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Laccases are polyphenol oxidase enzymes and form the enzyme complex known for their role in wood decomposition and lignin degradation. The present study aimed to systematically review the state-of-the-art trends in scientific publications on laccase enzymes of the last 10 years. The main aspects checked included the laccase-producing fungal genera, the conditions of fungal growth and laccase production, the methods of immobilization, and potential applications of laccase. After applying the systematic search method 177 articles were selected to compound the final database. Although various fungi produce laccase, most studies were Trametes and Pleurotus genera. The submerged fermentation (SmF) has been the most used, however, the use of solid-state fermentation (SSF) appeared as a promising technique to produce laccase when using agro-industrial residues as substrates. Studies on laccase immobilization showed the covalent bonding and entrapment methods were the most used, showing greater efficiency of immobilization and a high number of enzyme reuses. The main use of the laccase was in bioremediation, especially in the discoloration of dyes from the textile industry and the degradation of pharmaceutical waste. Implications and consequences of all these findings in biotechnology and environment, as well as the trends and gaps of laccase research were discussed.

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Fungicides are indispensable for high-quality crops, but the rapid emergence and evolution of fungicide resistance have become the most important issues in modern agriculture. Hence, the sustainability and profitability of agricultural production have been challenged due to the limited number of fungicide chemical classes. Resistance to site-specific fungicides has principally been linked to target and non-target site mechanisms. These mechanisms change the structure or expression level, affecting fungicide efficacy and resulting in different and varying resistance levels. This review provides background information about fungicide resistance mechanisms and their implications for developing anti-resistance strategies in plant pathogens. Here, our purpose was to review changes at the target and non-target sites of quinone outside inhibitor (QoI) fungicides, methyl-benzimidazole carbamate (MBC) fungicides, demethylation inhibitor (DMI) fungicides, and succinate dehydrogenase inhibitor (SDHI) fungicides and to evaluate if they may also be associated with a fitness cost on crop pathogen populations. The current knowledge suggests that understanding fungicide resistance mechanisms can facilitate resistance monitoring and assist in developing anti-resistance strategies and new fungicide molecules to help solve this issue. © 2023 Society of Chemical Industry.

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Plant-microbe interactions are critical for the sustainability of agricultural production. In this study, our aims were to characterize the genetic and functional diversity of the culturable bacterial community associated with the cacao rhizosphere and access their potential for growth promotion of cacao seedling. Culture-dependent and molecular methods were used to characterize the population densities and diversity of bacterial communities from soil and cacao plants at two locations and two plant ages. A total of 63 strains were identified through hsp60 sequencing. Pseudomonas and Enterobacter were the most abundant genera in association with the cacao rhizosphere, whereas Bacillus was more numerous in soil. Parameters of seedling growth promotion were evaluated 60 days after inoculation of seeds, with partition of the assessments into root and shoot weight. Each isolate showed beneficial, neutral or deleterious effects on plant growth, depending on the isolate and on the parts of plant assessed. Interestingly, although an apparent overall decrease in total biomass of seedlings (roots + shoots dry matters) was observed for the majority of isolates (89%), 94% of all isolates, in fact, revealed an increase in plant roots/shoots dry biomass ratio. Despite that part of the isolates (35%) appeared to significantly decrease plant height, and that 65% did not influence plant height (neutral effect), 18 had significantly increased root dry biomass; nevertheless, seven of these root growth-increasing isolates simultaneously decreased shoots-related growth parameters. The results of this study evidentiated the functional diversity of culturable cacao rhizobacteria and how the partitioning of roots and shoots in the assessment of plant growth parameters could reveal the biotechnological potential of these isolates for promoting growth of clones for rehabilitation of commercial cacao plantations. KEY POINTS: • The most common culturable bacteria in cacao roots were Pseudomonas and Enterobacter • Most culturable bacteria from cacao roots increased the root/shoot ratio • Roots and shoots should be examined separately to detect cacao beneficial bacteria.

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BACKGROUND: The combination of biocontrol agents is a desirable strategy to improve control efficacy against the root-knot nematode (RKN) Meloidogyne incognita under field conditions. However, strains compatibility is generally tested in vitro and incompatible combinations are normally not further examined in experiments in planta. Therefore, there is virtually no information on the performance of incompatible strains. In this study, we evaluated two Bacillus strains previously described as incompatible in vitro for effects on plant growth and suppression of M. incognita, pathogenic fungi and nematophagous fungi. RESULTS: Strains BMH and INV were shown to be closely related to Bacillus velezensis. These strains, when applied individually, reduced the number of galls and eggs of M. incognita by more than 90% in tomato roots. When BMH and INV were combined (BMH + INV), RKN suppression and tomato shoot weight were lower compared to single-strain applications. Additionally, metabolites in cell-free supernatants and volatile organic compounds (VOCs) from strains BMH and INV had strong effects against the plant pathogens M. incognita, Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsiii, but not against three species of nematophagous fungi. Although strain INV and the combination BMH + INV emitted fewer VOCs than strain BMH, they were still capable of killing second-stage juveniles of M. incognita. CONCLUSIONS: Bacillus strains BMH and INV inhibited M. incognita and fungal pathogens, and promoted tomato growth. However, strain INV emitted fewer VOCs and the combination BMH + INV did not enhance the activity of the biocontrol strains against the RKN or their capacity to promote plant growth. © 2021 Society of Chemical Industry.

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BACKGROUND: Intragenomic variability in 16S rDNA is a limiting factor for taxonomic and diversity characterization of Bacteria, and studies on its occurrence in natural/environmental populations are scarce. In this work, direct DNA amplicon sequencing coupled with frequent-cutter restriction analysis allowed detection of intragenomic 16S rDNA variation in culturable endophytic bacteria from cacao seeds in a fast and attractive manner. METHODS: Total genomic DNA from 65 bacterial strains was extracted and the 16S rDNA hyper variable V5-V9 regions were amplified for enzyme digestion and direct Sanger-type sequencing. The resulting electropherograms were visually inspected and compared to the corresponding AluI-restriction profiles, as well as to complete genome sequences in databases. Restriction analysis were employed to substitute the need of amplicon cloning and re-sequencing. A specifically improved polyacrylamide-gradient electrophoresis allowed to resolve 5-bp differences in restriction fragment sizes. Chi-square analysis on 2 × 2 contingency table tested for the independence between the 'number of AluI bands' and 'type of eletropherogram'. RESULTS: Two types of electropherograms were obtained: unique template, with single peaks per base (clean chromatograms), and heterogeneous template, with various levels of multiple peaks per base (mixed chromatograms). Statistics revealed significant interaction between number of restriction fragments and type of electropherogram for the same amplicons: clean or mixed ones associated to ≤5 or ≥6 bands, respectively. The mixed-template pattern combined with the AluI-restriction profiles indicated a high proportion of 49% of the culturable endophytes from a tropical environment showing evidence of intragenomic 16S rDNA heterogeneity. CONCLUSION: The approach presented here was useful for a rapid, first-tier detection of intragenomic variation in culturable isolates, which can be applied in studies of other natural populations; a preliminary view of intragenomic heterogeneity levels can complement culture-dependent and -independent methods. Consequences of these findings in taxonomic and diversity studies in complex bacterial communities are discussed.

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In this study, the full genome sequence of Bacillus velezensis strain UFLA258, a biological control agent of plant pathogens was obtained, assembled, and annotated. With a comparative genomics approach, in silico analyses of all complete genomes of B. velezensis and closely related species available in the database were performed. The genome of B. velezensis UFLA258 consisted of a single circular chromosome of 3.95 Mb in length, with a mean GC content of 46.69%. It contained 3,949 genes encoding proteins and 27 RNA genes. Analyses based on Average Nucleotide Identity and Digital DNA-DNA Hybridization and a phylogeny with complete sequences of the rpoB gene confirmed that 19 strains deposited in the database as Bacillus amyloliquefaciens were in fact B. velezensis. In total, 115 genomes were analyzed and taxonomically classified as follows: 105 were B. velezensis, 9 were B. amyloliquefaciens, and 1 was Bacillus siamensis. Although these species are phylogenetically close, the combined analyses of several genomic characteristics, such as the presence of biosynthetic genes encoding secondary metabolites, CRISPr/Cas arrays, Average Nucleotide Identity and Digital DNA-DNA Hybridization, and other information on the strains, including isolation source, allowed their unequivocal classification. This genomic analysis expands our knowledge about the closely related species, B. velezensis, B. amyloliquefaciens, and B. siamensis, with emphasis on their taxonomical status.

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OBJECTIVE: Trichoderma species are found in soil and in association with plants. They can act directly or indirectly in the biological control of plant diseases and in the promotion of plant growth, being among the most used fungi in the formulation of bioproducts applied to agricultural systems. The main objective of this study was to characterize at a first-tier level a collection of 67 Trichoderma isolates from various tropical sources, based solely on sequencing of the internal transcribed spacer (ITS) region of the rRNA genes. Our goal was to provide a preliminary idea of the baseline diversity in this collection, to combine this information later with an array of other isolate-specific physiological data. This study provides a required knowledge at molecular level for assessment of this germplasm potential as a source of biotechnological products for beneficial effects in plants. RESULTS: Sequencing of the ITS region showed that the 67 Trichoderma isolates belonged in 11 species: T. asperellum, T. atroviride, T. brevicompactum, T. harzianum, T. koningiopsis, T. longibrachiatum, T. pleuroticola, T. reesei, T. spirale, T. stromaticum and T. virens. A total of 40.3% of the isolates were very closely related to each other and similar to T. harzianum. The baseline genetic diversity found indicates that the collection has different genotypes, which can be exploited further as a source of bioproducts, aiming at providing beneficial effects to plants of interest to cope with biotic and abiotic stresses.

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