RESUMEN
Barley (Hordeum vulgare) is one of the most widely cultivated crops for feedstock and beer production, whereas lupins (Lupinus spp.) are grown as fodder and their seeds are a source of protein. Both species produce the allelopathic alkaloids gramine and hordenine. These plant-specialized metabolites may be of economic interest for crop protection, depending on their tissue distribution. However, in high concentrations they pose a health risk to humans and animals that feed on them. This study was carried out to develop and validate a new method for monitoring these alkaloids and their related metabolites using fluorescence detection. Separation was performed on an HSS T3 column using slightly acidified water-acetonitrile eluents. Calibration plots expressed linearity over the range 0.09-100 pmol/µL for gramine. The accuracy and precision ranged from 97.8 to 123.4%, <7% RSD. The method was successfully applied in a study of the natural range of abundance of gramine, hordenine and their related metabolites, AMI, tryptophan and tyramine, in 22 barley accessions and 10 lupin species. This method provides accurate and highly sensitive chromatographic separation and detection of tryptophan- and tyrosine-derived allelochemicals and is an accessible alternative to LC-MS techniques for routine screening.
RESUMEN
Cyclic 3',5'-adenosine monophosphate (cAMP) is a nucleotide derived from adenosine triphosphate that acts as a second messenger throughout all kingdoms. Intracellular cAMP levels are synthesized by a membrane-bound protein, the adenylyl cyclase. In order to analyze the function of this gene and the importance of cAMP in the life cycle of the cereal pathogen Fusarium graminearum, the adenylyl cyclase gene (FGSG_01234) was deleted by gene replacement (ΔFgac1). The ΔFgac1 mutant displayed a drastically reduced growth on agar medium which could be rescued by a cAMP analogon. Furthermore, the ΔFgac1 mutant was unable to produce perithecia on detached wheat nodes. However, artificial conditions like carrot agar allowed perithecia development. Pathogenicity towards wheat was drastically reduced in ΔFgac1 compared to the wild type. Point-inoculated spikelets showed only small lesions but no typical head blight disease symptoms. Fluorescence microscopy using dsRed-expressing strains revealed that the ΔFgac1 strain was unable to develop any complex infection structures like lobate appressoria and infection cushions. Instead, hyphal anastomosis occurs frequently. Scanning electron microscopy demonstrated the lack of fungal penetration. Hence, the formation of compound appressoria seems to be essential for infection of wheat. Hyphae on flower leaves produced huge amounts of new conidia, thereby circumventing the infection cycle. This abundant sporulation on wheat epidermis was not observed in wild type. Intriguingly, the Fgac1 deletion mutant was able to infect maize cobs as wild type, indicating that cAMP signaling is not important for maize infection. The ΔFgac1 mutant was unable to produce the mycotoxin deoxynivalenol both in vitro and during wheat infection. In this study, we show that cAMP signaling controls important cellular processes such as development of infection structures, pathogenicity, secondary metabolite production and sexual reproduction. For the first time, we show that cAMP regulates the switch from vegetative to pathogenic lifestyle of F. graminearum on wheat.