Dissecting host specificity in the pathogenesis of S- and C-biotype isolates of Moniliophthora perniciosa, the causal agent of cacao witches\' broom disease

Author(s)

Daniele Paschoal

• Document type: Doctoral Thesis
• Press: Piracicaba.
• Institution: Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC)
• Defense date: 2024-07-19

Examining board members

Antonio Vargas de Oliveira Figueira; Alessandra Alves de Souza; Daniela Paula de Toledo Thomazella

• Advisor: Antonio Vargas de Oliveira Figueira

Abstract

Witches\' broom disease (WBD) of cacao (Theobroma cacao), caused by the hemibiotrophic fungus Moniliophthora perniciosa, poses a significant global threat to cacao production. Isolates of M. perniciosa can be categorized into biotypes based on host specificity: the C-biotype infects cacao, the L-biotype colonizes wild lianas, and the S-biotype infects solanaceous species, with limited cross-infection. Typical symptoms include loss of apical dominance and hypertrophic growth of axillary shoots. The use of tomato (Solanum lycopersicum) cv. Micro-Tom (MT) as a model enables the study of M. perniciosa interactions. In Chapter VII, we investigated the hormonal changes associated with these symptoms in MT, demonstrating that the S-biotype produces cytokinins (CKs) in vitro, potentially disrupting cytokinin balance in infected plants, as evidenced by elevated CK levels in the thickened, infected stems. MT lines overexpressing the Arabidopsis CYTOKININ OXIDASE-2 (35S::AtCKX2), which reduces CK levels, showed decreased symptom severity and infection incidence. Additionally, synthetic CK treatments mimicked disease symptoms, while CK perception inhibitors reduced symptoms. In Chapter VI, we demonstrated that infection in MT enhances sink strength in symptomatic stems, an effect absent in 35S::AtCKX2 lines, implicating CKs in the formation of a nutrient sink. These symptoms led to significant metabolic and physiological changes, reducing fruit yield and potentially reinforcing secondary cell walls and producing lignin, likely serving as a nutritional resource for the pathogen during the necrotrophic phase. Chapter V explored the indirect effects of infection in root development. We observed reduced root biomass coinciding with increased stem diameter after infection, with lateral root elongation being primarily affected. Hormonal imbalances could not fully explain this impairment; infected roots showed reduced levels of carbohydrate and amino acids, and a 13C-CO2 fixation assay revealed decreased carbon allocation to roots after infection. Sucrose supplementation in vitro partially restored lateral root growth, suggesting that reduced root biomass results from decreased photoassimilate supply, which might impair water and nutrient uptake and contribute to reduced host yield. Building on symptom development, Chapter II utilized the MT model to investigate fungal pathogenesis and plant defense responses by comparing MT infection by either the S-biotype (compatible) or the C-biotype (incompatible) of M. perniciosa. Our findings revealed common early transcriptional immune responses, particularly in pattern-triggered immunity (PTI) signaling and cell wall reinforcement, with stronger expression during the incompatible interaction, indicating more robust defenses. This aligns with pre- and co-inoculation assays demonstrating a priming response to the compatible pathogen. Fungal gene expression revealed presumed effectors and other genes involved in pathogenesis in cacao during early infection of MT. Hormonal responses, particularly jasmonic acid (JA) and ethylene (ET), were implicated in the infection process, with ET remarkably playing a role in inducing resistance to S_biotype M. perniciosa in MT. In Chapter III, we introduced Nicotiana benthamiana as a novel model host for M. perniciosa and conducted comparative transcriptional and functional profiling. N. benthamiana is susceptible to both S- and C- biotypes, though one S-biotype isolate (Tiradentes) showed incompatibility, challenging the classical view of M. perniciosa biotype classification. Early transcriptional responses to inoculation revealed shared immune responses to both biotypes, with quicker and stronger defenses in the incompatible interaction. Suppression of immune responses was observed at 72 hours after inoculation, coinciding with the expression of several fungal genes also active in cacao infections. Functional analyses suggested that TIR-NLR-related effector-triggered immunity (ETI) may not be essential for infection by the incompatible biotype in N. benthamiana, and we suspect that PTI could be involved. Additionally, in Chapter IV, we identified four effector candidates expressed in N. benthamiana that exhibited antimicrobial activity, potentially modulating the host microbiota during colonization. Characterizing symptom development, identifying early defense response components, and understanding fungal pathogenesis are crucial steps toward developing strategies to enhance cacao resistance to M. perniciosa. (AU)

• FAPESP's process: 18/18711-4 - Functional analysis of genes potentially associated with resistance to the basidiomycete Moniliophthora perniciosa in tomato (Solanum lycopersicum) cv. 'Micro-Tom'
• Grantee: Daniele Paschoal
• Support Opportunities: Scholarships in Brazil - Doctorate