Contribution des actinobactéries (actinomycètes) dans la lutte biologique contre Pythium myriotylum, agent responsable de la pourriture des racines de macabo (Xanthosoma sagittifolium L. Schott)

Abstract

Cocoyam (Xanthosoma sagittifolium L. Schott) is an important nutritional and economic tuber crop. Cocoyam root rot disease (CRRD) caused by Pythium myriotylum is the major disease that limits growth and productivity of this crop. To reduce the usage of chemicals, an alternative for a substainable control method is necessary. Actinobacteria are known to produce antimicrobial compounds, are potential biocontrol agents and plant growth promoters. This study was carried out to isolate, evaluate and characterize actinobacteria from roots and rhizosphere of healthy cocoyam from localities around Mount Cameroon for their antagonistic activities against P. myriotylum and / or their ability to promote plant growth. Actinobacteria were isolated on culture media supplemented with antibiotics such as nystatin and nalixidic acid. Dual culture assay was used to screen the isolates against fungal phytopathogens including P. myriotylum and diffusion method was used for antibacterial activities. The antagonistic isolates were characterized morphologically, physiologically, biochemically and genetically. Furthermore, these isolates were characterized for production of extracellular hydrolytic enzymes, Plant Growth Promoting (PGP) traits and their effects were evaluated on tissue culture-derived cocoyam plantlets. Morever, biocontrol marker (gene chiC) and plant growth promotion marker (gene iaaM) were searched in the antagonistic strains using PCR technique. Finally, volatile and non volatile secondary bioactive metabolites produced by the antagonistic strains that showed ≥ 80 % percentage inhibition against P. myriotylum after dual assay, were partially characterized using GC-MS and LC-MS/MS analyses respectively. A total of 19 different actinobacterial isolates (07 endophytic and 12 rhizospheric) was obtained from roots and rhizosphere of healthy cocoyam plants based on morphological characteristics. After screening these isolates against P. myriotylum and other fungi, 07 isolates namely PERM1, PERM2, PERM4, PERM5, PFK4, PFBOT7 and PFEL2 showed high antagonistic activities against P. myriotylum, other fungal and bacteria phytopathogens. Phenotypic characters combined to molecular identification of antagonistic isolates indicated that all belonged to Streptomyces spp. genus. However, isolates PERM2 and PERM4 with 98 % and 97 % of similarities respectively with the closed strains retrieved from Genbank databases might represent novel species. The antagonistic Streptomyces strains showed abilities to produce cell wall degrading enzymes such as chitinase, β-1,3-glucanase, protease, lipase, cellulase and pectinase. They were also able to produce PGP traits like siderophores, Indole Acetic Acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, ammoniac (NH3), phosphate solubilization and nitrogen fixation (N2). However, PERM2 and PERM4 showed remarkable effects on cocoyam plantlets growth parameters (root length, fresh weight and secondary roots) and the gene (chiC) of chitinases was detected into these two strains. GC-MS analysis of Streptomyces sp. PFK4, PERM1, PERM2 and PERM4 ethyl acetate crude extracts, revealed the presence of several volatile bioactive metabolites which possess antimicrobial activities, PGP properties and the most representatives characterized by high peaks were linoleic acid (Z, Z), oleic acid, 1-Ascorbic acid 2,6-dihexadecanoate, trifluoroacetic acid, tetramethylphenazyn and 2,3-butanediol. Based on LC-MS/MS analysis, non-volatile bioactive metabolites belongs to antibiotics (amicoumarin, fungichromin, rapamycin), phenolic acids (3, 30-di-O-methylallegic acid, p-hydroxybenzoic acid, cafeic acid, ferulic acid, hydroxycinnamic acid, 7- oxooactadecanoic acid and vanilic acid) and amino acids (N-acetyl-D-phenylaline) with antifungal, antibacterial, antioxidant, PGP properties were identified in the crude extracts. Therefore, Streptomyces sp. PERM2, PERM4 and PFK4 could be developed as potential candidates in formulation of biofungicides and biofertilizers for a sustainable and environmentally friendly agriculture.