Selección y caracterización de cepas de "Streptomyces sp." como agentes de biocontrol frente a "Verticillium dahliae"

  1. Calvo Peña, Carla
Supervised by:
  1. Juan José Rubio Coque Director
  2. Rebeca Cobos Román Director

Defence university: Universidad de León

Fecha de defensa: 04 October 2024

Committee:
  1. Jesús Manuel Cantoral Fernández Chair
  2. Marcia Barquero Quirós Secretary
  3. Alexander Bernal Cabrera Committee member

Type: Thesis

Abstract

The fungus Verticillium dahliae is one of the main plant pathogens worldwide. It infects more than 200 species including such important crops as potato, sunflower, cotton and olive. Verticillium wilt of olive is currently considered the most serious biotic threat for olive cultivation, causing extreme worry in the Spanish olive sector. It is a vascular disease whose causal agent, V. dahliae, blocks the xylematic vessels causing, in turn, the wilting of the plant. This disease has been the subject of research for decades due to the difficulty of controlling it since this pathogen is characterized by the production of resistance structures called microsclerotia that allow its prolonged survival in the soil. This Thesis project is based on the isolation and characterization of actinomycetes belonged to the Streptomyces genus from both the rhizosphere and the root system of olive trees and their application in a putative “suppressive mixture” as a novel and environmentally friendly alternative to control infections caused by V. dahliae. Thus, 202 actinomycetes (96 rhizosphere and 106 endophytic strains) were isolated. Twelve of them were selected as promising Biological Control Agents (BCAs) according to in vitro assays against V. dahliae. These bioassays allowed us to quantify the antifungal activity of the actinomycetes in solid culture media. The selected strains were molecularly characterized using multilocus sequence analysis (MLSA) with the aim of obtain a species-level identification. This analysis confirmed that rhizosphere isolates OR6, OR14 and OR92 were conspecific and possibly belong to a new species. Isolate OR6 was further characterized and albocycline was found to be the main antifungal compound produced by this strain. Isolates OR58, OR67 and OR96 are also putative new species as they did not show identity with any other sequence in the databases. With regard to the endophytic isolates, MLSA analysis suggested that isolates OE79 and OE103 were identical and could represent a putative new species, isolate OE69 is indistinguishable from S. castelarensis or S. modarskii, isolate OE99 was identified as S. violaceochromogenes, and isolate OE54 was unequivocally identified as the rapamycinproducing S. iranensis. Molecular identification together with cross interactions analysis led to the selection of six isolates for the suppressive mixture: 3 rhizosphere strains (OR6, OR58 and OR96) and 3 endophytic strains (OE54, OE57 and OE69). Molecular markers for qPCR actinomycetes detection were designed from poorly conserved regions of the genome using two methods: RAPD amplification for isolates OR6 and OR96 and in silico analysis of non-coding regions in the genome for OE54. The effectiveness of the selected actinomycetes against the fungus V. dahliae was also demonstrated in small-scale soil tests. All the strains exhibited strong antifungal activity, especially isolate OR6, whose efficacy was close to 100%. HPLC-MS techniques allowed us to elucidate the main compounds involved in antifungal activity of some isolates. Thus, albocycline was found to be the main antifungal compound produced by isolate OR6 while rapamycin is the main bioactive compound for the antifungal activity of isolates OE57 and OE54, although anthranilic acid production was also detected in OE54. On the other hand, we carried out assays to evaluate the biocontrol agents as a preventive tool for the management of Verticillium wilt on olive trees. Unfortunately, we were no able to assess the efficacy due to the difficulty of infection with this pathogen and the time required for show symptoms of the disease. However, the effect on the composition of soil microbial communities in the field was evaluated by applying the mixtures to the soil of an olive orchard and then performing a metataxonomic analysis of the existing microorganisms. Finally, it should be highlighted the detection of the species Pleurostoma richardsiae during the search for phytopathogenic fungi in olive wood with apparent symptoms of Verticillium Wilt. It was the first report of this fungus associated with twig and branch dieback of olive trees in Spain. In summary, in this Thesis Project have been isolated actinomycetes as promising biocontrol agents for the fungal phytopathogen Verticillium dahliae. These microorganisms have been characterized and some of the most important bioactive compounds have been identified. In addition, -----------------------------CONFIDENTIAL------------------------------ and the fungus Pleurostoma richardsiae has been detected in olive wood for the first time in Spain.