A systematic study on the characteristics and applications of laccases produced by fungi: insights on their potential for biotechnologies.

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.

More than just an insect killer: The non-insecticidal activities of Bacillus thuringiensis with biotechnological potential.

Bacillus thuringiensis (Bt) is known for the biological control of important insect pests, but scientific advances have revealed several interesting characteristics, in addition to this classical function as a bioinsecticide. To investigate the current knowledge about these non-insecticidal activities, a systematic research on primary data in the scientific literature was conducted on alternative functions of Bt with biotechnological potential. Out of a total of 140 articles selected, 15 non-insecticidal Bt activities were found. Publications related to this topic are available since 1971, and different metadata were reported, such as biomolecules and genes involved in Bt performances in non-insecticidal bioactivities. A total of 11 Bt activities with different effect measures (response variables) were identified, with an average of 48 distinct Bt strains evaluated per activity. Approximately 81.2% of all identified experiments/tests deal with the direct effects of Bt on target cells/organisms, with 36.3% of the strains within these studies tested for antibacterial action; of all microbial targets tested, 92.8% are bacteria, which led to 75.2% of the experimental conditions for all direct activities being performed in vitro. Regarding indirect Bt activities, 67.6% of these studies reported tritrophic Bt-plant-pathogen interactions. Bioremediation also appears as a relevant Bt activity being investigated in-depth. Alternative Bt activities offer innovative ways of developing biotechnology for different areas of anthropic interest; hence, we also focus on the possibility of finding multifunctional strains of Bt, as this may be advantageous from a bioeconomic point of view. Our findings are discussed in terms of research trends, aspects, details and depth of the current knowledge on alternative non-insecticidal Bt traits. We also discuss the potential application of this science for useful technological developments, aiming at solving issues related to human health, sustainable agriculture and environmental preservation/restoration.

Target and non-target site mechanisms of fungicide resistance and their implications for the management of crop pathogens.

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.

Assessing the functional diversity of rhizobacteria from cacao by partitioning root and shoot biomasses.

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.

Histidine kinase two-component response regulators Ssk1, Skn7 and Rim15 differentially control growth, developmental and volatile organic compounds emissions as stress responses in Trichoderma atroviride.

The Skn7, Ssk1 and Rim15 proteins are response regulators involved in osmotic, oxidative and nutritional stress in fungi. In order to verify the involvement of these genes in Trichoderma atroviride IMI206040's growth, conidiation, direct antagonism against plant pathogens (Rhizoctonia solani and Sclerotinia sclerotiorum), production of volatile organic compounds (VOCs) with fungistatic effect, and interaction with plants (growth promotion), single mutants were generated, and the phenotypic patterns were analysed in comparison to the wild-type (wt) strain. The mutants were submitted to osmotic, oxidative, membrane and cell wall stress conditions in vitro. The Δskn7 and Δrim15 mutants did not show either significant differences at morphological level, or marked decreases in mycelial growth and conidiation in relation to wt, whereas Δssk1 had altered phenotypes in most conditions tested. The plant-growth promotion of Arabidopsis thaliana seedlings induced by VOCs was not quantitatively modified by any of the mutants in relation to the wt strain, although possible differences in secondary root hairs was noticed for Δrim15. The fungistatic activity was significantly altered for Δssk1 and Δrim15. Overall, the Δssk1 strain showed remarkable morphological differences, with decrease in mycelial growth and conidiation, being also affected in the antagonistic capacity against plant pathogens. The impacts demonstrated by the deletion of ssk1 suggest this gene has a relevant participation in the signalling response to different stresses in T. atroviride and in the interactive metabolism with phytopathogens and plants. On the other hand, unlike other fungal models, Skn7 did not appear to have a critical participation in the above-mentioned processes; Rim15 seemed to confirm its involvement in modulating cellular responses to nutritional status, although with a possible cross-talk with other cellular processes. Our results suggest that Ssk1 likely plays a key regulatory role, not only in basic metabolisms of T. atroviride, but also in biocontrol-related characteristics.

The combination of two Bacillus strains suppresses Meloidogyne incognita and fungal pathogens, but does not enhance plant growth.

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.

First-tier detection of intragenomic 16S rRNA gene variation in culturable endophytic bacteria from cacao seeds.

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.

Characterization of genetic diversity on tropical Trichoderma germplasm by sequencing of rRNA internal transcribed spacers.

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.

Complete Genome Sequence of the Biocontrol Agent Bacillus velezensis UFLA258 and Its Comparison with Related Species: Diversity within the Commons.

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.

The NADPH Oxidases Nox1 and Nox2 Differentially Regulate Volatile Organic Compounds, Fungistatic Activity, Plant Growth Promotion and Nutrient Assimilation in Trichoderma atroviride.

In eukaryotic systems, membrane-bound NADPH oxidases (Nox) generate reactive oxygen species (ROS) as a part of normal physiological functions. In the soil-borne mycoparasitic and plant facultative symbiont Trichoderma atroviride, Nox1 and the regulator NoxR are involved in differentiation induced by mechanical damage, while the role of Nox2 has not been determined. The knock-out strains Δnox1, ΔnoxR and Δnox2 were compared to the parental strain (WT) in their ability to grow and conidiate under a series of stress conditions (osmotic, oxidative, membrane, and cell-wall stresses). All three genes were differentially involved in the stress-response phenotypes. In addition, several interactive experiments with biotic factors (plant seedlings and other fungi) were performed comparing the mutant phenotypes with the WT, which was used as the reference strain. Δnox1 and ΔnoxR significantly reduced the antagonistic activity of T. atroviride against Rhizoctonia solani and Sclerotinia sclerotiorum in direct confrontation assays, but Δnox2 showed similar activity to the WT. The Δnox1, ΔnoxR, and Δnox2 mutants showed quantitative differences in the emission of several volatile organic compounds (VOCs). The effects of a blend of these volatiles on plant-growth promotion of Arabidopsis thaliana seedlings were determined in closed-chamber experiments. The increase in root and shoot biomass induced by T. atroviride VOCs was significantly lowered by ΔnoxR and Δnox1, but not by Δnox2. In terms of fungistatic activity at a distance, Δnox2 had a significant reduction in this trait against R. solani and S. sclerotiorum, while fungistasis was highly increased by ΔnoxR and Δnox1. Identification and quantification of individual VOCs in the blends emitted by the strains was performed by GC-MS and the patterns of variation observed for individual volatiles, such as 6-Pentyl-2H-pyran-2-one (6PP-1) and (E)-6-Pent-1-enylpyran-2-one (6PP-2) were consistent with their negative effects in plant-growth promotion and positive effects in fungistasis at a distance. Nox1 and NoxR appear to have a ubiquitous regulatory role of in a variety of developmental and interactive processes in T. atroviride either as positive or negative modulators. Nox2 may also have a role in regulating production of VOCs with fungistatic activity.