Bacillus methylotrophicus DCS1: Production of Different Lipopeptide Families, In Vitro Antifungal Activity and Suppression of Fusarium Wilt in Tomato Plants.

The present work aims to quantitatively and qualitatively monitor the production of lipopeptide mixtures by Bacillus methylotrophicus DCS1 strain in Landy medium and to investigate the antifungal activities of DCS1 strain and its produced lipopeptides. The in vitro activities were tested by the direct confrontation and agar well diffusion methods, while the in vivo study was carried out in order to test the efficiency of DCS1 bacterial suspension in the control of Fusarium wilt in tomato plants. Identification of lipopeptides by mass spectrometry (LC/MSD-TOF) showed that lipopeptide isoforms produced during the first 24 h and 48 h of fermentation are identical, belonging to bacillomycin D and fengycins A and B homologues with a difference in the yield of production. After 72 h of fermentation corresponding to the end of incubation period, B. methylotrophicus DCS1 is able to produce a mixture of surfactin, pumilacidin, iturin A/mycosubtilin, iturin C1, bacillomycin D and fengycins A and B isoforms. The results of in vitro antifungal experiments suggest that B. methylotrophicus DCS1 has a significant potential as a biocontrol agent, owing to lipopeptides produced, endowed with antifungal activity against several phytopathogenic fungi. The curative treatment of tomato plants with DCS1 bacterial suspension was more effective in the protection against Fusarium oxysporum f. sp. radicis-lycopersici (FORL) than the preventive treatment by comparing the average number of leaves remaining healthy after 30 days of each treatment and the appearance of tomato plants roots. The results indicate that B. methylotrophicus DCS1 exhibit a significant suppression of Fusarium wilt symptoms in tomato plants comparable to that of commercial fungicides and could be an alternative to chemically synthesized pesticides.

Olea europaea L. Root Endophyte Bacillus velezensis OEE1 Counteracts Oomycete and Fungal Harmful Pathogens and Harbours a Large Repertoire of Secreted and Volatile Metabolites and Beneficial Functional Genes.

Oomycete and fungal pathogens, mainly Phytophthora and Fusarium species, are notorious causal agents of huge economic losses and environmental damages. For instance, Phytophthora ramorum, Phytophthora cryptogea, Phytophthora plurivora and Fusarium solani cause significant losses in nurseries and in forest ecosystems. Chemical treatments, while harmful to the environment and human health, have been proved to have little or no impact on these species. Recently, biocontrol bacterial species were used to cope with these pathogens and have shown promising prospects towards sustainable and eco-friendly agricultural practices. Olive trees prone to Phytophthora and Fusarium disease outbreaks are suitable for habitat-adapted symbiotic strategies, to recover oomycetes and fungal pathogen biocontrol agents. Using this strategy, we showed that olive trees-associated microbiome represents a valuable source for microorganisms, promoting plant growth and healthy benefits in addition to being biocontrol agents against oomycete and fungal diseases. Isolation, characterization and screening of root microbiome of olive trees against numerous Phytophthora and other fungal pathogens have led to the identification of the Bacillus velezensis OEE1, with plant growth promotion (PGP) abilities and strong activity against major oomycete and fungal pathogens. Phylogenomic analysis of the strain OEE1 showed that B. velezensis suffers taxonomic imprecision that blurs species delimitation, impacting their biofertilizers' practical use. Genome mining of several B. velezensis strains available in the GenBank have highlighted a wide array of plant growth promoting rhizobacteria (PGPR) features, metals and antibiotics resistance and the degradation ability of phytotoxic aromatic compounds. Strain OEE1 harbours a large repertoire of secreted and volatile secondary metabolites. Rarefaction analysis of secondary metabolites richness in the B. velezenis genomes, unambiguously documented new secondary metabolites from ongoing genome sequencing efforts that warrants more efforts in order to assess the huge diversity in the species. Comparative genomics indicated that B. velezensis harbours a core genome endowed with PGP features and accessory genome encoding diverse secondary metabolites. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of OEE1 Volatile Organic Compounds (VOCs) and Liquid Chromatography High Resolution Mas Spectrometry (LC-HRMS) analysis of secondary metabolites identified numerous molecules with PGP abilities that are known to interfere with pathogen development. Moreover, B. velezensis OEE1 proved effective in protecting olive trees against F. solani in greenhouse experiments and are able to inhabit olive tree roots. Our strategy provides an effective means for isolation of biocontrol agents against recalcitrant pathogens. Their genomic analysis provides necessary clues towards their efficient implementation as biofertilizers.

Morphological and molecular characterization of Fusarium spp. associated with olive trees dieback in Tunisia.

Dieback and wilting symptoms caused by complex soilborne fungi are nowadays the most serious threatening disease affecting olive trees (Olea europaea) in Tunisia and presumably in many Mediterranean basin countries. Fusarium is one of the important phytopathogenic genera associated with dieback symptoms of olive trees. The objective of the present study was to confirm the pathogenicity of Fusarium spp. isolated from several olive-growing areas in Tunisia. According to the pathogenic test done on young olive trees (cv. Chemlali), 23 out of 104 isolates of Fusarium spp. were found to be pathogenic and the others were weakly or not pathogenic. The pathogenic Fusarium spp. isolates were characterized using molecular methods based on ITS PCR. Isolation results revealed the predominance of Fusarium solani (56.5%) and F. oxysporum species (21.7%) compared to F. chalmydosporum (8.7%), F. brachygibbosum (8.7%) and F. acuminatum (4.34%). Based on pathogenicity test, disease severity was highly variable among the 23 pathogenic isolates tested (P < 0.05) where F. solani was the most aggressive dieback agent. To the best of our knowledge, this is the first work that shows that Fusarium spp. might be a major agent causing dieback disease of olive trees in Tunisia.

The use of newly isolated Streptomyces strain TN258 as potential biocontrol agent of potato tubers leak caused by Pythium ultimum.

An actinomycete strain designated TN258, was isolated from Tunisian Sahara soil and selected for its antagonistic activity, especially against Pythium ultimum (P. ultimum) causing potato tubers leak. Based on the results of cultural characteristic of TN258 strain, the 16S rRNA gene nucleotide sequence (1433 bp, accession n° HE600071) and the phylogenetic analysis, we propose the assignment of our new isolate bacterium as Streptomyces TN258 strain. After culture optimization, the inhibitory effect of TN258 free cell supernatant against P. ultimum was evaluated. As result, by application of 50% (v/v) from 25 mg ml-1 of concentration, mycelial growth was totally inhibited with hyphal destruction. At the same concentration, the oospores were distorted and the germination was completely stopped. In potato tubers, Streptomyces TN258 filtrated supernatant, applied 24 h before inoculation by P. ultimum (preventive treatment group) was able to significantly decrease pathogen penetration by 62% and to reduce the percentage of weight loss by 59.43%, in comparison with non-treated group.