Cold-adapted strains as plant growth-promoting bacteria on soybean seeds and biocontrol agents against Macrophomina phaseolina.

AIM: The aim was to characterize cold-adapted bacteria by testing their PGP features and antagonistic activity against Macrophomina phaseolina, both in vitro and coating soybean seeds (Glycine max [L.] Merr.). METHODS AND RESULTS: Burkholderia gladioli MB39, Serratia proteamaculans 136 and Serratia proteamaculans 137 were evaluated. In vitro tests showed that S. proteamaculans 136 and 137 produce siderophore and indole-acetic acid (IAA), solubilize phosphate and fix nitrogen. Additionally, B. gladioli MB39 and S. proteamaculans 137 showed hydrolase activity and potent antifungal effects. The biocontrol efficacy over soybean seeds was evaluated using in vitro and greenhouse methods by immersing seeds into each bacterial suspension. As a result, S. proteamaculans 136 has improved the performance in all the seed germination evaluated parameters. In addition, S. proteamaculans 137 and B. gladioli MB39 strongly inhibited M. phaseolina, reducing the infection index values to 10% and 0%, respectively. CONCLUSION: Serratia proteamaculans 136, 137 and Burkholderia gladioli MB39 showed plant growth promotion features and inhibition of Macrophomina phaseolina infection by producing different antifungal compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results reinforce the application of cold-adapted Serratia proteamaculans and Burkholderia gladioli bacterial strains as candidates for developing microbial formulation to promote plant growth and guarantee antifungal protection in soybean crops.

Burkholderia gladioli MB39 an Antarctic Strain as a Biocontrol Agent.

Bioprospecting sub-explored environments such as Antarctic locations leads to finding out diverse activities, reducing harmful chemical usage that affects both human health and the environment. In this study, ~ 7000 cold-adapted bacterial strains were isolated from samples around Melchior Antarctic Base at 5 °C and more than 13,000 at 15 °C. Out of them, 900 different colony morphotypes were evaluated for antimicrobial production, and 13 isolates demonstrated antibacterial and antifungal activities. One isolate, closely related to Burkholderia gladioli according to 16S rDNA (99.8%), gyrB (99.6%) and Cpn60 (99.4%) gene sequence analysis, showed a consistent, broad antimicrobial spectrum against both pathogenic and phytopathogenic bacteria. Its potent antifungal activity inhibits the growth of various plant pathogenic fungi, whereas it was mainly studied against Penicillium digitatum and Macrophomina phaseolina, the causal agents of blue mould in postharvest fruits and charcoal rot in soybean crops, respectively. The antibacterial compound exhibited low molecular weight (< 6000 Da), resistance to lytic enzymes and stability in a broad range of temperature and pHs. Observations of the B. gladioli MB39 antifungal effects over M. phaseolina mycelia by scanning electron microscopy showed alterations in hyphal structures, reduced hyphal extension, and severe cell morphology changes such as cytoplasmic leakage, flattened and empty mycelia. Here we report the isolation and identification of a cold-adapted B. gladioli strain. The results describe the effectiveness of the antarctic strain for bacterial and fungal phytopathogens biocontrol and its potential for crop protection plans.

Isolation of Sphaerotilus-Leptothrix strains from iron bacteria communities in Tierra del Fuego wetlands.

Sheath-forming iron- and manganese-depositing bacteria belonging to the Sphaerotilus-Leptothrix group (SLG) are widespread in natural and artificial water systems. Known requirements for their growth include the presence of organic substrates and molecular oxygen. High concentrations of reduced iron or manganese, although not necessary for most species, make their growth a noticeable phenomenon. Such microbial communities have been studied mostly in the Northern Hemisphere. Here, we present descriptions of diverse ochre-depositing microbial communities in Tierra del Fuego, Argentina, using a combined approach of microscopical examination, clone library construction and cultivation focused on SLG bacteria. To date, only few SLG type strains are available. The present work increases the number and diversity of cultivated SLG bacteria by obtaining isolates from biofilms and sediment samples of wetlands in Tierra del Fuego. Thirty isolates were selected based on morphological features such as sheath formation and iron/manganese deposition. Five operational taxonomic units (OTUs) were deduced. Sequencing of 16S rRNA genes showed that one OTU is identical to the Leptothrix mobilis Feox-1(T) -sequence while the four remaining OTUs show similarity values related to previously described type strains. Similarity values ranged from 96.5% to 98.8%, indicating possible new species and subspecies.

Gluconic acid produced by Gluconacetobacter diazotrophicus Pal5 possesses antimicrobial properties.

Gluconic acid is produced in large quantities by the endophytic and diazotrophic bacterium Gluconacetobacter diazotrophicus Pal5. This organic acid derives from direct oxidation of glucose by a pyrroloquinoline-quinone-linked glucose dehydrogenase in this plant growth-promoting bacterium. In the present article, evidence is presented showing that gluconic acid is also responsible for the antimicrobial activity of G. diazotrophicus Pal5. The broad antagonistic spectrum includes Gram-positive and -negative bacteria. Eukaryotic microorganisms are more resistant to growth inhibition by this acid. Inhibition by gluconic acid can be modified through the presence of other organic acids. In contrast to other microorganisms, the Quorum Sensing system of G. diazotrophicus Pal5, a regulatory mechanism that plays a key role in several microbe-microbe interactions, is not related to gluconic acid production and the concomitant antagonistic activity.

Andrimid production at low temperature by a psychrotolerant Serratia proteamaculans strain.

Andrimid, a known non-ribosomal pseudo-peptide antibiotic, was isolated from a psychrotolerant Serratia proteamaculans strain. The antibiotic peptide was produced at low temperature (8 °C) in a 7.5 l BIOFLO 101 bioreactor under batch culture mode. Andrimid activity from S. proteamaculans culture was only detected at 25 °C and below and potent antibacterial activity was revealed against both, pathogenic and non-pathogenic bacteria. Minimal inhibitory concentration values determined by microdilution experiments varied in the range between 0.01 and 0.78 µg/ml. Antimicrobial purification and structure elucidation were carried out by LC-MS/MS and ¹H/¹³C NMR approaches. The effects on the ultrastructure of sensitive Escherichia coli 35,218 cells were observed by transmission electron microscopy at different inhibition stages. This work demonstrated the significance of bioprospection from cold environments through the screening of microorganisms with ability to produce cold-active biomolecules of biotechnological interest. S. proteamaculans 136 was revealed as a novel microbial source for andrimid production at low temperatures, showing biotechnological potential to be applied in cryopreservation, food or cosmetic industries against pathogenic bacteria.

Lactic acid bacteria isolated from apples are able to catabolise arginine.

We investigated the potentiality of lactic acid bacteria (LAB) isolated from two apples variety to utilize arginine at different initial pH values. Apples surface contained average levels of bacteria ranging from log 2.49 ± 0.53 to log 3.73 ± 0.48 cfu/ml for Red Delicious and Golden Delicious varieties, respectively. Thirty-one strains able to develop in presence of arginine at low pH were phenotypically and genotipically identified as belonging to Lactobacillus, Pediococcus and Leuconostoc genera. In general, they did not produce ammonia from arginine when cultivated in basal medium with arginine (BMA) at pH 4.5 or 5.2. When this metabolite was quantified only six strains belonging to Leuconostoc dextranicum, Lactobacillus brevis and Lactobacillus plantarum species formed higher ammonia amounts in BMA as compared to control. This was correlated with arginine utilization and it was more pronounced at pH 4.5 than 5.2. Analysis of citrulline production confirmed the arginine utilization in these bacteria by the arginine deiminase (ADI) pathway. Maxima citrulline production was observed for Lactobacillus brevis M15 at the two pH values. In this strain ammonia was formed at higher rate than citrulline, which was detected in concentration lower than 1 mM. Thus, main LAB species found on apple surfaces with abilities to degrade arginine by the ADI pathway under different conditions were reported here at the first time. The results suggested that the ADI pathway in apples LAB might not be mainly relevant for their survival in the acid natural environmental, despite leading to the ammonia formation, which may contribute to the increase in pH, coping the acid stress.

Cold-adapted microorganisms as a source of new antimicrobials.

Thirty out of 8,000 different colony morphotypes isolated from soil samples of Isla de los Estados were selected based on their ability to produce antimicrobials. The significant influence of culture media and incubation temperature on antimicrobial production was proved, being LB medium and 8 degrees C the conditions of choice. Most of the psychrotolerant isolates were phylogenetically related to Serratia proteamaculans (96.4-97.9%) while the psychrophilic isolated 8H1 was closely related to Pseudomonas sp. (90-94% similarity). Produced antimicrobials showed a promising wide spectrum of activity both against gram-positive and gram-negative pathogenic bacteria. They were suspected to be microcin-like compounds (Mw <2,000 Da) and showed a marked tolerance to heat (1 h in boiling water bath) and pH-treatments (1-12). Antimicrobial compounds also showed to partially keep their activity even after overnight freezing at -20 and -80 degrees C and displayed a negative net charge at pH 8.0, a common feature of class II microcins.