Pseudomonas cyclic lipopeptides (CLPs) are encoded non-ribosomally by biosynthetic gene clusters (BGCs) and possess diverse biological activities. In this study, we conducted chemical structure and BGC analyses with antimicrobial activity assays for two CLPs produced by Pseudomonas strains isolated from the cocoyam rhizosphere in Cameroon and Nigeria. LC-MS and NMR analyses showed that the Pseudomonas sp. COR52 and A2W4.9 produce pseudodesmin and viscosinamide, respectively. These CLPs belong to the Viscosin group characterized by a nonapeptidic moiety with a 7-membered macrocycle. Similar to other Viscosin-group CLPs, the initiatory non-ribosomal peptide synthetase (NRPS) gene of the viscosinamide BGC is situated remotely from the other two NRPS genes. In contrast, the pseudodesmin genes are all clustered in a single genomic locus. Nano- to micromolar levels of pseudodesmin and viscosinamide led to the hyphal distortion and/or disintegration of Rhizoctonia solani AG2-2 and Pythium myriotylum CMR1, whereas similar levels of White Line-Inducing Principle (WLIP), another member of the Viscosin group, resulted in complete lysis of both soil-borne phytopathogens. In addition to the identification of the biosynthetic genes of these two CLPs and the demonstration of their interaction with soil-borne pathogens, this study provides further insights regarding evolutionary divergence within the Viscosin group.

The potential use of Bacillus velezensis FZB42 for biological control of various phytopathogens has been documented over the past few years, but its antagonistic interactions with xanthomonads has not been studied in detail. Novel aspects in this study consist of close observation of the death of Xanthomonas campestris pv. campestris cells in a co-culture with B. velezensis FZB42, and quantification of lipopeptides and a siderophore, bacillibactin, involved in the killing process. A new robust Xcc-SU isolate tolerating high concentrations of ferric ions was used. In a co-culture with the antagonist, the population of Xcc-SU was entirely destroyed within 24-48 h, depending on the number of antagonist cells used for inoculation. No inhibitory effect of Xcc-SU on B. velezensis was observed. Bacillibactin and lipopeptides (surfactin, fengycin, and bacillomycin) were present in the co-culture and the monoculture of B. velezensis. Except for bacillibactin, the maximum contents of lipopeptides were higher in the antagonist monoculture compared with the co-culture. Scanning electron microscopy showed that the death of Xcc-SU bacteria in co-culture was caused by cell lysis, leading to an enhanced occurrence of distorted cells and cell ghosts. Analysis by mass spectrometry showed four significant compounds, bacillibactin, surfactin, fengycin, and bacillomycin D amongst a total of 24 different forms detected in the co-culture supernatant: Different forms of surfactin and fengycin with variations in their side-chain length were also detected. These results demonstrate the ability of B. velezensis FZB42 to act as a potent antagonistic strain against Xcc.

Pepper bacterial spot is one of the most severe plant diseases in terms of infection persistence and economic losses when it comes to fresh pepper fruits used in nutrition and industrial processing. In this study, Bacillus velezensis IP22 isolated from fresh cheese was used as a biocontrol agent of pepper bacterial spot, whose main causal agent is the cosmopolitan pathogen Xanthomonas euvesicatoria. After optimization of the cultivation medium composition aimed at maximizing of the antimicrobial activity against X. euvesicatoria and validation of the optimized medium at the scale of a laboratory bioreactor, in planta tests were performed. The results have showed significant suppression of bacterial spot symptoms in pepper plants by the produced biocontrol agent, as well as reduction of disease spreading on the healthy (uninoculated) pepper leaves. Furthermore, HPLC-MS (high pressure liquid chromatography-mass spectrometry) analysis was employed to examine antimicrobial metabolites produced by B. velezensis IP22, where lipopeptides were found with similar m/z values compared to lipopeptides from fengycin and locillomycin families. The bioprocess solution developed at the laboratory scale investigated in this study represents a promising strategy for production of pepper bacterial spot biocontrol agent based on B. velezensis IP22, a food isolate with a great perspective for application in plant protection.

Verticillium wilt, caused by the pathogen Verticillium dahliae, is extremely devastating to olive trees (Olea europea). Currently, no successful control measure is available against it. The objective of this work was to evaluate the antifungal activity of Bacillus velezensis XT1, a well-characterized salt-tolerant biocontrol strain, against the highly virulent defoliating V. dahliae V024. In vitro, strain XT1 showed to reduce fungal mycelium from 34 to 100%, depending on if the assay was conducted with the supernatant, volatile compounds, lipopeptides or whole bacterial culture. In preventive treatments, when applied directly on young olive trees, it reduced Verticillium incidence rate and percentage of severity by 54 and ~80%, respectively. It increased polyphenol oxidase (PPO) activity by 395%, indicating an enhancement of disease resistance in plant tissues, and it decreased by 20.2% the number of fungal microsclerotia in soil. In adult infected trees, palliative inoculation of strain XT1 in the soil resulted in a reduction in Verticillium symptom severity by ~63%. Strain XT1 is biosafe, stable in soil and able to colonize olive roots endophytically. All the traits described above make B. velezensis XT1 a promising alternative to be used in agriculture for the management of Verticillium wilt.

Bacillus strains can produce various lipopeptides, known for their antifungal properties. This makes them attractive metabolites for applications in agriculture. Therefore, identification of productive wild-type strains is essential for the development of biopesticides. Bacillus velezensis FZB42 is a well-established strain for biocontrol of plant pathogens in agriculture. Here, we characterized an alternative strain, B. velezensis UTB96, that can produce higher amounts of all three major lipopeptide families, namely surfactin, fengycin, and iturin. UTB96 produces iturin A. Furthermore, UTB96 showed superior antifungal activity towards the soybean fungal pathogen Diaporthe longicolla compared to FZB42. Moreover, the additional provision of different amino acids for lipopeptide production in UTB96 was investigated. Lysine and alanine had stimulatory effects on the production of all three lipopeptide families, while supplementation of leucine, valine and isoleucine decreased the lipopeptide bioproduction. Using a 45-litre bioreactor system for upscaling in batch culture, lipopeptide titers of about 140 mg/L surfactin, 620 mg/L iturin A, and 45 mg/L fengycin were achieved. In conclusion, it becomes clear that B. velezensis UTB96 is a promising strain for further research application in the field of agricultural biological controls of fungal diseases.

This study aimed to isolate lactic acid bacteria (LAB) from wheat rhizosphere, to characterize their in vitro plant growth promoting activities and to differentiate plant-associated LAB from those associated with foods or human disease through comparative genomic analysis. Lactococcus lactis subsp. lactis and Enterococcus faecium were isolated using de Man-Rogosa-Sharpe (MRS) and Glucose Yeast Peptone (GYP) as enrichment culture media. Comparative genomic analyses showed that plant-associated LAB strains were enriched in genes coding for bacteriocin production when compared to strains from other ecosystems. Isolates of L. lactis and E. faecium did not produce physiologically relevant concentrations of the phyto-hormone indolacetic acid. All isolates solubilized high amount of phosphate and 12 of 16 strains solubilized potassium. E. faecium LB5, L. lactis LB6, LB7, and LB9 inhibited the plant pathogenic Fusarium graminearum to the same extent as two strains of Bacillus sp. However, the antifungal activity of the abovementioned LAB strains depended on the medium of cultivation and a low pH while antifungal activity of Bacillus spp. was independent of the growth medium and likely relates to antifungal lipopeptides. This study showed the potential of rhizospheric LAB for future application as biofertilizers in agriculture.

Some bacteria (notably the genera Bacillus and Clostridium) have the capacity to form endospores that can survive for millions of years in isolated habitats. The genomes of such ancient bacteria provide unique opportunities to understand bacterial evolution and metabolic capabilities over longer time scales. Herein, we sequenced the genome of a 2000-year-old bacterial strain (Mal05) isolated from intact apple seeds recovered during archaeological excavations of a Roman villa in Italy. Phylogenomic analyses revealed that this strain belongs to the species Bacillus stercoris and that it is placed in an early-branching position compared to most other strains of this species. Similar to other Bacillus species, B. stercoris Mal05 had been previously shown to possess antifungal activity. Its genome encodes all the genes necessary for the biosynthesis of fengycin and surfactin, two cyclic lipopeptides known to play a role in the competition of Bacilli with other microorganisms due to their antimicrobial activity. Comparative genomics and analyses of selective pressure demonstrate that these genes are present in all sequenced B. stercoris strains, despite the fact that they are not under strong purifying selection. Hence, these genes may not be essential for the fitness of these bacteria, but they can still provide a competitive advantage against other microorganisms present in the same environment.

Many aspects regarding the role of lipopeptides (LPs) in bacterial interaction with plants are not clear yet. Of particular interest is the LP family of surfactin, immunogenic molecules involved in induced systemic resistance (ISR) and the bacterial colonization of plant surfaces. We hypothesize that the concentration of surfactin produced by a strain correlates directly with its ability to colonize and persist on different plant surfaces, which conditions its capacity to trigger ISR. We used two Bacillus velezensis strains (BBC023 and BBC047), whose antagonistic potential in vitro is practically identical, but not on plant surfaces. The surfactin production of BBC047 is 1/3 higher than that of BBC023. Population density and SEM images revealed stable biofilms of BBC047 on leaves and roots, activating ISR on both plant surfaces. Despite its lower surfactin production, strain BBC023 assembled stable biofilms on roots and activated ISR. However, on leaves only isolated, unstructured populations were observed, which could not activate ISR. Thus, the ability of a strain to effectively colonize a plant surface is not only determined through its production of surfactin. Multiple aspects, such as environmental stressors or compensation mechanisms may influence the process. Finally, the importance of surfactin lies in its impacts on biofilm formation and stable colonization, which finally enables its activity as an elicitor of ISR.

Polymyxin-producing bacteria within the Paenibacillus polymyxa complex have broad-spectrum activities against fungi and bacteria. Their antibacterial activities against soft rot Dickeya and Pectobacterium phytopathogens containing multiple polymyxin-resistant genes were not clear. Here, we selected nine strains within the P. polymyxa complex having broad-spectrum antagonistic activities against phytopathogenic fungi and a polymyxin-resistant D. dadantii strain causing stem and root rot disease of sweet potato and did antagonistic assays on nutrient agar and sweet potato tuber slices. These strains within the P. polymyxa complex showed clear antagonistic activities against D. dadantii in vitro and in vivo. The most effective antagonistic strain P. polymyxa ShX301 showed broad-spectrum antagonistic activities against all the test Dickeya and Pectobacterium strains, completely eliminated D. dadantii from sweet potato seed tubers, and promoted the growth of sweet potato seedlings. Cell-free culture filtrate of P. polymyxa ShX301 inhibited D. dadantii growth, swimming motility, and biofilm formation and disrupted D. dadantii plasma membranes, releasing nucleic acids and proteins. Multiple lipopeptides produced by P. polymyxa ShX301 may play a major role in the bactericidal and bacteriostatic actions. This study clarifies that the antimicrobial spectrum of polymyxin-producing bacteria within the P. polymyxa complex includes the polymyxin-resistant Dickeya and Pectobacterium phytopathogens and strengthens the fact that bacteria within the P. polymyxa complex have high probability of being effective biocontrol agents and plant growth promoters.

In this study, 58 endophytic bacterial strains were isolated from pods of two hybrid vanilla plants from Madagascar, Manitra ampotony and Tsy taitra. They were genetically characterized and divided into four distinct phylotypes. Three were associated to genus Bacillus species, and the fourth to the genus Curtobacterium. A selection of twelve strains corresponding to the identified genetic diversity were tested in vitro for four phytobeneficial capacities: phosphate solubilisation, free nitrogen fixation, and phytohormone and siderophore production. They were also evaluated in vitro for their ability to biocontrol the growth of the vanilla pathogenic fungi, Fusarium oxysporum f. sp. radicis vanillae and Cholletotrichum orchidophilum. Three bacteria of phylotype 4, m62a, m64 and m65, showed a high nitrogen fixation capacity in vitro, similar to the Pseudomonas florescens F113 bacterium used as a control (phospate solubilizing efficiency respectively 0.50 ± 0.07, 0.43 ± 0.07 and 0.40 ± 0.06 against 0.48 ± 0.03). Strain t2 related to B. subtilis showed a higher siderophore production than F113 (respectively 1.40 ± 0.1 AU and 1.2 ± 0.1 AU). The strain m72, associated with phylotype 2, showed the highest rate of production of Indole-3-acetic acid (IAA) in vitro. Bacteria belonging to the pylotype 4 showed the best capacity to inhibit fungal growth, especially the strains m62b m64 and t24, which also induced a significant zone of inhibition, suggesting that they may be good candidates for controlling fungal diseases of vanilla. This competence was highlighted with spectral imaging showing the production of lipopeptides (Iturin A2 and A3, C16 and C15-Fengycin A and C14 and C15-Surfactin) by the bacterial strains m65 confronted with the pathogenic fungi of vanilla.