to pathogen might have triggered the down regulation of fungal CaM gene which confirms that deciphering the role of CaM through HIGS lead to resistance to Marssonina blotch in apple.

Draft genome sequence of Marssonina coronaria, causal agent of apple blotch, and comparisons with the Marssonina brunnea and Marssonina rosae genomes

Marssonina coronaria Ellis & Davis is a filamentous fungus in the class Leotiomycetes that causes apple blotch, an economically important disease of apples worldwide. Here, we sequenced the whole genome of M. coronaria strain NL1. The genome contained 50.3 Mb with 589 scaffolds and 9,622 protein-coding genes. A phylogenetic analysis using multiple loci and a whole-genome alignment revealed that M. coronaria is closely related to Marssonina rosae and Marssonina brunnea. A comparison of the three genomes revealed 90 species-specific carbohydrate-active enzymes, 19 of which showed atypical distributions, and 12 species-specific secondary metabolite biosynthetic gene clusters, two of which have the potential to synthesize products analogous to PR toxin and swainsonine, respectively. We identified 796 genes encoding for small secreted proteins in Marssonina spp., many encoding for unknown hypothetical proteins. In addition, we revealed the genetic architecture of the MAT1-1 and MAT1-2 mating-type loci of M. coronaria, as well as 16 tested isolates carrying either MAT1-1 idiomorph (3) or MAT1-2 idiomorph (13). Our results showed a series of species-specific carbohydrate-active enzyme, secondary metabolite biosynthetic gene clusters and small-secreted proteins that may be involved in the adaptation of Marssonina spp. to their distinct hosts. We also confirmed that M. coronaria possesses a heterothallic mating system and has outcrossing potential in nature.

Marssonina coronaria, NUEVO AGENTE CAUSAL DE MANCHAS FOLIARES EN MANZANOS DE BAJOS REQUERIMIENTOS DE FRIO DEL CENTRO DE ARGENTINA

Durante los años 2017-2018, una enfermedad fúngica causó una importante defoliación temprana en manzanos de los cvs `Caricia ́, `Princesa ́ y `Eva ́ del centro-este de la Provincia de Santa Fe. La sintomatología fue coincidente con la reportada para Marssonina spp. Con el objetivo de caracterizar la etiología de la enfermedad se obtuvieron 9 aislados, siendo 4 caracterizados morfométrica y molecularmente. Luego de 60 días, las colonias alcanzaron 10 mm de diámetro, con márgenes irregulares y coloración marrón. Los conidios, hialinos, ligeramente curvos, ovoides y bi-celulares midieron 15,23 ± 1,49 μm de largo y 5,47 ± 0,52 μm de ancho. Las secuencias de ITS indicaron 100% de identidad con Marssonina coronaria. Se confi rmó la patogenicidad de los aislados. Todas las inoculaciones produjeron síntomas y el patógeno fue re-aislado coincidiendo sus características con las inicialmente mencionadas. Este estudio constituye el primer reporte de M. coronaria afectando hojas de manzano en Argentina y proporciona información útil para diseñar estrategias de manejo.