Artemisia, an ark of bioactive endophytic fungi

  1. Andreea Daniela Cosoveanu
Supervised by:
  1. Raimundo Manuel Cabrera Pérez Director
  2. Beatrice Michaela Iacomi Director
  3. Ionela Dobrin Director

Defence university: Universidad de La Laguna

Year of defence: 2017

Department:
  1. Botánica, Ecología y Fisiología Vegetal

Type: Thesis

Teseo: 494080 DIALNET

Abstract

Artemisia is a plant genus highly studied for its medicinal applications. The studies on the associated fungal endophytes are scarce. We have conveyed a survey on 77 plants of 14 species of Artemisia collected from the Canary Islands, Cape Verde, Sardinia, Romania and China. 518 endophytic fungal strains were isolated and classified into 105 fungal species and 48 fungal genera. Among the dominant endophytic fungal species, we observed taxa such as Alternaria, Fusarium, Aspergillus, Neofusicoccum, Botryosphaeria and Curvularia. In this study, the number of the fragments plated did not influence the number of the endophytic fungal isolates (p = 0.778). These preliminary data of our study revealed a possible fungal endophytic specificity towards host plant and plant species. Artemisia thuscula had the highest diversity of fungal endophytes confirmed by all indices, in both species and genera taxa. Diversity compared between localities showed the plants from San Bartolo (La Palma) to have the highest species richness. In terms of regions, La Palma plants proved to be the most diverse, although a slightly higher species richness value was observed in the plants from the Romanian coast (6.70 for Romania versus 6.23 in La Palma). In the case study of Artemisia thuscula, among the rare species were found Aplosporella prunicola, Camarosporium sp., Macrophomina phaseolina, Chaetomium sp., Nectria mauritiicola, Neofusicoccum australe, Pestalotiopsis sp., Phoma sp. and Stachybotrys longispora. Artemisia thuscula was the plant host species with the highest number of endophytes with antagonist activity against fungal pathogens. Of 30 extracts obtained from endophytic fungi and tested against bacterial growth, the most effective one was found to be 1053 (Alternaria sp. strain HTF52) which highly inhibited eight of the nine tested bacterial strains. 48 endophytic fungi selected according to their activity shown in preliminary dual culture assays of antagonism were cultivated on rice, solvent extracted and their extracts tested against fungal pathogenic strains. Seven endophytic fungi were solvent extracted twice. Extracts obtained from Alternaria sp. strain HTF58 and Fusarium sp. strain HRO8 maintained their activity against Botrytis cinerea and Alternaria alternata, respectively, during at least two cycles of extraction. Extracts obtained from HTF58 maintained their activity against Botrytis cinerea with low variations IC50 = 0.23 mg ml-1 versus IC50 = 0.07 mg ml-1, respectively. Five extracts were obtained from HRO8 using different solvents. The activity of the extracts against fungal growth of Alternaria alternata ranged between 63% and 100% at 0.5 mg ml-1. Five extracts obtained from Thielavia sp. strain HCV15, Alternaria sp. strain HTF52, Fusarium sp. strain HRO8, Stemphylium sp. strain HTF83, Neoplatysporoides sp. strain HLP44 were tested for their potential to protect Raphanus sativus seeds from Alternaria brassicicola (Abra43). Overall all extracts showed a potential to protect seedlings. Using nephelometry technique we have detected the effects of Fusarium sp. strain HRO8 extract 1057 and its fractions on fungal pathogen Abra43 and its null mutants AbNik1, AbHog1, AbSch9 and AbAp1. The mutants were highly susceptible to the polar fraction F6. The sensitivity of these null mutants to 1057F6 compounds indicates toxicity exerted in A. brassicicola through oxidative stress. In addition, H2DCFDA assay revealed 1057F6-induced ROS accumulation in A. brassicicola fungal cells. The in vivo bioassay tomato plants- fungal endophyte-fungal pathogen indicate that the fungal endophyte Fusarium sp. strain HRO8 caused no harm to the tomato plants when directly applied to the soil and also protected the host against Alternaria alternata.