Project description:Epidemics of coffee leaf rust (CLR) lead to great yield losses and huge depreciation of coffee marketing values, if no control measures are applied. Societal expectations of a more sustainable coffee production are increasingly imposing the replacement of pesticide treatments by alternative solutions. A good protection strategy is to take advantage of the plant immune system by eliciting its constitutive defenses. Based on such concept, plant resistance inducers (PRIs) have been developed. The Greenforce CuCa formulation made by UFLA (Brazil) is, in addition to acibenzolar-S-methyl (ASM), showing promising results in the control of CLR (Hemileia vastatrix) in Coffea arabica. In order to improve our understanding of the molecular mechanisms of the PRIs, proteomic (2DE-MALDI/TOF/TOF-MS/MS), physiological (leaf gas-exchange) and biochemical (enzymatic) analyses of coffee leaves treated with Greenforce CuCa and ASM and inoculation with H. vastatrix were performed. Proteomic data showed metabolic adjustments mainly related with photosynthesis, protein metabolism and stress responses but, the proteins modulated by the two PRIs were different. Greenforce CuCa, on its own, increased photosynthesis and stomatal conductance, while ASM caused a decrease in these parameters. Upon H. vastratix infection, the Greenforce CuCa showed a higher protective effect on the leaf physiology than ASM. The enzymatic analyses indicated that Greenforce CuCa reinforces the redox homeostasis of the leaf, while ASM seems to increase the involvement of secondary metabolism. So, the PRIs prepare the plant to resist CLR but, inducing different defense mechanisms upon pathogen infection. The data also evidenced the existence of a link between the primary metabolism and defense responses. Furthermore, Greenforce CuCa emerged as a significant agent for CLR management. The identification of components of the plant primary metabolism, essential for plant growth and development that, simultaneously, participate in the plant defense responses can open new perspectives for plant breeding programs.
Project description:Coffee leaf rust (CLR), caused by the fungal pathogen Hemileia vastatrix, has plagued coffee production worldwide for over 150 years. Hemileia vastatrix produces urediniospores, teliospores, and the sexual basidiospores. Infection of coffee by basidiospores of H. vastatrix has never been reported and thus far, no alternate host, capable of supporting an aecial stage in the disease cycle, has been found. Due to this, some argue that an alternate host of H. vastatrix does not exist. Yet, to date, the plant pathology community has been puzzled by the ability of H. vastatrix to overcome resistance in coffee cultivars despite the apparent lack of sexual reproduction and an aecidial stage. The purpose of this study was to introduce a new method to search for the alternate host(s) of H. vastatrix. To do this, we present the novel hypothetical alternate host ranking (HAHR) method and an automated text mining (ATM) procedure, utilizing comprehensive biogeographical botanical data from the designated sites of interests (Ethiopia, Kenya and Sri Lanka) and plant pathology insights. With the HAHR/ATM methods, we produced prioritized lists of potential alternate hosts plant of coffee leaf rust. This is a first attempt to seek out an alternate plant host of a pathogenic fungus in this manner. The HAHR method showed the highest-ranking probable alternate host as Psychotria mahonii, Rubus apetalus, and Rhamnus prinoides. The cross-referenced results by the two methods suggest that plant genera of interest are Croton, Euphorbia, and Rubus. The HAHR and ATM methods may also be applied to other plant-rust interactions that include an unknown alternate host or any other biological system, which rely on data mining of published data.
Project description:Population genetic studies of Hemileia vastatrix have been conducted in order to describe the evolutionary dynamics of the pathogen and the disease epidemiology as consequence of changes in disease management and host distribution occurred in Peru after the 2013 epidemic. These analyses were performed by sequencing the internal transcribed spacers of the nuclear ribosomal DNA (rDNA-ITS) of H. vastatrix collected from two coffee growing areas in 2014 and 2018. H. vastatrix population showed high haplotype diversity (Hd = 0.9373 ± 0.0115) with a low nucleotide diversity (π = 0.00322 ± 0.00018). Likewise, AMOVA indicated that fungus population has behaved as a large population without structuring by geographical origin and sampling years (FST = 0.00180, P = 0.20053 and FST = 0.00241, P = 0.19693, respectively). Additionally, the haplotype network based on intraspecific phylogenetic analysis of H. vastatrix using Peruvian and NCBI sequences revealed that Peruvian ancestral haplotypes, which were maintained in time and space, would correspond to the reported sequences of the races II and XXII. This result suggests that no substantial changes have occurred through time in Peruvian Hemileia vastatrix population.
Project description:A proteomic analysis of the apoplastic fluid (APF) of coffee leaves was conducted to investigate the cellular processes associated with incompatible (resistant) and compatible (susceptible) Coffea arabica-Hemileia vastatrix interactions, during the 24-96 hai period. The APF proteins were extracted by leaf vacuum infiltration and protein profiles were obtained by 2-DE. The comparative analysis of the gels revealed 210 polypeptide spots whose volume changed in abundance between samples (control, resistant and susceptible) during the 24-96 hai period. The proteins identified were involved mainly in protein degradation, cell wall metabolism and stress/defense responses, most of them being hydrolases (around 70%), particularly sugar hydrolases and peptidases/proteases. The changes in the APF proteome along the infection process revealed two distinct phases of defense responses, an initial/basal one (24-48 hai) and a late/specific one (72-96 hai). Compared to susceptibility, resistance was associated with a higher number of proteins, which was more evident in the late/specific phase. Proteins involved in the resistance response were mainly, glycohydrolases of the cell wall, serine proteases and pathogen related-like proteins (PR-proteins), suggesting that some of these proteins could be putative candidates for resistant markers of coffee to H. vastatrix. Antibodies were produced against chitinase, pectin methylesterase, serine carboxypeptidase, reticuline oxidase and subtilase and by an immunodetection assay it was observed an increase of these proteins in the resistant sample. With this methodology we have identified proteins that are candidate markers of resistance and that will be useful in coffee breeding programs to assist in the selection of cultivars with resistance to H. vastatrix.
Project description:Epidemics of coffee leaf rust (CLR) leads to great yield losses and huge depreciation of coffee marketing values, if no control measures are applied. Societal expectations of a more sustainable coffee production are increasingly imposing the replacement of fungicide treatments by alternative solutions. A protection strategy is to take advantage of the plant immune system by eliciting constitutive defenses. Based on such concept, plant resistance inducers (PRIs) have been developed. The Greenforce CuCa formulation, similarly to acibenzolar-S-methyl (ASM), shows promising results in the control of CLR (Hemileia vastatrix) in Coffea arabica cv. Mundo Novo. The molecular mechanisms of PRIs action are poorly understood. In order to contribute to its elucidation a proteomic, physiological (leaf gas-exchange) and biochemical (enzymatic) analyses were performed. Coffee leaves treated with Greenforce CuCa and ASM and inoculation with H. vastatrix were considered. Proteomics revealed that both PRIs lead to metabolic adjustments but, inducing distinct proteins. These proteins were related with photosynthesis, protein metabolism and stress responses. Greenforce CuCa increased photosynthesis and stomatal conductance, while ASM caused a decrease in these parameters. It was further observed that Greenforce CuCa reinforces the redox homeostasis of the leaf, while ASM seems to affect preferentially the secondary metabolism and the stress-related proteins. So, the PRIs prepare the plant to resist CLR but, inducing different defense mechanisms upon pathogen infection. The existence of a link between the primary metabolism and defense responses was evidenced. The identification of components of the plant primary metabolism, essential for plant growth and development that, simultaneously, participate in the plant defense responses can open new perspectives for plant breeding programs.