Pseudomonas protegens MP12: A plant growth-promoting endophytic bacterium with broad-spectrum antifungal activity against grapevine phytopathogens

Abstract Pseudomonas sp. MP12 was isolated from a soil sample collected in a typical warm-temperate deciduous forest near Brescia, Northern Italy. Phylogenetic analysis identified the species as Pseudomonas protegens. We evidenced in this strain the presence of the genes phlD, pltB and prnC responsible for the synthesis of the antifungal compounds 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin and pyrrolnitrin, respectively. P. protegens MP12 was also shown to produce siderophores and ammonia, yielded positive results with the indole-3-acetic acid test, and was capable of phosphate solubilization. Moreover, P. protegens MP12 exhibited inhibitory effects on in vitro mycelial growth of prominent grapevine (Vitis vinifera) phytopathogens such as Botrytis cinerea, Alternaria alternata, Aspergillus niger, Penicillium expansum and Neofusicoccum parvum. The strain showed activity even against Phaeomoniella chlamydospora and Phaeoacremonium aleophilum, which cause the devastating tracheomycosis/esca disease of grapevine trunks for which no efficacious control methods have been demonstrated so far. Furthermore, the MP12 strain manifested in vivo antifungal activity against B. cinerea on grapevine leaves. Culture-dependent and culture-independent analysis revealed the ability of P. protegens MP12 to efficiently and permanently colonize inner grapevine tissues. These results suggest that P. protegens MP12 could be worth of exploitation as an antifungal biocontrol agent for applications in viticulture

Influence of bacterial physiology on processing of selenite, biogenesis of nanomaterials and their thermodynamic stability

We explored how Ochrobactrum sp. MPV1 can convert up to 2.5 mM selenite within 120 h, surviving the challenge posed by high oxyanion concentrations. The data show that thiol-based biotic chemical reaction(s) occur upon bacterial exposure to low selenite concentrations, whereas enzymatic systems account for oxyanion removal when 2 mM oxyanion is exceeded. The selenite bioprocessing produces selenium nanomaterials, whose size and morphology depend on the bacterial physiology. Selenium nanoparticles were always produced by MPV1 cells, featuring an average diameter ranging between 90 and 140 nm, which we conclude constitutes the thermodynamic stability range for these nanostructures. Alternatively, selenium nanorods were observed for bacterial cells exposed to high selenite concentration or under controlled metabolism. Biogenic nanomaterials were enclosed by an organic material in part composed of amphiphilic biomolecules, which could form nanosized structures independently. Bacterial physiology influences the surface charge characterizing the organic material, suggesting its diverse biomolecular composition and its involvement in the tuning of the nanomaterial morphology. Finally, the organic material is in thermodynamic equilibrium with nanomaterials and responsible for their electrosteric stabilization, as changes in the temperature slightly influence the stability of biogenic compared to chemogenic nanomaterials

Microbial-Based Bioremediation of Selenium and Tellurium Compounds

The chalcogens selenium (Se) and tellurium (Te) are rare earth elements, which are mainly present in the environment as toxic oxyanions, due to the anthropogenic activities. Thus, the increased presence of these chalcogen-species in the environment and the contamination of wastewaters nearby processing facilities led to the necessity in developing remediation strategies aimed to detoxify waters, soils and sediments. Among the different decontamination approaches, those based on the ability of microorganisms to bioaccumulate, biomethylate or bioconvert Se- and/or Te-oxyanions are considered the leading strategy for achieving a safe and eco-friendly bioremediation of polluted sites. Recently, several technologies based on the use of bacterial pure cultures, bacterial biofilms or microbial consortia grown in reactors with different configurations have been explored for Se- and Te-decontamination purposes. Further, the majority of microorganisms able to process chalcogen-oxyanions have been described to generate valuable Se- and/or Te-nanomaterials as end-products of their bioconversion, whose potential applications in biomedicine, optoelectronics and environmental engineering are still under investigation. Here, the occurrence, the use and the toxicity of Se- and Te-compounds will be briefly overviewed, while the microbial mechanisms of chalcogen-oxyanions bioprocessing, as well as the microbial-based strategies used for bioremediation approaches will be extensively described

Physical–chemical properties of biogenic selenium nanostructures produced by Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1

Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1 were isolated from the rhizosphere soil of the selenium-hyperaccumulator legume Astragalus bisulcatus and waste material from a dumping site for roasted pyrites, respectively. Here, these bacterial strains were studied as cell factories to generate selenium-nanostructures (SeNS) under metabolically controlled growth conditions. Thus, a defined medium (DM) containing either glucose or pyruvate as carbon and energy source along with selenite (www.frontiersin.org) was tested to evaluate bacterial growth, oxyanion bioconversion and changes occurring in SeNS features with respect to those generated by these strains grown on rich media. Transmission electron microscopy (TEM) images show extra- or intra-cellular emergence of SeNS in SeITE02 or MPV1 respectively, revealing the presence of two distinct biological routes of SeNS biogenesis. Indeed, the stress exerted by www.frontiersin.org upon SeITE02 cells triggered the production of membrane vesicles (MVs), which surrounded Se-nanoparticles (SeNPsSeITE02-G_e and SeNPsSeITE02-P_e with average diameter of 179 ± 56 and 208 ± 60 nm, respectively), as highlighted by TEM and scanning electron microscopy (SEM), strongly suggesting that MVs might play a crucial role in the excreting mechanism of the SeNPs in the extracellular environment. On the other hand, MPV1 strain biosynthesized intracellular inclusions likely containing hydrophobic storage compounds and SeNPs (123 ± 32 nm) under pyruvate conditioning, while the growth on glucose as the only source of carbon and energy led to the production of a mixed population of intracellular SeNPs (118 ± 36 nm) and nanorods (SeNRs; average length of 324 ± 89). SEM, fluorescence spectroscopy, and confocal laser scanning microscopy (CLSM) revealed that the biogenic SeNS were enclosed in an organic material containing proteins and amphiphilic molecules, possibly responsible for the high thermodynamic stability of these nanomaterials. Finally, the biogenic SeNS extracts were photoluminescent upon excitation ranging from 380 to 530 nm, whose degree of fluorescence emission (λem = 416–640 nm) was comparable to that from chemically synthesized SeNPs with L-cysteine (L-cys SeNPs). This study offers novel insights into the formation, localization, and release of biogenic SeNS generated by two different Gram-negative bacterial strains under aerobic and metabolically controlled growth conditions. The work strengthens the possibility of using these bacterial isolates as eco-friendly biocatalysts to produce high quality SeNS targeted to possible biomedical applications and other biotechnological purposes

Untargeted Metabolomics Investigation on Selenite Reduction to Elemental Selenium by Bacillus mycoides SeITE01

Bacillus mycoides SeITE01 is an environmental isolate that transforms the oxyanion selenite ( SeO3 2- ) into the less bioavailable elemental selenium (Se0 ) forming biogenic selenium nanoparticles (Bio-SeNPs). In the present study, the reduction of sodium selenite (Na2SeO3) by SeITE01 strain and the effect of SeO3 2- exposure on the bacterial cells was examined through untargeted metabolomics. A time-course approach was used to monitor both cell pellet and cell free spent medium (referred as intracellular and extracellular, respectively) metabolites in SeITE01 cells treated or not with SeO3 2- . The results show substantial biochemical changes in SeITE01 cells when exposed to SeO3 2- . The initial uptake of SeO3 2- by SeITE01 cells (3 h after inoculation) shows both an increase in intracellular levels of 4-hydroxybenzoate and indole-3-acetic acid, and an extracellular accumulation of guanosine, which are metabolites involved in general stress response adapting strategies. Proactive and defensive mechanisms against SeO3 2- are observed between the end of lag (12h) and beginning of exponential (18h) phases. Glutathione and N-acetyl-L-cysteine are thiol compounds that would be mainly involved in Painter-type reaction for the reduction and detoxification of SeO3 2- to Se0 . In these growth stages, thiol metabolites perform a dual role, both acting against the toxic and harmful presence of the oxyanion and as substrate or reducing sources to scavenge ROS production. Moreover, detection of the amino acids L-threonine and ornithine suggests changes in membrane lipids. Starting from stationary phase (24 and 48h), metabolites related to the formation and release of SeNPs in the extracellular environment begin to be observed. 5-hydroxyindole acetate, D-[+]-glucosamine, 4-methyl-2-oxo pentanoic acid, and ethanolamine phosphate may represent signaling strategies following SeNPs release from the cytoplasmic compartment, with consequent damage to SeITE01 cell membranes. This is also accompanied by intracellular accumulation of trans-4-hydroxyproline and L-proline, which likely represent osmoprotectant activity. The identification of these metabolites suggests the activation of signaling strategies that would protect the bacterial cells from SeO3 2- toxicity while it is converting into SeNPs

Biogenic selenium and tellurium nanoparticles synthesized by environmental microbial isolates efficaciously inhibit bacterial planktonic cultures and biofilms

The present study deals with Se(0)- and Te(0)-based nanoparticles bio-synthesized by two selenite- and tellurite-reducing bacterial strains, namely Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1, isolated from polluted sites. We evidenced that, by regulating culture conditions and exposure time to the selenite and tellurite oxyanions, differently sized zero-valent Se and Te nanoparticles were produced. The results revealed that these Se(0) and Te(0) nanoparticles possess antimicrobial and biofilm eradication activity against Escherichia coli JM109, Pseudomonas aeruginosa PAO1, and Staphylococcus aureus ATCC 25923. In particular, Se(0) nanoparticles exhibited antimicrobial activity at quite low concentrations, below that of selenite. Toxic effects of both Se(0) and Te(0) nanoparticles can be related to the production of reactive oxygen species upon exposure of the bacterial cultures. Evidence so far achieved suggests that the antimicrobial activity seems to be strictly linked to the dimensions of the nanoparticles: indeed, the highest activity was shown by nanoparticles of smaller sizes. In particular, it is worth noting how the bacteria tested in biofilm mode responded to the treatment by Se(0) and Te(0) nanoparticles with a susceptibility similar to that observed in planktonic cultures. This suggests a possible exploitation of both Se(0) and Te(0) nanoparticles as efficacious antimicrobial agents with a remarkable biofilm eradication capacity

Assisted phytostabilization of soils affected by mining activities using Lolium perenne L.

We studied the application of Lolium per-enne L. and of different organic residues in the assisted phytostabilization of mine-contaminated soils (Aljustrel Mine Area, Iberian Pyrite Belt). Although the applica-tion of the organic residues led to immobilization of Cu, Pb, and Zn in the soil, decreas-ing their effective available fraction, their concentrations in the above-ground plant material did not change. The accumulation factors (AF) obtained were AF(Cu) and AF(Pb) <<1, and AF(Zn) <1, allowing its election as a possible candidate for phytos-tabilization purposes. However, Pb and Cu concentrations in ryegrass shoots were con-sidered toxic, which suggests that there is a risk of contamination of the human food chain. Multivariate statistical analysis em-phasized sewage sludge as the organic resi-due with the greatest capacity to correct soil acidity, and to improve soil characteristics, followed by the compost obtained from municipal solid waste residues. However, an impaired growth was obtained when using the highest application rate (100 Mg ha-1) of both residues. All garden waste compost rates were unable to correct general soil constraints to plant growth

Utilização de Lolium Perenne L. na fitoestabilização controlada de solos degradados por actividades mineiras

Foi estudada a utilização de Lolium perenne L. e de diferentes resíduos orgânicos (RO) na fitoestabilização controlada de solos degradados por atividades mineiras (Mina de Aljustrel, Faixa Piritosa Ibérica). Apesar da aplicação dos RO ter permitido a imobilização de Cu, Pb e Zn no solo, baixando a fracção efetivamente disponível, os teores foliares destes elementos não diminuíram significativamente. Os factores de acumulação (FA) obtidos, FA(Cu) e FA(Pb) <<1 e FA(Zn) <1, permitem considerar esta espécie adequada para a fitoestabilização deste tipo de solos. Os teores foliares de Pb e Cu atingiram níveis considerados tóxicos, indicando risco de entrada destes metais na cadeia alimentar humana. Métodos estatísticos multivariados evidenciaram a lama residual urbana como o RO com maior capacidade para corrigir a acidez do solo e para melhorar as características deste, seguida do composto de resíduos sólidos urbanos. Porém,observou-se um crescimento reduzido por aplicação do nível mais elevado (100 Mg ha-1) destes RO. Todos os níveis do composto de resíduos verdes foram insuficientes para corrigir as principais restrições do solo ao crescimento vegetal

Physical–Chemical Properties of Biogenic Selenium Nanostructures Produced by Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1

Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1 were isolated from the rhizosphere soil of the selenium-hyperaccumulator legume Astragalus bisulcatus and waste material from a dumping site for roasted pyrites, respectively. Here, these bacterial strains were studied as cell factories to generate selenium-nanostructures (SeNS) under metabolically controlled growth conditions. Thus, a defined medium (DM) containing either glucose or pyruvate as carbon and energy source along with selenite () was tested to evaluate bacterial growth, oxyanion bioconversion and changes occurring in SeNS features with respect to those generated by these strains grown on rich media. Transmission electron microscopy (TEM) images show extra- or intra-cellular emergence of SeNS in SeITE02 or MPV1 respectively, revealing the presence of two distinct biological routes of SeNS biogenesis. Indeed, the stress exerted by upon SeITE02 cells triggered the production of membrane vesicles (MVs), which surrounded Se-nanoparticles (SeNPsSeITE02-G_e and SeNPsSeITE02-P_e with average diameter of 179 ± 56 and 208 ± 60 nm, respectively), as highlighted by TEM and scanning electron microscopy (SEM), strongly suggesting that MVs might play a crucial role in the excreting mechanism of the SeNPs in the extracellular environment. On the other hand, MPV1 strain biosynthesized intracellular inclusions likely containing hydrophobic storage compounds and SeNPs (123 ± 32 nm) under pyruvate conditioning, while the growth on glucose as the only source of carbon and energy led to the production of a mixed population of intracellular SeNPs (118 ± 36 nm) and nanorods (SeNRs; average length of 324 ± 89). SEM, fluorescence spectroscopy, and confocal laser scanning microscopy (CLSM) revealed that the biogenic SeNS were enclosed in an organic material containing proteins and amphiphilic molecules, possibly responsible for the high thermodynamic stability of these nanomaterials. Finally, the biogenic SeNS extracts were photoluminescent upon excitation ranging from 380 to 530 nm, whose degree of fluorescence emission (λem = 416–640 nm) was comparable to that from chemically synthesized SeNPs with L-cysteine (L-cys SeNPs). This study offers novel insights into the formation, localization, and release of biogenic SeNS generated by two different Gram-negative bacterial strains under aerobic and metabolically controlled growth conditions. The work strengthens the possibility of using these bacterial isolates as eco-friendly biocatalysts to produce high quality SeNS targeted to possible biomedical applications and other biotechnological purposes

Antimicrobial activity of biogenically produced spherical Se-nanomaterials embedded in organic material against Pseudomonas aeruginosa and\ua0Staphylococcus aureus strains on hydroxyapatite-coated surfaces

In an effort to prevent the formation of pathogenic biofilms on hydroxyapatite (HA)-based clinical devices and surfaces, we present a study evaluating the antimicrobial efficacy of Spherical biogenic Se-Nanostructures Embedded in Organic material (Bio Se-NEMO-S) produced by Bacillus mycoides SelTE01 in comparison with two different chemical selenium nanoparticle (SeNP) classes. These nanomaterials have been studied as potential antimicrobials for eradication of established HA-grown biofilms, for preventing biofilm formation on HA-coated surfaces and for inhibition of planktonic cell growth of Pseudomonas aeruginosa NCTC 12934 and Staphylococcus aureus ATCC 25923. Bio Se-NEMO resulted more efficacious than those chemically produced in all tested scenarios. Bio Se-NEMO produced by B.\ua0mycoides SelTE01 after 6 or 24\ua0h of Na2 SeO3 exposure show the same effective antibiofilm activity towards both P.\ua0aeruginosa and S.\ua0aureus strains at 0.078\ua0mg\ua0ml(-1) (Bio Se-NEMO6 ) and 0.3125\ua0mg\ua0ml(-1) (Bio Se-NEMO24 ). Meanwhile, chemically synthesized SeNPs at the highest tested concentration (2.5\ua0mg\ua0ml(-1) ) have moderate antimicrobial activity. The confocal laser scanning micrographs demonstrate that the majority of the P.\ua0aeruginosa and S.\ua0aureus cells exposed to biogenic SeNPs within the biofilm are killed or eradicated. Bio Se-NEMO therefore displayed good antimicrobial activity towards HA-grown biofilms and planktonic cells, becoming possible candidates as new antimicrobials