Austropuccinia psidii, the causal agent of myrtle rust, is a biotrophic pathogen whose growth and development depends on the host tissues. The uredospores of A. psidii infect Eucalyptus by engaging in close contact with the host surface and interacting with the leaf cuticle that provides important chemical and physical signals to trigger the infection process. In this study, the cuticular waxes of Eucalyptus spp. were analyzed to determine their composition or structure and correlation with susceptibility/resistance to A. psidii. Twenty-one Eucalyptus spp. in the field were classified as resistant or susceptible. The resistance/susceptibility level of six Eucalyptus spp. were validated in controlled conditions using qPCR, revealing that the pathogen can germinate on the eucalyptus surface of some species without multiplying in the host. CG-TOF-MS analysis detected 26 compounds in the Eucalyptus spp. cuticle and led to the discovery of the role of hexadecanoic acid in the susceptibility of Eucalyptus grandis and Eucalyptus phaeotricha to A. psidii. We characterized the epicuticular wax morphology of the six previously selected Eucalyptus spp. using scanning electron microscopy and observed different behavior in A. psidii germination during host infection. It was found a correlation of epicuticular morphology on the resistance to A. psidii. However, in this study, we provide the first report of considerable interspecific variation in Eucalyptus spp. on the susceptibility to A. psidii and its correlation with cuticular waxes chemical compounds that seem to play a synergistic role as a preformed defense mechanism.

Eucalyptus rust caused by Puccinia psidii is responsible for losses of approximately 20% of young Eucalyptus plants, depending on the environmental conditions and the geographic location. Despite its economic importance, there are few studies describing the genetic variability in P. psidii populations that infect different host plants. In the present study, we evaluated the ribosomal DNA internal transcribed spacer region (rDNA-ITS) using polymerase chain reaction denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism to assess the genetic variability in P. psidii populations infecting different Eucalyptus spp. and hybrids, as well as guava, jabuticaba, and syzygium. These culture-independent methods were efficient in differentiating populations based on the host species from which they were collected. In general, the results from both techniques showed that the populations collected from guava, jabuticaba, and syzygium were different from and had a greater level of diversity than the Eucalyptus rust populations. The sequencing of cloned rDNA-ITS fragments confirmed that the vast majority of the profiles generated were from P. psidii. This analysis also revealed interesting single-nucleotide polymorphisms. Therefore, these culture-independent methods are suitable for the rapid assessment of genetic variability within and between populations of this biotrophic fungus on a variety of host species and could be a tool to study the evolution of this pathogen and its interactions with host plants.

Puccinia psidii sensu lato (s.l.) is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to P. psidii dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava) and eucalyptus leaves (PpEucalyptus). NanoUPLC-MSE was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database) resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.

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