The main purpose of this study was to set up a new pathosystem that could be used to analyze molecular factors that mediate the transmission of fungal pathogens to seeds. Arabidopsis thaliana reproductive organs were inoculated with the seedborne pathogen A. brassicicola. Some general conclusions can be drawn from the findings of experiments designed to define the optimal conditions for studying fungal transmission to seeds of A. thaliana. First, concerning the seed transmission routes, our results did not reveal whether A. brassicicola was able to cause seed infection via flowers, since floral development was interrupted due to inoculation with a virulent isolate. Oliver et al. demonstrated, via glasshouse inoculation trials and examination of naturally infected Cakile seeds, that seed infection could occur through the flowers, though less efficiently than the fruit route. Indeed, the floral route seems to be more specific to opportunistic pathogenic strains of Alternaria alternata, with this phenomenon being explained by the weakness of flower defence mechanisms. By contrast, we found that the fruit route was efficient and the seed contamination levels (30%) thus observed in A. thaliana under our experimental conditions were comparable to those obtained by Oliver et al. for Cakile maritima seeds in field and laboratory conditions, with the frequency of infection positively correlated with the lesion density on the fruit surface.
Our results also confirmed data obtained on Brassica seed crops in field conditions, indicating a vertical disease gradient on fruits, with the infection first affecting the lowest siliques and then spreading slowly upwards. This was explained by the observation that conidia are often disseminated short distances and retained within the canopy. As in our laboratory conditions, inoculum was distributed equally on all siliques, the more efficient transmission to seeds from the lower siliques could be mainly due to silique maturity differences, as Alternaria development is favored on senescent tissues.
The mode of seed infection by A. brassicicola and its localization on the reproductive organ tissues was already described several years ago. We used our model pathosystem to perform a microscopic analysis of contaminated seeds, but also of contaminated siliques. Concerning seeds, surface colonization via conidia and hyphae was observed and resulted in desquamation of the seed surface, in accordance with previously published data. Such intensive fungal development could be favored by the release of carbohydrates at the surface of damaged seeds as proposed by Knox-Davies. Conidiogenesis has frequently been observed on contaminated seeds, with spores preferentially located in seed coat folds and cracks, which may provide protection against harsh environments. Superficial hyphae also penetrated within the seed, as confirmed by SEM and confocal laser scanning microscopy. Our observations did not allow us to determine if A. brassicicola was limited to the seed coat or also present inside the endosperm and embryo. However, according to Singh & Mathur, whereas biotrophic fungi are often located in the embryo, necrotrophic fungi like A. brassicicola are generally located on seed and fruit coats, and deeper penetration is uncommon. In line with this, A. brassicicola was found to be confined to the testa of naturally infected Brussels sprout seeds while being mainly on the surface cabbage seeds. The extent of testa colonization could not be accurately determined and would require transmission electronic microscopy analysis of ultra-thin seed cuts or confocal laser scanning microscopy analysis of seed cuts. Nevertheless, hyphal penetration of A. thaliana seed coats was evidently accompanied by seed surface damage. This phenomenon could be due to the production of plant tissue degradation enzymes, for example cutinases, as already described for A. brassicicola, and it could potentially accompany the penetration of cabbage tissues[21, 22]. The same hypothesis was put forward by Vaughan et al. with respect to soybean seed colonization by several Alternaria sp.. To our knowledge, no detailed microscopic analysis has ever been reported concerning silique colonization. A few days before seed harvest, typical black-spot symptoms developed on fruits at inoculation sites where intensive development of the fungus was observed on the outer surface of siliques. Penetration inside the fruit was either via stomata, intercellular spaces or replum, with occasional differentiation of appressoria-like structures.
The model pathosystem thus defined was then used to specify the molecular factors impacting the seed colonization process by using mutant genotypes of the pathogen and host plant. In maturing seeds, the water content dramatically decreases, leading to a reduction in the metabolism necessary for seed conservation. During seed colonization, fungi could thus be exposed to severe water and osmotic stresses that they have to overcome to be efficiently transmitted to seeds and complete their infection cycle. Then the ability to cope with water and osmotic stress could be a factor that determines their transmission to seeds. Arabidopsis thaliana silique inoculation in controlled conditions allowed us to repeatedly show a lower seed transmission capacity for nik1Δ3, a disruption mutant deficient in a group III histidine kinase involved in the osmotic stress response, by comparison with its wild-type parental strain Abra43. The contamination level was significantly lower at every silique stage, with a gradient from the oldest to the youngest silique, as observed after Abra43 inoculation. This confirms previous findings indicating that A. brassicicola null mutants in the AbNIK1 gene, although their virulence on host vegetative tissues remained intact, were affected in their transmission to radish seeds after artificial inoculation in field conditions.
Using our model pathosystem, we took advantage of the availability of A. thaliana transparent testa (tt) mutants, to assess the influence of flavonoids present in seed testa on their susceptibility to fungal infection. The selected mutant line tt4 is altered in the chalcone synthase gene and therefore disrupted in the first step of the flavonoid biosynthesis pathway. It produces pale yellow seeds and does not produce any of the flavonoid compounds present in WT seeds, i.e. proanthocyanidins and flavonols. Based on the well-documented plant protection functions of flavonoids against abiotic and biotic stresses, and previously published data showing that peas with dark seed coats were less susceptible to fungal infections than those with light-colored coats, we anticipated that A. brassicicola would colonize seeds of the tt4-1 allele more efficiently than the wild type. Unexpectedly, although a downward vertical gradient of seed infection was still observed for both wild-type and mutant genotypes, significantly lower A. brassicicola infection probabilities were obtained with the flavonoid deficient mutant. A recent metabolomic study characterized several compounds over-accumulating in seeds of tt4-1 plants compared to those of wild-type Ler, as phenolic choline esters, sinapate-derived metabolites and glucosinolate breakdown products. Although the antimicrobial properties of the former compounds have yet to be characterized, glucosinolate derivatives have been well documented as potentially toxic for pathogens, including A. brassicicola, and such metabolomic changes in tt4-1 seeds might explain our observations. These hypotheses can be tested by investigating the transmission behaviour of Arabidopsis mutants disrupted in sinapate and glucosinolate metabolisms. Complementary investigations using different tt mutants and different tt4 alleles are required to reliably conclude on the role of flavonoids in the seed transmission process of A. brassicicola to A. thaliana.