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Seeds are involved in the vertical transmission of microorganisms from one plant generation to another and consequently act as reservoirs for the plant microbiota. However, little is known about the structure of seed-associated microbial assemblages and the regulators of assemblage structure. In this work, we have assessed the response of seed-associated microbial assemblages of Raphanus sativus to invading phytopathogenic agents, the bacterial strain Xanthomonas campestris pv. campestris (Xcc) 8004 and the fungal strain Alternaria brassicicola Abra43. According to the indicators of bacterial (16S rRNA gene and gyrB sequences) and fungal (ITS1) diversity employed in this study, seed transmission of the bacterial strain Xcc 8004 did not change the overall composition of resident microbial assemblages. In contrast seed transmission of Abra43 strongly modified the richness and structure of fungal assemblages without affecting bacterial assemblages. The sensitivity of seed-associated fungal assemblage to Abra43 is mostly related to changes in relative abundance of closely related fungal species that belong to the Alternaria genus. Variation in stability of the seed microbiota in response to Xcc and Abra43 invasions could be explained by differences in seed transmission pathways employed by these micro-organisms, which ultimately results in divergence in spatio-temporal colonization of the seed habitat.

Alternaria brassicicola field isolates highly resistant to dicarboximide fungicides (DCF) and carrying non-sense codons or micro-deletions in the AbNIK1 gene encoding a group III histidine kinase were characterized at the protein level using specific antibodies. These isolates, as well as laboratory mutants expressing the same resistant phenotype, were shown to correspond to null mutants. The fitness of these isolates was compared to that of wild-type isolates by measuring i) mycelial growth, ii) sporulation and iii) conidial germination under standard conditions. None of these factors were affected in the resistant isolates. Mycelial growth was also measured under stress conditions. Unlike suboptimal incubation temperatures or exposure to the superoxide ion generator menadione, osmotic stress generated by high sorbitol concentrations in the culture medium significantly affected the growth of AbNIK1-null mutants compared to wild-types. The effects of these mutations on parasitic fitness were estimated under both controlled and field conditions after inoculation of radish with individual isolates, to measure their aggressiveness, or with mixed conidial suspensions, to measure their competitive ability against wild-type isolates. These tests on radish seedlings revealed that DCF-resistant isolates were still aggressive. Despite this, the data obtained in the competition experiments showed that the loss of the ability to synthesize AbNIK1p in A. brassicicola DCF-resistant mutants was linked to a severe decrease in their competitiveness.

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