Characterization of copper-resistant bacteria and bacterial communities from copper-polluted agricultural soils of central Chile.

BACKGROUND: Copper mining has led to Cu pollution in agricultural soils. In this report, the effects of Cu pollution on bacterial communities of agricultural soils from Valparaiso region, central Chile, were studied. Denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA genes was used for the characterization of bacterial communities from Cu-polluted and non-polluted soils. Cu-resistant bacterial strains were isolated from Cu-polluted soils and characterized. RESULTS: DGGE showed a similar high number of bands and banding pattern of the bacterial communities from Cu-polluted and non-polluted soils. The presence of copA genes encoding the multi-copper oxidase that confers Cu-resistance in bacteria was detected by PCR in metagenomic DNA from the three Cu-polluted soils, but not in the non-polluted soil. The number of Cu-tolerant heterotrophic cultivable bacteria was significantly higher in Cu-polluted soils than in the non-polluted soil. Ninety two Cu-resistant bacterial strains were isolated from three Cu-polluted agricultural soils. Five isolated strains showed high resistance to copper (MIC ranged from 3.1 to 4.7 mM) and also resistance to other heavy metals. 16S rRNA gene sequence analyses indicate that these isolates belong to the genera Sphingomonas, Stenotrophomonas and Arthrobacter. The Sphingomonas sp. strains O12, A32 and A55 and Stenotrophomonas sp. C21 possess plasmids containing the Cu-resistance copA genes. Arthrobacter sp. O4 possesses the copA gene, but plasmids were not detected in this strain. The amino acid sequences of CopA from Sphingomonas isolates (O12, A32 and A55), Stenotrophomonas strain (C21) and Arthrobacter strain (O4) are closely related to CopA from Sphingomonas, Stenotrophomonas and Arthrobacter strains, respectively. CONCLUSIONS: This study suggests that bacterial communities of agricultural soils from central Chile exposed to long-term Cu-pollution have been adapted by acquiring Cu genetic determinants. Five bacterial isolates showed high copper resistance and additional resistance to other heavy metals. Detection of copA gene in plasmids of four Cu-resistant isolates indicates that mobile genetic elements are involved in the spreading of Cu genetic determinants in polluted environments.

Identification of Exoenzymes Secreted by Entomopathogenic Fungus Beauveria pseudobassiana RGM 2184 and Their Effect on the Degradation of Cocoons and Pupae of Quarantine Pest Lobesia botrana.

Beauveria pseudobassiana RGM 2184 has shown 80% maximum efficacy against the pest Lobesia botrana in the autumn and winter seasons. This suggests that the strain possesses an interesting battery of enzymes that are cold-adapted to penetrate the thick and hydrophobic cocoon of L. botrana. In this study, screening of the proteolytic, lipolytic, and chitinolytic activity of enzyme extracts secreted by the RGM 2184 strain was carried out in various culture media. The enzyme extracts with the highest activity were subjected to zymography and mass spectrometry. These analyses allowed the identification of two proteases, two lipases, and three chitinases. Comparative analysis indicated that the degree of similarity between these enzymes was substantially reduced when the highest degree of taxonomic relatedness between RGM 2184 and the entomopathogenic fungus strain was at the family level. These results suggest that there is a wide variety of exoenzymes in entomopathogenic fungi species belonging to the order Hypocreales. On the other hand, exoenzyme extract exposure of cocoons and pupae of L. botrana provoked damage at 10 °C. Additionally, an analysis of the amino acid composition of the RGM 2184 exoenzyme grouped them close to the cold-adapted protein cluster. These results support the use of this strain to control pests in autumn and winter. Additionally, these antecedents can form a scaffold for the future characterization of these exoenzymes along with the optimization of the strain's biocontrol ability by overexpressing them.

Genomic and Experimental Analysis of the Biostimulant and Antagonistic Properties of Phytopathogens of Bacillus safensis and Bacillus siamensis.

The B. safensis RGM 2450 and B. siamensis RGM 2529 strains were isolated from the rhizosphere of plants presenting resilience to abiotic and biotic stress conditions. To understand the implications of bacteria in resilience, a genomic and experimental analysis was carried out on their biostimulant and phytopathogenic antagonist properties. Genome analyses of both strains indicated that they have the potential to synthesize bioactive compounds such as the battery of non-ribosomal peptides, polyketides, extracellular enzymes and phytohormones. These results were consistent with the antagonistic activities of both strains against the phytopathogens Botrytis cinerea, Colletotrichum acutatum, Fusarium oxysporum and Phytophtora cinnamomi. They also showed the capacity to solubilize phosphorus, fix nitrogen and produce indole acetic acid. This was observed in tomato seedlings grown from seeds inoculated with the mixture of strains which presented significantly greater length as well as wet and dry weight in comparison with the treatments individually inoculated with each strain and the control. Accordingly, the combination of B. safensis RGM 2450 and B. siamensis RGM 2529 showed synergistic biostimulant activity. These findings contribute new knowledge of the genomic and metabolomic properties taking part in the symbiotic interactions between these strains and the plants and uphold the combined use of both strains as a biostimulant.

Genomic and Experimental Analysis of the Insecticidal Factors Secreted by the Entomopathogenic Fungus Beauveria pseudobassiana RGM 2184.

The entomopathogenic fungus Beauveria pseudobassiana strain RGM 2184 can reach a maximum efficacy of 80% against the quarantine pest Lobesia botrana in field assays. In this study, the RGM 2184 genome was sequenced, and genome mining analyses were performed to predict the factors involved in its insecticidal activity. Additionally, the metabolic profiling of the RMG 2184 culture's supernatants was analyzed by mass spectrometry, and the insecticidal activity from one of these extracts was evaluated in Galleria mellonella larvae. The genome analysis resulted in 114 genes encoding for extracellular enzymes, four biosynthetic gene clusters reported as producers of insecticidal and bactericidal factors (oosporein, beauvericin, desmethylbassianin, and beauveriolide), 20 toxins, and at least 40 undescribed potential biocontrol factors (polyketides and nonribosomal peptides). Comparative genomic analysis revealed that 65-95% of these genes are Beauveria genus-specific. Metabolic profiling of supernatant extracts from RGM 2184 cultures exhibited secondary metabolites such as beauveriolide, oosporein, inflatin C, and bassiatin. However, a number of detected metabolites still remain undescribed. The metabolite extract caused 79% mortality of Galleria mellonella larvae at 28 days. The results of this research lay the groundwork for the study of new insecticidal molecules.

Lobesia botrana: A Biological Control Approach with a Biopesticide Based on Entomopathogenic Fungi in the Winter Season in Chile.

Lobesia botrana (Denis and Shiffermüller) (Lepidoptera: Tortricidae) is one of the main pests that affect the production and export of table grapes in Chile. Because this pest has quarantine status, the fruit must be fumigated with methyl bromide, which reduces the fruit's export competitiveness in the destination market. In the present study, to help resolve this issue, six native entomopathogenic fungi were identified through multilocus analysis, including three Beauveria pseudobassiana and three Metarhizium robertsii. These fungi were evaluated in the laboratory to control L. botrana in its pupal stage in a silk cocoon and compared against a biological control product. Formulations with additional carbon sources improved the performance of the fungi. The treatments with outstanding performance contained the fungal strains B. pseudobassiana RGM 2184 and M. robertsii RGM 678. These strains were evaluated in the field during the winter season in two different regions of the country; the strains reached maximum efficacies of 80% and 88%, respectively, at 21 days post first application. Therefore, entomopathogenic fungi can contribute to reducing pupal populations in winter, thereby decreasing the moth population in spring-summer.

Potential biological control of the pupal stage of the European grapevine moth Lobesia botrana by the entomopathogenic fungus Beauveria pseudobassiana in the winter season in Chile.

OBJECTIVE: Lobesia botrana, the European grapevine moth, affects Vitis vinifera L. and other species of economic importance in a number of countries through damage caused by its larvae in berries and associated secondary diseases such as Botrytis cinerea. Control of the moth in urban areas is difficult due to poor chemical management of infested plants in houses. Additionally, in winter, L. botrana is in its pupal stage covered with a cocoon that prevents the penetration of chemical pesticides. For this reason, the objective of this work was to control the pupal stage with a formulation based on the entomopathogenic fungus Beauveria pseudobassiana in urban areas. RESULTS: The strain RGM 1747 was identified as B. pseudobassiana by multilocus sequence analysis. The biocontrol activity of this formulated fungus against the infestation of vines with breeding pupae without cocoons showed 100% infection 21 days after inoculation under winter conditions. Finally, the biocontrol activity of the formulated fungus against natural infestations of L. botrana in winter in urban areas reached an efficacy of 51%. This result suggests that the B. pseudobassiana formulation is able to penetrate the cocoon and contributes to the integrated pest management of L. botrana.

Candidate nematicidal proteins in a new Pseudomonas veronii isolate identified by its antagonistic properties against Xiphinema index.

The nematode Xiphinema index affects grape vines and transmits important viruses associated with fanleaf degeneration. Pseudomonas spp. are an extensive bacterial group in which important biodegradation and/or biocontrol properties can occur for several strains in the group. The aim of this study was to identify new Pseudomonas isolates with antagonist activity against X. index. Forty bacterial isolates were obtained from soil and root samples from Chilean vineyards. Thirteen new fluorescent pseudomonads were found and assessed for their antagonistic capability. The nematicide Pseudomonas protegens CHA0 was used as a control. Challenges of nematode individuals in King's B semi-solid agar Petri dishes facilitated the identification of the Pseudomonas veronii isolate R4, as determined by a 16S rRNA sequence comparison. This isolate was as effective as CHA0 as an antagonist of X. index, although it had a different lethality kinetic. Milk-induced R4 cultures exhibited protease and lipase activities in cell supernatants using both gelatin/tributyrin Petri dish assays and zymograms. Three proteins with these activities were isolated and subjected to mass spectrometry. Amino acid partial sequences enabled the identification of a 49-kDa protease similar to metalloprotease AprA and two lipases of 50 kDa and 69 kDa similar to LipA and ExoU, respectively. Electron microscopy analyses of challenged nematodes revealed degraded cuticle after R4 supernatant treatment. These results represent a new and unexplored property in this species associated with the presence of secretable lipases and protease, similar to characterized enzymes present in biocontrol pseudomonads.

A draft genome sequence of Pseudomonas veronii R4: a grapevine (Vitis vinifera L.) root-associated strain with high biocontrol potential.

A new plant commensal Pseudomonas veronii isolate (strain R4) was identified from a Xiphinema index biocontrol screen. Isolated from grapevine roots from vineyards in central Chile, the strain R4 exhibited a slower yet equivalently effective nematicide activity as the well-characterized P. protegens CHA0. Whole genome sequencing of strain R4 and comparative analysis among the available Pseudomonas spp. genomes allowed for the identification of gene clusters that encode putative extracellular proteases and lipase synthesis and secretion systems, which are proposed to mediate-at least in part-the observed nematicidal activity. In addition, R4 strain presented relevant gene clusters related to metal tolerance, which is typical in P. veronii. Bioinformatics analyses also showed gene clusters associated with plant growth promoting activity, such as indole-3-acetic acid synthesis. In addition, the strain R4 genome presented a metabolic gene clusters associated with phosphate and ammonia biotransformation from soil, which could improve their availability for plants.