Potential of Metarhizium spp. isolates in the alternative control against Asian soybean rust

Author(s)

Sabrina Holz

• Document type: Master's Dissertation
• Press: Piracicaba.
• Institution: Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC)
• Defense date: 2020-06-22

Examining board members

Sergio Florentino Pascholati; Italo Delalibera Junior; Edson Luiz Furtado; Cláudia Vieira Godoy

• Advisor: Sergio Florentino Pascholati

Abstract

The production of soybean crop is under constant threat of different fungal pathogens, including Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR). Thus, alternative control measures, such as biological control and resistance induction, have been integrated into the management system of this disease. On the other hand, the entomopathogenic fungus Metarhizium sp. is used in the biological control of pests and shows association with plants through endophytic activity or rhizosphere colonization exibiting potential to be used against plant pathogens. Thus, the objective of this work was to evaluate the potential of three diferent species of the fungus (M. anisopliae BR-01, M. robertsii BR-02 and M. humberii BR-03) in the control of soybean rust under in vitro (direct effect) and in vivo (indirect effect) assays. Besides that, the biologically active molecules present in the Metarhizium spp. culture filtrates free of fungal structures (CFFFS) were characterized in terms of molecular weight, heat and proteinase K activity stabilities. The in vitro tests were carried out inside of polystyrene Petri dishes, by using micro drops to evaluate the direct effect of conidial suspensions of Metarhizium (2x105 conidia/mL) and of the CFFFS filtrates in germination of the rust spores. By using detached soybean leaves (kept under laboratory conditions), they were first sprayed by using the preparations (CFFFS filtrates and conidial suspensions) and after 72 h they were spray-inoculated with the rust spores and the number of pustules per cm2 was evaluated. In plants maintained under greenhouse conditions (cultivar DS 5916 IPRO), two experiments were carried out. In the first assay, Metarhizium conidia were sprayed onto two trefoil of the soybean plants and after 72 h they were spray-inoculated with urediniospores suspensions. In the second one, CFFFS filtrates were sprayed onto two trefoil of the plants and later on spray-inoculated with the rust spores in three trefoil, aiming to evaluate a possible signal translocation (systemic resistance) and the severity of the disease was measured by using a diagrammatic scale. In the other experiment, Metarhizium spp. was evaluated via soil inoculation (\"soil drench\") by adding 2 mL of the spore suspension (1x108 conidia/mL), and after 20 days, a urediniospore suspension was spray-inoculated in two trefoil of the plants and the severity mensured. For all the mentioned experiments, the species Metarhizium (BR-01, BR-02 e BR-03) were used. In the results for the in vitro tests, only the CFFFS filtrate treatments showed inhibition, where the M. robertsii BR-02 was the best one, with inhibition around 66% in the lowest final concentration (12,5%) and around 99% in the highest one (50%). In the detached leaves assays, by using the conidial suspensions, the M. anisopliae BR-01 and M. humberii BR-03 isolates showed the best performance based upon reduction in pustule number. For the CFFFS filtrates both in detached leaves and in whole plants, the M. humberii BR-03 isolate was the best one, by showing a significant reduction in rust symptoms in the three trefoils, and in the third trefoil that did not receive the inducers. In the assay regarding Metarhizium as inoculant via soil, the M. anisopliae BR-01 isolate was the best one in reducing the symtptoms of rust in the plants. During the intership at Louisiana State University - USA, assays were performed to quantify the rust fungal biomass inside soybean leaves by using real time PCR, in soybean plants (Pioneer 93M60) treated with CFFFS filtrates (M. humberii BR-03, M. anisopliae BR-01 and M. robertsii US-04). The results were consistent as well as in reducing the disease severity and the relative expression of the target P. pachyrhizi gene with the three species. Although, the assay was carried out under different conditions from Brazil, the CFFFS filtrates showed anti-rust activity. Regarging to characterization of the biologically active molecules, the active compounds in the CFFFS filtrates are in the range of 3 to 10 kDa, heat stable, and most likely non-protein in nature, mainly for the isolates M. anisopliae US-02 and M. humberii BR-03. Therefore, the conclusion is that different Metarhizium isolates show anti-rust activity (directly and indirectly, probably by induction of resistance in soybean plants) and the mode of action needs to studied through biochemical and molecular approaches. Finally, there are not previous reports in the literature by using Metarhizium for this pathosystem, and the results from this study are novel and have a high potencial for the control of the Asian soybean rust. (AU)

• FAPESP's process: 18/18393-2 - Potential of different isolates of Metarhizium sp in the alternative control (induced resistance) of Phakopsora pachyrhizi in soybean plants
• Grantee: Sabrina Holz
• Support Opportunities: Scholarships in Brazil - Master