During their life cycles, pathogens have to adapt to many biotic and abiotic environmental stresses to maximize their overall fitness. Morphological transitions are one of the least understood of the many strategies employed by fungal plant pathogens to adapt to constantly changing environments, even though different morphotypes may play important biological roles. Here, we first show that blastospores (the “yeast-like” form of the pathogen typically known only under laboratory conditions) can form from germinated pycnidiospores (asexual spores) on the surface of wheat leaves, suggesting that this morphotype can play an important role in the natural history of Z. tritici . Next, we characterized the morphological responses of this fungus to a series of environmental stresses to understand the effects of changing environments on fungal morphology and adaptation. All tested stresses induced morphological changes, but different responses were found among four strains. We discovered that Z. tritici forms chlamydospores and demonstrated that these structures are better able to survive extreme cold, heat and drought than other cell types. Finally, a transcriptomic analysis showed that morphogenesis and the expression of virulence factors are co-regulated in this pathogen. Our findings illustrate how changing environmental conditions can affect cellular morphology and lead to the formation of new morphotypes, with each morphotype having a potential impact on both pathogen survival and disease epidemiology.

Background: The ability of fungal cells to undergo cell-to-cell communication and anastomosis, the process of vegetative hyphal fusion, allows them to maximize their overall fitness. Previous studies in a number of fungal species have identified the requirement of several signaling pathways for anastomosis, including the so far best characterized soft (So) gene, and the MAPK pathway components MAK-1 and MAK-2 of Neurospora crassa. Despite the observations of hyphal fusions' involvement in pathogenicity and host adhesion, the connection between cell fusion and fungal lifestyles is still unclear. Here, we address the role of anastomosis in fungal development and asexual reproduction in Zymoseptoria tritici, the most important fungal pathogen of wheat in Europe. Results: We show that Z. tritici undergoes self-fusion between distinct cellular structures, and its mechanism is dependent on the initial cell density. Contrary to other fungi, cell fusion in Z. tritici only resulted in cytoplasmic mixing but not in multinucleated cell formation. The deletion of the So orthologous ZtSof1 disrupted cell-to-cell communication affecting both hyphal and germling fusion. We show that Z. tritici mutants for MAPK-encoding ZtSlt2 (orthologous to MAK-1) and ZtFus3 (orthologous to MAK-2) genes also failed to undergo anastomosis, demonstrating the functional conservation of this signaling mechanism across species. Additionally, the ΔZtSof1 mutant was severely impaired in melanization, suggesting that the So gene function is related to melanization. Finally, we demonstrated that anastomosis is dispensable for pathogenicity, but essential for the pycnidium development, and its absence abolishes the asexual reproduction of Z. tritici. Conclusions: We demonstrate the role for ZtSof1, ZtSlt2, and ZtFus3 in cell fusions of Z. tritici. Cell fusions are essential for different aspects of the Z. tritici biology, and the ZtSof1 gene is a potential target to control septoria tritici blotch (STB) disease.

Combining different biocontrol agents (BCA) is an approach to increase efficacy and reliability of biological control. If several BCA are applied together, they have to be compatible and ideally work together. We studied the interaction of a previously selected BCA consortium of entomopathogenic pseudomonads (Pseudomonas chlororaphis), nematodes (Steinernema feltiae associated with Xenorhabdus bovienii), and fungi (Metarhizium brunneum). We monitored the infection course in a leaf- (Pieris brassicae) and a root-feeding (Diabrotica balteata) pest insect after simultaneous application of the three BCA as well as their interactions inside the larvae in a laboratory setting. The triple combination caused the highest mortality and increased killing speed compared to single applications against both pests. Improved efficacy against P. brassicae was mainly caused by the pseudomonad-nematode combination, whereas the nematode-fungus combination accelerated killing of D. balteata. Co-monitoring of the three BCA and the nematode-associated Xenorhabdus symbionts revealed that the four organisms are able to co-infect the same larva. However, with advancing decay of the cadaver there is increasing competition and cadaver colonization is clearly dominated by the pseudomonads, which are known for their high competitivity in the plant rhizosphere. Altogether, the combination of the three BCA increased killing efficacy against a Coleopteran and a Lepidopteran pest which indicates that this consortium could be applied successfully against a variety of insect pests. Graphical Abstract

26The ability of fungal cells to undergo cell fusion allows them to maximize their overall 27 fitness. In this study, we characterized the role of the so gene orthologous in 28Zymoseptoria tritici and the biological contribution of vegetative cell fusions in the 29 lifestyle of this latent necrotrophic fungus. Firstly, we show that Z. tritici undergoes 30 self-fusion between distinct cellular structures and its mechanism is dependent on the 31 initial cell density. Next, the deletion of ZtSo resulted in the loss of cell-to-cell 32 communication affecting both hyphal and germlings fusion. We show that Z. tritici 33 mutants for MAP kinase-encoding ZtSlt2 (orthologous MAK-1) and ZtFus3 34 (orthologous MAK-2) genes also fail to undergo self-stimulation and self-fusion, 35demonstrating the functional conservation of this signaling mechanism across 36 species. Additionally, the DZtSo mutant was severely impaired in melanization, which 37 leads us to identify a trade-off between fungal growth and melanization. Though it has 38 been proposed that So is a scaffold protein for MAP kinase genes from the CWI 39 pathway, its deletion did not affect the cell wall integrity of the fungus. Finally, we 40 demonstrated that anastomose is dispensable for pathogenicity, but essential for the 41 fruiting body development and its absence abolish the asexual reproduction of Z. tritici. 42Taken together, our data show that ZtSo is required for fungal development, while 43 vegetative cell fusions are essential for fungal fitness. 44 45 46 47 48 49 50