Differential carbohydrate-active enzymes and secondary metabolite production by the grapevine trunk pathogen Neofusicoccum parvum Bt-67 grown on host and non-host biomass.

Neofusicoccum parvum is one of the most aggressive Botryosphaeriaceae species associated with grapevine trunk diseases. This species may secrete enzymes capable of overcoming the plant barriers, leading to wood colonization. In addition to their roles in pathogenicity, there is an interest in taking advantage of N. parvum carbohydrate-active enzymes (CAZymes), related to plant cell wall degradation, for lignocellulose biorefining. Furthermore, N. parvum produces toxic secondary metabolites that may contribute to its virulence. In order to increase knowledge on the mechanisms underlying pathogenicity and virulence, as well as the exploration of its metabolism and CAZymes for lignocellulose biorefining, we evaluated the N. parvum strain Bt-67 capacity in producing lignocellulolytic enzymes and secondary metabolites when grown in vitro with two lignocellulosic biomasses: grapevine canes (GP) and wheat straw (WS). For this purpose, a multiphasic study combining enzymology, transcriptomic, and metabolomic analyses was performed. Enzyme assays showed higher xylanase, xylosidase, arabinofuranosidase, and glucosidase activities when the fungus was grown with WS. Fourier transform infrared (FTIR) spectroscopy confirmed the lignocellulosic biomass degradation caused by the secreted enzymes. Transcriptomics indicated that the N. parvum Bt-67 gene expression profiles in the presence of both biomasses were similar. In total, 134 genes coding CAZymes were up-regulated, where 94 of them were expressed in both biomass growth conditions. Lytic polysaccharide monooxygenases (LPMOs), glucosidases, and endoglucanases were the most represented CAZymes and correlated with the enzymatic activities obtained. The secondary metabolite production, analyzed by high-performance liquid chromatography-ultraviolet/visible spectophotometry-mass spectrometry (HPLC-UV/Vis-MS), was variable depending on the carbon source. The diversity of differentially produced metabolites was higher when N. parvum Bt-67 was grown with GP. Overall, these results provide insight into the influence of lignocellulosic biomass on virulence factor expressions. Moreover, this study opens the possibility of optimizing the enzyme production from N. parvum with potential use for lignocellulose biorefining.

Botryosphaeriaceae gene machinery: Correlation between diversity and virulence.

The Botryosphaeriaceae family comprises numerous fungal pathogens capable of causing economically meaningful diseases in a wide range of crops. Many of its members can live as endophytes and turn into aggressive pathogens following the onset of environmental stress events. Their ability to cause disease may rely on the production of a broad set of effectors, such as cell wall-degrading enzymes, secondary metabolites, and peptidases. Here, we conducted comparative analyses of 41 genomes representing six Botryosphaeriaceae genera to provide insights into the genetic features linked to pathogenicity and virulence. We show that these Botryosphaeriaceae genomes possess a large diversity of carbohydrate-active enzymes (CAZymes; 128 families) and peptidases (45 families). Botryosphaeria, Neofusicoccum, and Lasiodiplodia presented the highest number of genes encoding CAZymes involved in the degradation of the plant cell wall components. The genus Botryosphaeria also exhibited the highest abundance of secreted CAZymes and peptidases. Generally, the secondary metabolites gene cluster profile was consistent in the Botryosphaeriaceae family, except for Diplodia and Neoscytalidium. At the strain level, Neofusicoccum parvum NpBt67 stood out among all the Botryosphaeriaceae genomes, presenting a higher number of secretome constituents. In contrast, the Diplodia strains showed the lowest richness of the pathogenicity- and virulence-related genes, which may correlate with their low virulence reported in previous studies. Overall, these results contribute to a better understanding of the mechanisms underlying pathogenicity and virulence in remarkable Botryosphaeriaceae species. Our results also support that Botryosphaeriaceae species could be used as an interesting biotechnological tool for lignocellulose fractionation and bioeconomy.