RESUMEN
BACKGROUND: The diamondback moth (DBM) (Plutella xylostella) causes large losses to global crop production. Conventional insecticides are losing effectiveness due to resistance. Consequently, there is a growing interest in sustainable control methods like entomopathogenic fungi (EPF) in Integrated Pest Management. However, the field efficacy of fungi varies due to environmental influences. In this study, a group of 50 Beauveria strains sourced from different locations were characterized by genotype and phenotype with respect to their conidial production, temperature and UV-B radiation tolerance, and virulence against DBM. RESULTS: Phylogenetic analysis revealed two distinct species: Beauveria bassiana (84%) and B. pseudobassiana (16%). Most strains showed optimal growth between 25 °C and 28 °C, with germination severely affected at 10 °C and 33 °C. Notably, 44% displayed high resistance to UV-B radiation (5.94 kJ m-2), with germination rates between 60.9% and 88.1%. Geographical origin showed no correlation with temperature or UV radiation tolerance. In virulence experiments, 52% of strains caused mortality rates exceeding 80% in DBM second instars at 7 days after exposure to a 4 mL conidial suspension (107 conidia/mL). CONCLUSION: Survival under environmental conditions is crucial for EPF-based commercial products against DBM. Results suggest strain tolerance to environmental stressors is more tied to specific micro-climatic factors than geographical origin. Each strain exhibited unique characteristics; for example, the most virulent strain (#29) was highly UV-sensitive. Therefore, characterizing diverse strains provides essential genotypic and phenotypic insights, which are fundamental for understanding their role as biocontrol agents while facilitating efficient biopesticide product development and uptake. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
RESUMEN
Although several bacterial lignin-oxidising enzymes have been discovered in recent years, it is not yet clear whether different lignin-degrading bacteria use similar mechanisms for lignin oxidation and degradation of lignin fragments. Genome sequences of 13 bacterial lignin-oxidising bacteria, including new genome sequences for Microbacterium phyllosphaerae and Agrobacterium sp., were analysed for the presence of lignin-oxidising enzymes and aromatic degradation gene clusters that could be used to metabolise the products of lignin degradation. Ten bacterial genomes contain DyP-type peroxidases, and ten bacterial strains contain putative multi-copper oxidases (MCOs), both known to have activity for lignin oxidation. Only one strain lacks both MCOs and DyP-type peroxidase genes. Eleven bacterial genomes contain aromatic degradation gene clusters, of which ten contain the central ß-ketoadipate pathway, with variable numbers and types of degradation clusters for other aromatic substrates. Hence, there appear to be diverse metabolic strategies used for lignin oxidation in bacteria, while the ß-ketoadipate pathway appears to be the most common route for aromatic metabolism in lignin-degrading bacteria.
Asunto(s)
Bacterias/enzimología , Bacterias/genética , Genoma Bacteriano , Lignina/metabolismo , Agrobacterium/enzimología , Agrobacterium/genética , Proteínas Bacterianas/metabolismo , Fenómenos Bioquímicos , Genómica , Ingeniería Metabólica , Microbacterium/enzimología , Microbacterium/genética , Oxidación-Reducción , Oxidorreductasas/metabolismo , Peroxidasas/metabolismoRESUMEN
CopA is a protein formed as part of a copper resistance operon in Pseudomonas syringae pv tomato, but CopA has also been identified from gene library screening as a potential lignin-oxidising enzyme. Few bacterial homologues for bacterial multi-copper laccases have been identified that can assist in lignin degradation. Bioinformatic analysis revealed that copA and copC genes were found in the genomes of bacterial strains capable of lignin oxidation. In this study, CopA enzymes from bacterial strains with lignin oxidation activity, Pseudomonas putida and P. fluorescens, were heterologously expressed and characterised kinetically, and expression of bacterial CopC proteins was also investigated. Purified CopA enzymes were dependent upon exogenous copper (II) ions for activity when expressed under fully aerated conditions, however after expression under microaerobic conditions with copper reconstitution, the activity was independent of copper addition. The CopA enzymes showed activity towards the laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS); syringaldazine (SGZ); guaiacol; 2,6-dimethoxyphenol (DMP) and 2,4-dichlorophenol (DCP). Moreover, CopA proteins were able to oxidise the lignin model compounds guaiacylglycerol-beta-guaiacyl (GGE) and 2,2'-dihydroxy-3,3'-dimethoxy-5,5'-dicarboxybiphenyl (DDVA), giving oxidised dimerised products; and they were active towards Ca-lignosulfonate, giving vanillic acid as product. A double gene deletion of copA-I and copA-II genes in Pseudomonas putida KT2440 was constructed, and this mutant showed diminished growth capability on different small aromatic compounds related with lignin degradation, when copper salts were present in the media.
Asunto(s)
Proteínas Bacterianas/metabolismo , Lignina/metabolismo , Pseudomonas fluorescens/enzimología , Pseudomonas putida/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Eliminación de Gen , Cinética , Oxidación-ReducciónRESUMEN
The identification of enzymes responsible for oxidation of lignin in lignin-degrading bacteria is of interest for biotechnological valorization of lignin to renewable chemical products. The genome sequences of two lignin-degrading bacteria, Ochrobactrum sp., and Paenibacillus sp., contain no B-type DyP peroxidases implicated in lignin degradation in other bacteria, but contain putative multicopper oxidase genes. Multi-copper oxidase CueO from Ochrobactrum sp. was expressed and reconstituted as a recombinant laccase-like enzyme, and kinetically characterized. Ochrobactrum CueO shows activity for oxidation of ß-aryl ether and biphenyl lignin dimer model compounds, generating oxidized dimeric products, and shows activity for oxidation of Ca-lignosulfonate, generating vanillic acid as a low molecular weight product. The crystal structure of Ochrobactrum CueO (OcCueO) has been determined at 1.1 Å resolution (PDB: 6EVG), showing a four-coordinate mononuclear type I copper center with ligands His495, His434 and Cys490 with Met500 as an axial ligand, similar to that of Escherichia coli CueO and bacterial azurin proteins, whereas fungal laccase enzymes contain a three-coordinate type I copper metal center. A trinuclear type 2/3 copper cluster was modeled into the active site, showing similar structure to E. coli CueO and fungal laccases, and three solvent channels leading to the active site. Site-directed mutagenesis was carried out on amino acid residues found in the solvent channels, indicating the importance for residues Asp102, Gly103, Arg221, Arg223, and Asp462 for catalytic activity. The work identifies a new bacterial multicopper enzyme with activity for lignin oxidation, and implicates a role for bacterial laccase-like multicopper oxidases in some lignin-degrading bacteria. DATABASE: Structural data are available in the PDB under the accession number 6EVG.