A multi-faceted approach for determining the reproductive biology of the causal agent of frosty pod rot of cacao Moniliophthora roreri
Moniliophthora roreri causes cacao frosty pod rot disease rendering seeds useless for chocolate production. Before the 1950s it was confined to north-western South America but currently the disease has spread north as far as Mexico and south to Bolivia. Currently it poses a real threat to cocoa growers in Brazil and also in West Africa and Asia, where over 85% of world production now occurs. Moniliophthora roreri belongs to the mushroom-forming fungi in the family Marasmiaceae but no basidiocarp has ever been observed for this fungus, although billions of spores are produced directly from fungal mycelium on pod surfaces. Since meiosis and production of sexual propagules are produced in mushrooms, the lack of such structures in M. roreri raises the question of whether it carries out sexual reproduction at all. Thus in this study we looked for evidence of cryptic sex via multiple approaches. First, we examined the nuclear condition of hyphae and spores and determined the method of sporogenesis. We found M. roreri produces a random number of nuclei per cell in a manner typical of monokaryotic fungi and that the spores were produced via the thallic, rhexolytic manner, as for asexual spores in some other Marasmioid species. Dikaryotic hyphae were never observed. Secondly, we produced a draft genome of M. roreri to identify potential A (homeodomain transcription factors HD1 and HD2) and B (pheromones and receptors) mating genes. After screening a set of 47 geographically distinct isolates, two A and two B alleles but only two mating types were identified. Next, a microsatellite analysis across the same pool of isolates revealed no sexual recombination between both mating types, suggesting M. roreri possesses a tetrapolar mating system but does not outcross. Finally, we examined cultural characteristics and vegetative compatibility. Distinct mating type isolates behaved differently in terms of growth and sporulation, especially with the addition of activated charcoal to the medium, suggesting that previously observed morphological variation within the species might be attributed to phenotypic differences conferred by mating type, and not to the presence of subspecies varieties as previously interpreted. Ultimately, these results will provide cacao programs with cutting-edge knowledge about M. roreri to make the best decisions concerning management and breeding against frosty pod rot.
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