MAJOR ADVANCES ON COFFEE RUST RESEARCH

Transcription

1 MAJOR ADVANCES ON COFFEE RUST RESEARCH Maria do Céu Silva

2 CIFC Centro Inv. Ferrugens do Cafeeiro (Coffee Rusts Research Centre) The importance of coffee leaf rust as a threat to world production led, in 1955, the Governments of the USA and Portugal (Agreement FO- PO- 5, Project D. O. A ), to provide for the foundation of CIFC in Portugal Labs Greenhouses More than 3.000m 2 of heated greenhouses CIFC s main objectives To provide a centre for international cooperation on coffee leaf rust (CLR) research and pre- breeding outside the coffee growing regions and so avoid inadvertently spreading of new virulent races to coffee countries

3 CIFC Collaborations Coffee Leaf Rust Research Researchers: Ana Paula Pereira, Dora Batista, Helena Azinheira, Leonor Guimarães, Maria do Céu Silva, Pedro Talhinhas, Vitor Várzea Octávio Paulo Cândido P. Ricardo Carla Pinheiro Rita Abranches Fernando Cardoso Inês Chaves Vagner T. Queiroz Laércio Zambolim Luxembourg Institute of Science and Technology Jenny Renaut Sébastien Planchon Mário L. Resende Kátia Possa Danielle R. Barros Anne- Sophie Petitot Diana Fernandez Michel Nicole Philippe Lashermes Sebastien Duplessis Ralf Voegele Tobias Link Elijah Gichuru Alvaro Gaitan Dr. Y. Raghuramulu Nayani Prakash Uthai Noppakoonwong Chatnapa Khomarwut Hongbo Zhang Li Jinhong James Teri D. Kilambo

4 CIFC Research Topics Pathogenicity surveys Spectrum of rust virulence on a set of coffee differentials Screening of disease resistance Spectrum of coffee resistance to CIFC s rust races Molecular markers associated with Hv patotypes Population genomics Hv Pathogenicity Microscopy, transcriptomics, functional genomics H. vastatrix (Hv) and coffee unique world collections Coffee Resistance Microscopy, biochemistry/proteomics, transcriptomics

5 Pathogenicity Surveys & Disease Resistance Screening Main Results of CIFC activities Ø The gene- for- gene theory is applied to coffee - H. vastatrix (Hv) interactions Ø Identification of nine resistance genes (S H 1 S H 9) on coffee and the matching of virulence genes (v 1 v 9 ) in Hv Ø A decisive step forward in the research programme of the CIFC was the discovery, in the late 1950 s, of the Timor Hybrid (HDT), a spontaneous natural hybrid between Arabica and Robusta Some HDTs, with resistance to all known rust races, were used as sources of resistance in the breeding programmes originating varieties such as CATIMOR, SARCHIMOR CATIMOR = Variety Caturra x HDT (CIFC 832/1) SARCHIMOR = Variety Villa Sarchix HDT (CIFC 832/2) Both HDT and its derivatives as well as all the available coffee materials of CIFC have been provided free of charge to all coffee growing countries of the world CATIMOR, China More than 90% of Arabica coffee varieties resistant to rust grown in different coffee growing countries were createdfrom studies carried out at CIFC Rodrigues et al. (1975) Annual Review of Phytopathology 13: Bettencourt & Rodrigues Jr. (1988). In R. J. Clarke, & R. Macrae (Eds.), Coffee Agronomy Vol. 4 (pp ).

6 Pathogenicity Surveys & Disease Resistance Screening Recent findings Ø The breakdown of resistance of several resistant cultivars in some coffee growing countries is associated with the increase of virulence in rust populations (new rust races) Ø More than 50 rust races were already characterized Ø Some rust races (from Timor), recently characterized, revealed ability to attack all the sources of resistance used in the creation ofthe majority of resistant cultivars (Catimor and Sarchimor) including the original clone of HDT (CIFC s collection) Perspectives In the near future new sources of resistance must be identified Varzea & Marques (2005) (In L. Zambolim, E. Zambolim, & V. M. P. Várzea (Eds.), Durable resistance to coffee leaf rust (pp.53 74). Várzea et al. (2009). Proc. 22nd Int. Conf Coffee Sci (ASIC), Diniz et al. (2012) European Journal of Plant Pathology 133:

7 Identification of molecular markers associated with H. vastatrix race- specific pathotypes Different approaches are being applied, mainly: Genotyping of SSR (microsatellites) and AFLP (Amplified Fragment Length Polymorphism) loci Analysis of population genotypic profiles Population genomics using Next Generation Sequencing (NGS) tools Detection of SNP markers (Single Nucleotide Polymorphism) and analysis of population genotypic profiles Ongoing analysis of 110 Hv isolates from 20 countries, different virulence profiles, collection years and coffee hosts Results Very low SSR polymorphism No correlation found for AFLP markers Test for statistically significant correlations between specific marker genotypes and virulence profiles Validation Markers putatively associated Preliminary results within a sub- sample Identification of SNPs differentiating Hv population groups Silva et al. (2015). Proc. 25 th Int. Conf Coffee Sci (ASIC), pb248

8 H. vastatrix Pathogenicity Rust fungi are biotrophic pathogens with a complex life cycle. They have evolved specialized structures, haustoria, formed within the living host cells to efficiently acquire nutrients. Germinated uredospore and appressorium Appressorium over A stomata Large sporulating pustules 21 days after inoculation Uredosporic sorum Penetration hypha Susceptibility: in the majority of the infection sites (70-80%) the fungus grows without apparent inhibition Intercellular hyphae with haustoria Haustorial mother cell with haustoria Anchor Rijo et al. (1990) Revista de Ciências Agrárias XIII: ; Silva et al. (1999) Int. Journal Plant Sciences 160 (1): Silva et al. (2006) Braz J Plant Physiol18:

9 H. vastatrix Pathogenicity Transcriptomic analysis From 454 Pyrosequencing and qpcr (quantitative polymerase chain reaction) Intense signalling, transport and secretory activity were identified in germ tubes; this suggests the onset of a plant- fungus dialogue as early as at the germ tube stage Up- regulation of biogenesis, lipid transport and energy production during apressoriumformation DNA replication and cell division transcripts increase at the onset of sporulation From 9234 genes reported, 516 were predicted to encode secreted proteins (candidate effectors), particularly in germ tubes and haustoria Effectors are pathogen- produced proteins that induce or suppress plant responses These candidate effectors will be further validated and used to identify the respective host targets for marker assisted selection Fernandez et al. (2012) Molecular Plant Pathology 13: Vieira et al. (2012) European Journal of Plant Pathology 133: Gonçalves et al. (2013) Acta Phytopathologica Sinica 43 (Suppl): 335. Talhinhas et al. (2014) Frontiers in Plant Science 5: 88. Loureiro et al. (2015) Proc. 25 th Int. Conf Coffee Sci (ASIC), Colombia, pb250

10 Coffee Resistance Microscopic analysis Resistance Restricted fungal growth Germinated uredospore and appressorium Appressorium A over stomata Reaction flt Prehaustorial Resistance Large sporulating pustules Uredosporic sorum Penetration hypha Intercellular hyphae with haustoria Haustorial mother cell with haustoria Anchor Previous cytological studies have shown that host plants with high levels of prehaustorial defences to rust fungi may be sources of more durable resistance Niks and Rubiales (2002) Euphytica 124: Fernandez- Aparicio et al. (2011) Annals of Applied Biology 159: Diniz etal. (2012) European Journal of Plant Pathology 133: Posthaustorial Resistance

11 Coffee Resistance Microscopic analysis Pre- and Posthaustorial Resistanceisassociated with rapid plantcell death (Hypersensitive Reaction - HR) C I1 I2 A A %infection sites with HR A 41 hai 66 hai LM: Epifluorescence test A - Appressorium Days after inoculation C= Compatible interaction =Susceptibility I1= Incompatible interaction - Prehaustorial Resistance I2= Incompatible interaction - Posthaustorial Resistance Ø Early accumulation of phenolic- like compounds Silva et al. (2002) Physiol Mol Plant Pathol 60 (4): ; Guerra- Guimarães L. (2004). PhD Thesis, FCUL

12 Coffee Resistance Biochemical analysis Phenylalanine ammonia-lyase (PAL) activity First enzyme of the phenylpropanoid pathway A A 41 hai 66 hai LM: Epifluorescence test A - Appressorium Ø Early accumulation of phenolic- like compounds Silva et al. (2002) Physiol Mol Plant Pathol 60 (4): Activity of PAL (mg trans- cinnamic acid h - 1 g - 1 dry weight) 9 8 C T 7 6 H I1 T 6 5 H I2 T H Hours after inoculation

13 Coffee Resistance Biochemical analysis Other enzymes involved in the resistance response: Ø Superoxide dismutases Ø Lipoxigenases Ø Peroxidases Ø Chitinases Ø Glucanases 36 hai 96 hai Maxemiuc- Naccache et al. (1992). Rev Bras Bot; 15: Rojas et al. (1993). Physiol Mol Plant Pathol 43: Silva et al. (2008) Physiol Mol Plant Pathol 72: 29-38; Guerra- Guimarães et al. (2009), Proceedings ASIC, pp ; Guerra- Guimarães et al. (2009) Biol. Plantarum 53:702-6

14 Coffee Resistance Proteomic analysis Principal Component Analysis (PCA) Control Resistant Susceptible (24, 48, 72 and 96hai) Proteins oftheextracellular space vacuum infiltration S C R performed for the spots whose volume significantly changed in abundance 2- DE electrophoresis 116 proteins were identify by MS (Mass Spectrometry) Circled spots changed in abundance between samples (Anova p- value < 0,05 and fold change >1,5) Proteins up- regulated in the resistant genotype and identified as: Chitinases, Pectin methylesterase, Serine carboxypeptidase, Reticuline oxidase, Subtilisin protease were selected as antigens for antibody production against these putative protein biomarkers for resistance. Guerra- Guimarães et al. (2015). Front. Plant Sci. 6:478.

15 Coffee Resistance Proteomic analysis Higher level of detection in the resistant samples The reliability of these putative resistant biomarkers are being tested in different coffee genotypes from CIFC collection. These antibodies will be used to develop an ELISA kit assay, as a potential diagnostic tool to assist in the selection of varieties with resistance to H. vastatrix. Guerra- Guimarães et al. (2015) Front. Plant Sci. 6:478

16 Coffee Resistance Transcriptomic analysis Different approaches (e.g. suppression subtractive hybridisationmethod, 454 pyrosequencingand qpcr) allowed the identification of several genes putatively involved in coffee resistance (e.g. receptor- like kinases, WRKY transcription factors, chitinases, PRs, lipoxygenase, germin- like) Relative expression Incompatible Compatible Expression profile of Ca_22853 a Germin- likeprotein previously described (proteomic studies) as up- regulated in incompatible interaction Time after inoculation Fernandez et al. (2004). Molecular Plant Pathology 5: Ganesh et al Plant Science 170: Fernandez et al. (2004). Molecular Plant Pathology5: Ganesh et al Plant Science 170: Azinheira et al 2013; XIII Congresso Luso- Espanhol de Fisiologia Vegetal, Lisboa.

17 Conclusions and Perspectives Pathogenicity Surveys More than 50 rust races were characterized (time consuming work) Hv Pathogenicity Discovery of fungal effectorsas tools to uncover R- genes Coffee Resistance Prehaustorial resistance = = more durable resistance Early plant responses associated: HR, accumulation of phenolic- like compounds and up- regulation of genes and proteins putatively involved in plant defenses THANK YOU Molecular Markers Associated with Hv patotypes Identification of SNPs differentiating Hv virulence profiles H. vastatrix (Hv) and coffee unique world collections Disease Resistance Screening The breakdown of resistance of HDT derivatives is associated with the appearance of new rust races Inthe near future new sources of resistance must be identified and characterized combining the screening tests with the study of coffee resistance mechanisms along with the characterization of pathogen effectors and genetic variability