Plant Tissue Culture: Industrial Relevance and Future Directions.

Plant tissue culture has evolved in the last decades with several types of cultures being developed to promote a more sustainable food production system. Moreover, these cultures can be applied for the production of relevant metabolites with medicinal potential, thus contributing to nutrition and healthcare. Importantly, plant micropropagation has enabled agricultural expansion and tissue culture has emerged as a promising production alternative for several plants and their metabolites in the food, cosmetic, and pharmaceutical industries. Plant tissue cultures present several advantages over conventional propagation techniques as they are season independent, enabling a continuous supply of the plants/compounds of interest, with the guarantee of high phytosanitary quality. In addition, genetic uniformity is generally maintained, thus reducing chemical variability that can compromise safety and efficacy. Nevertheless, despite their undeniable potential, with many researchers focusing on new strategies to improve production yield in cell cultures, such as with the use of elicitors or resorting to metabolomics engineering, an effective and lucrative large-scale production has yet to be obtained. Indeed, only a few compounds with market value are produced in this regard and several limitations such as contaminations, low culture yield and production costs still need to be overcome in order to take advantage of the full potential of these techniques.

On Interactions of Sulfamerazine with Cyclodextrins from Coupled Diffusometry and Toxicity Tests.

This scientific study employs the Taylor dispersion technique for diffusion measurements to investigate the interaction between sulfamerazine (NaSMR) and macromolecular cyclodextrins (ß-CD and HP-ß-CD). The results reveal that the presence of ß-CD influences the diffusion of the solution component, NaSMR, indicating a counterflow of this drug due to solute interaction. However, diffusion data indicate no inclusion of NaSMR within the sterically hindered HP-ß-CD cavity. Additionally, toxicity tests were conducted, including pollen germination (Actinidia deliciosa) and growth curve assays in BY-2 cells. The pollen germination tests demonstrate a reduction in sulfamerazine toxicity, suggesting potential applications for this antimicrobial agent with diminished adverse effects. This comprehensive investigation contributes to a deeper understanding of sulfamerazine-cyclodextrin interactions and their implications for pharmaceutical and biological systems.

Ethylene Action Inhibition Improves Adventitious Root Induction in Adult Chestnut Tissues.

Phase change refers to the process of maturation and transition from the juvenile to the adult stage. In response to this shift, certain species like chestnut lose the ability to form adventitious roots, thereby hindering the successful micropropagation of adult plants. While auxin is the main hormone involved in adventitious root formation, other hormones, such as ethylene, are also thought to play a role in its induction and development. In this study, experiments were carried out to determine the effects of ethylene on the induction and growth of adventitious roots. The analysis was performed in two types of chestnut microshoots derived from the same tree, a juvenile-like line with a high rooting ability derived from basal shoots (P2BS) and a line derived from crown branches (P2CR) with low rooting responses. By means of the application of compounds to modify ethylene content or inhibit its signalling, the potential involvement of this hormone in the induction of adventitious roots was analysed. Our results show that ethylene can modify the rooting competence of mature shoots, while the response in juvenile material was barely affected. To further characterise the molecular reasons underlying this maturation-derived shift in behaviour, specific gene expression analyses were developed. The findings suggest that several mechanisms, including ethylene signalling, auxin transport and epigenetic modifications, relate to the modulation of the rooting ability of mature chestnut microshoots and their recalcitrant behaviour.

Drought-Stress-Induced Changes in Chloroplast Gene Expression in Two Contrasting Strawberry Tree (Arbutus unedo L.) Genotypes.

This study investigated the effect of drought stress on the expression of chloroplast genes in two different genotypes (A1 and A4) of strawberry tree plants with contrasting performances. Two-year-old plants were subjected to drought (20 days at 18% field capacity), and the photosynthetic activity, chlorophyll content, and expression levels of 16 chloroplast genes involved in photosynthesis and metabolism-related enzymes were analyzed. Genotype-specific responses were prominent, with A1 displaying wilting and leaf curling, contrasting with the mild symptoms observed in A4. Quantification of damage using the net CO2 assimilation rates and chlorophyll content unveiled a significant reduction in A1, while A4 maintained stability. Gene expression analysis revealed substantial downregulation of A1 (15 out of 16 genes) and upregulation of A4 (14 out of 16 genes). Notably, psbC was downregulated in A1, while it was prominently upregulated in A4. Principal Component Analysis (PCA) highlighted genotype-specific clusters, emphasizing distinct responses under stress, whereas a correlation analysis elucidated intricate relationships between gene expression, net CO2 assimilation, and chlorophyll content. Particularly, a positive correlation with psaB, whereas a negative correlation with psbC was found in genotype A1. Regression analysis identified potential predictors for net CO2 assimilation, in particular psaB. These findings contribute valuable insights for future strategies targeting crop enhancement and stress resilience, highlighting the central role of chloroplasts in orchestrating plant responses to environmental stressors, and may contribute to the development of drought-tolerant plant varieties, which are essential for sustaining agriculture in regions affected by water scarcity.

Primary Metabolite Screening Shows Significant Differences between Embryogenic and Non-Embryogenic Callus of Tamarillo (Solanum betaceum Cav.).

Tamarillo is a solanaceous tree that has been extensively studied in terms of in vitro clonal propagation, namely somatic embryogenesis. In this work, a protocol of indirect somatic embryogenesis was applied to obtain embryogenic and non-embryogenic callus from leaf segments. Nuclear magnetic resonance spectroscopy was used to analyze the primary metabolome of these distinct calli to elucidate possible differentiation mechanisms from the common genetic background callus. Standard multivariate analysis methods were then applied, and were complemented by univariate statistical methods to identify differentially expressed primary metabolites and related metabolic pathways. The results showed carbohydrate and lipid metabolism to be the most relevant in all the calli assayed, with most discriminant metabolites being fructose, glucose and to a lesser extent choline. The glycolytic rate was higher in embryogenic calli, which shows, overall, a higher rate of sugar catabolism and a different profile of phospholipids with a choline/ethanolamine analysis. In general, our results show that a distinct primary metabolome between embryogenic and non-embryogenic calli occurs and that intracellular levels of fructose and sucrose and the glucose to sucrose ratio seem to be good candidates as biochemical biomarkers of embryogenic competence.

Ethylene Inhibition Reduces De Novo Shoot Organogenesis and Subsequent Plant Development from Leaf Explants of Solanum betaceum Cav.

In de novo shoot organogenesis (DNSO) plant cells develop into new shoots, without the need of an existing meristem. Generally, this process is triggered by wounding and specific growth regulators, such as auxins and cytokinins. Despite the potential significance of the plant hormone ethylene in DNSO, its effect in regeneration processes of woody species has not been thoroughly investigated. To address this gap, Solanum betaceum Cav. was used as an experimental model to explore the role of this hormone on DNSO and potentially extend the findings to other woody species. In this work it was shown that ethylene positively regulates DNSO from tamarillo leaf explants. Ethylene precursors ACC and ethephon stimulated shoot regeneration by increasing the number of buds and shoots regenerated. In contrast, the inhibition of ethylene biosynthesis or perception by AVG and AgNO3 decreased shoot regeneration. Organogenic callus induced in the presence of ethylene precursors showed an upregulated expression of the auxin efflux carrier gene PIN1, suggesting that ethylene may enhance shoot regeneration by affecting auxin distribution prior to shoot development. Additionally, it was found that the de novo shoot meristems induced in explants in which ethylene biosynthesis and perception was suppressed were unable to further develop into elongated shoots. Overall, these results imply that altering ethylene levels and perception could enhance shoot regeneration efficiency in tamarillo. Moreover, we offer insights into the possible molecular mechanisms involved in ethylene-induced shoot regeneration.

Morpho-Physiological Evaluation of Solanum betaceum Cav. In Vitro Cloned Plants: A Comparison of Different Micropropagation Methods.

Tamarillo (Solanum betaceum Cav.) is a subtropical solanaceous tree with increasing agronomic interest due to its nutritious edible fruits. Growing demand for tamarillo plants and fruits requires optimization of existing propagation methods and scaled-up systems for large-scale cloning of selected germplasm. Three in vitro protocols have been used to micropropagate tamarillo: (1) axillary shoot proliferation in a semisolid medium, (2) organogenesis, and (3) somatic embryogenesis procedures. Variables such as the age of the established shoot cultures and rooting treatments were also analyzed. The morphological and physiological quality of acclimatized plants derived from all the methodologies were compared, with seed-derived plants used as a control group. Overall, the results show that in vitro-derived plants have a similar development to seed-derived plants. Micropropagation by axillary shoot proliferation was highly efficient, with rooting rates above 80% in most treatments. Organogenesis induction was more effective from lamina explants using MS media with 2.0 mg·L-1 6-benzylaminopurine. Both organogenesis and somatic embryogenesis-derived plants were also morphologically and physiologically equivalent to seed and axillary shoot-derived plants. The specificities of each micropropagation method are discussed.

Proteomic and Metabolic Analysis of Pinus halepensis Mill. Embryonal Masses Induced under Heat Stress.

Understanding the physiological and molecular adjustments occurring during tree stress response is of great importance for forest management and breeding programs. Somatic embryogenesis has been used as a model system to analyze various processes occurring during embryo development, including stress response mechanisms. In addition, "priming" plants with heat stress during somatic embryogenesis seems to favor the acquisition of plant resilience to extreme temperature conditions. In this sense, Pinus halepensis somatic embryogenesis was induced under different heat stress treatments (40 °C for 4 h, 50 °C for 30 min, and 60 °C for 5 min) and its effects on the proteome and the relative concentration of soluble sugars, sugar alcohols and amino acids of the embryonal masses obtained were assessed. Heat severely affected the production of proteins, and 27 proteins related to heat stress response were identified; the majority of the proteins with increased amounts in embryonal masses induced at higher temperatures consisted of enzymes involved in the regulation of metabolism (glycolysis, the tricarboxylic acid cycle, amino acid biosynthesis and flavonoids formation), DNA binding, cell division, transcription regulation and the life-cycle of proteins. Finally, significant differences in the concentrations of sucrose and amino acids, such as glutamine, glycine and cysteine, were found.

An Efficient Agrobacterium-Mediated Genetic Transformation Method for Solanum betaceum Cav. Embryogenic Callus.

Somatic embryogenesis in Solanum betaceum (tamarillo) has proven to be an effective model system for studying morphogenesis, since optimized plant regeneration protocols are available, and embryogenic competent cell lines can be induced from different explants. Nevertheless, an efficient genetic transformation system for embryogenic callus (EC) has not yet been implemented for this species. Here, an optimized faster protocol of genetic transformation using Agrobacterium tumefaciens is described for EC. The sensitivity of EC to three antibiotics was determined, and kanamycin proved to be the best selective agent for tamarillo callus. Two Agrobacterium strains, EHA105 and LBA4404, both harboring the p35SGUSINT plasmid, carrying the reporter gene for ß-glucuronidase (gus) and the marker gene neomycin phosphotransferase (nptII), were used to test the efficiency of the process. To increase the success of the genetic transformation, a cold-shock treatment, coconut water, polyvinylpyrrolidone and an appropriate selection schedule based on antibiotic resistance were employed. The genetic transformation was evaluated by GUS assay and PCR-based techniques, and a 100% efficiency rate was confirmed in the kanamycin-resistant EC clumps. Genetic transformation with the EHA105 strain resulted in higher values for gus insertion in the genome. The protocol presented provides a useful tool for functional gene analysis and biotechnology approaches.

An Efficient Method to Prepare Barcoded cDNA Libraries from Plant Callus for Long-Read Sequencing.

Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full-length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies when compared to former sequencing methods. However, woody plant tissues are challenging to the successful preparation of cDNA libraries, thus, impairing further cutting-edge sequencing analyses. Here, a detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies® is described. This method was used to prepare eight barcoded cDNA libraries from two Solanum betaceum cell lines: one with compact morphology and embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The libraries were successfully sequenced, and data quality assessment showed high mean quality scores. Using this method, long-read sequencing will allow a comprehensive analysis of plant transcriptomes.

Antioxidant Potential of Tamarillo Fruits-Chemical and Infrared Spectroscopy Analysis.

Native to South America, tamarillo (Solanum betaceum Cav.) is a small tree cultivated as a fruit crop in several regions of the world. Known for its sweet and sour taste, tamarillo fruits are very nutritious due to the presence of health-beneficial components such as fiber, vitamins, and antioxidants. Despite its nutritional value, tamarillo remains poorly known in global markets. The present work aims to study the antioxidant activity of four genotypes of tamarillo. Several chemical assays were performed to assess the antioxidant components and antioxidant activity of aqueous ethanolic extracts from each genotype. Overall, the Mealhada genotype (a red cultivar) showed the most interesting results, displaying the highest amount of total phenolic, flavonoids, and anthocyanin contents, as well as higher antioxidant activity. To evaluate the composition of the extract, Fourier-transform infrared spectroscopy (FTIR) was used to characterize important components in aqueous ethanolic extracts of the fruits, having revealed the presence of high amounts of phenols (the main compounds responsible for antioxidant activity), as well as triterpenoids and polysaccharides. The present results highlight the potential nutraceutical importance of tamarillo fruits.

Testing Explant Sources, Culture Media, and Light Conditions for the Improvement of Organogenesis in Pinus ponderosa (P. Lawson and C. Lawson).

Pinus. ponderosa (P. Lawson and C. Lawson) is a commercial tree and one of the most important forest species in North America. Ponderosa pine suffers hardship when going through vegetative propagation and, in some cases, 15-30 years are needed to achieve full reproductive capacity. Based on previous works on P. ponderosa regeneration through in vitro organogenesis and trying to improve the published protocols, our objective was to analyze the influence of different types of explants, basal culture media, cytokinins, auxins, and light treatments on the success of shoot multiplication and rooting phases. Whole zygotic embryos and 44 µΜ 6-benzyladenine showed the best results in terms of explants survival. For shoot organogenesis, whole zygotic embryos and half LP (LP medium, Quoirin and Lepoivre, 1977, modified by Aitken-Christie et al., 1988) macronutrients were selected. A significant positive interaction between whole zygotic embryos and half LP macronutrients was found for the percentage of explants forming shoots. Regarding the light treatments applied, a significantly higher percentage of shoots elongated enough to be rooted was detected in shoots growing under blue LED at a light intensity of 61.09 µmol m-2 s-1. However, the acclimatization percentage was higher in shoots previously cultivated under fluorescent light at a light intensity of 61.71 µmol m-2 s-1. Anatomical studies using light microscopy and scanning electron microscopy showed the light treatments promoted differences in anatomical aspects in in vitro shoots; needles of plantlets exposed to red and blue LEDs revealed less stomata compared with needles from plantlets exposed to fluorescent light.

Enhancing the Production of Hydrolytic Enzymes in Elicited Tamarillo (Solanum betaceum Cav.) Cell Suspension Cultures.

Plant cell suspension cultures are widely used as a tool for analyzing cellular and molecular processes, metabolite synthesis, and differentiation, bypassing the structural complexity of plants. Within the range of approaches used to increase the production of metabolites by plant cells, one of the most recurrent is applying elicitors capable of stimulating metabolic pathways related to defense mechanisms. Previous proteomics analysis of tamarillo cell lines and cell suspension cultures have been used to further characterize and optimize the growth and stress-related metabolite production under in vitro controlled conditions. The main objective of this work was to develop a novel plant-based bioreactor system to produce hydrolytic enzymes using an elicitation approach. Based on effective protocols for tamarillo micropropagation and plant cell suspension culture establishment from induced callus lines, cell growth has been optimized, and enzymatic activity profiles under in vitro controlled conditions characterized. By testing different sucrose concentrations and the effects of two types of biotic elicitors, it was found that 3% (w/v) sucrose concentration in the liquid medium enhanced the production of hydrolytic enzymes. Moreover, casein hydrolysate at 0.5 and 1.5 g/L promoted protein production, whereas yeast extract (0.5 g/L) enhanced glycosidase activity. Meanwhile, chitosan (0.05 and 0.1 g/L) enhanced glycosidases, alkaline phosphates, and protease activities.

RNA methyltransferases in plants: Breakthroughs in function and evolution.

Each day it is becoming increasingly difficult not to notice the completely new, fast growing, extremely intricate and challenging world of epitranscriptomics as the understanding of RNA methylation is expanding at a hasty rate. Writers (methyltransferases), erasers (demethylases) and readers (RNA-binding proteins) are responsible for adding, removing and recognising methyl groups on RNA, respectively. Several methyltransferases identified in plants are now being investigated and recent studies have shown a connection between RNA-methyltransferases (RNA-MTases) and stress and development processes. However, compared to their animal and bacteria counterparts, the understanding of RNA methyltransferases is still incipient, particularly those located in organelles. Comparative and systematic analyses allowed the tracing of the evolution of these enzymes suggesting the existence of several methyltransferases yet to be characterised. This review outlines the functions of plant nuclear and organellar RNA-MTases in plant development and stress responses and the comparative and evolutionary discoveries made on RNA-MTases across kingdoms.

Genotype determines Arbutus unedo L. physiological and metabolomic responses to drought and recovery.

Strawberry tree (Arbutus unedo) is a small resilient species with a circum-Mediterranean distribution, high ecological relevance in southern European forests and with several economical applications. As most orchards are usually installed on marginal lands where plants usually face severe drought, selecting plants that can better cope with water restriction is critical, and a better understanding of the tolerance mechanisms is required. Strawberry tree plants under drought follow a typical isohydric strategy, by limiting transpiration through stomata closure. However, the contribution of genotype and its bio-geographic origin on plant performance needs clarification, as well as the involvement of a specific metabolic reactions associated with the mechanical response. To test this hypothesis, several eco-physiological and biochemical parameters were assessed on different genotypes, and the metabolic profiles studied, including important stress-related phytohormones, on plants under different water regimes (plants watered to 70% and 18% field capacity) and a recovery assay. A contrasting drought tolerance was found in plants from different genotypes, associated with physiological and metabolic responses. Metabolomics revealed more than 500 metabolic features were differentially accumulated, including abscisic and salicylic acids, for the genotype with better performance under drought (A4). This genotype also recovered faster when the imposed stress was interrupted, thus indicating the relevance of metabolic adaptation under water deficit conditions. By correlating carbon assimilation with identified metabolites, some proved to be satisfactory predictors of plant performance under drought and might be used for marker assisted breeding. Therefore, our study proves the importance of genotype as a major selection criterion of resistant plants to drought and provides empirical knowledge of the metabolic response involved. We also hypothesized the involvement of phenolics on response mechanisms under drought, which is worth to be explored to shed light on the metabolic pathways involved in plant response to water stress.

Regulatory non-coding RNAs: Emerging roles during plant cell reprogramming and in vitro regeneration.

Plant regeneration is a well-known capacity of plants occurring either in vivo or in vitro. This potential is the basis for plant micropropagation and genetic transformation as well as a useful system to analyse different aspects of plant development. Recent studies have proven that RNA species with no protein-coding capacity are key regulators of cellular function and essential for cell reprogramming. In this review, the current knowledge on the role of several ncRNAs in plant regeneration processes is summarized, with a focus on cell fate reprogramming. Moreover, the involvement/impact of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and small-interfering RNAs (siRNAs) in the regulatory networks of cell dedifferentiation, proliferation and differentiation is also analysed. A deeper understanding of plant ncRNAs in somatic cell reprogramming will allow a better modulation of in vitro regeneration processes such as organogenesis and somatic embryogenesis.

Valorisation Potential of Invasive Acacia dealbata, A. longifolia and A. melanoxylon from Land Clearings.

Acacia spp. are invasive in Southern Europe, and their high propagation rates produce excessive biomass, exacerbating wildfire risk. However, lignocellulosic biomass from Acacia spp. may be utilised for diverse biorefinery applications. In this study, attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR), high-performance anion-exchange chromatography pulsed amperometric detection (HPAEC-PAD) and lignin content determinations were used for a comparative compositional characterisation of A. dealbata, A. longifolia and A. melanoxylon. Additionally, biomass was treated with three white-rot fungi species (Ganoderma lucidum, Pleurotus ostreatus and Trametes versicolor), which preferentially degrade lignin. Our results showed that the pre-treatments do not significantly alter neutral sugar composition while reducing lignin content. Sugar release from enzymatic saccharification was enhanced, in some cases possibly due to a synergy between white-rot fungi and mild alkali pretreatments. For example, in A. dealbata stems treated with alkali and P. ostreatus, saccharification yield was 702.3 nmol mg-1, which is higher than the samples treated only with alkali (608.1 nmol mg-1), and 2.9-fold higher than the non-pretreated controls (243.9 nmol mg-1). By characterising biomass and pretreatments, generated data creates value for unused biomass resources, contributing to the implementation of sustainable biorefining systems. In due course, the generated value will lead to economic incentives for landowners to cut back invasive Acacia spp. more frequently, thus reducing excess biomass, which exacerbates wildfire risk.

Thermopriming-associated proteome and sugar content responses in Pinus radiata embryogenic tissue.

Improving the capacity of plants to face adverse environmental conditions requires a deep understanding of the molecular mechanisms governing stress response and adaptation. Proteomics, combined with metabolic analyses, offers a wide resource of information to be used in plant breeding programs. Previous studies have shown that somatic embryogenesis in Pinus spp. is a suitable tool not only to investigate stress response processes but also to modulate the behaviour of somatic plants. Based on this, the objective of this study was to analyse the protein and soluble sugar profiles of Pinus radiata embryonal masses after the application of high temperatures to unravel the mechanisms involved in thermopriming and memory acquisition at early stages of the somatic embryogenesis process. Results confirmed that heat provokes deep readjustments in the life cycle of proteins, together with a significant reduction in the carbon-flux of central-metabolism pathways. Heat-priming also promotes the accumulation of proteins involved in oxidative stress defence, in the synthesis of specific amino acids such as isoleucine, influences cell division, the organization of the cytoskeleton and cell-walls, and modifies the levels of free soluble sugars like glucose or fructose. All this seems to be regulated by proteins linked with epigenetic, transcriptional and post-transcriptional mechanisms.

Induction of Somatic Embryogenesis in Tamarillo (Solanum betaceum Cav.) Involves Increases in the Endogenous Auxin Indole-3-Acetic Acid.

Somatic embryogenesis (SE) is a complex biological process regulated by several factors, such as the action of plant growth regulators, namely auxins, of which the most physiologically relevant is indole-3-acetic acid (IAA). In tamarillo, an optimized system for induction of SE creates, after an induction process, embryogenic (EC) and non-embryogenic callus (NEC). In this work the endogenous levels of auxin along the induction phase and in the calli samples were investigated using chemical quantifications by colorimetric reactions and HPLC as well as immunohistochemistry approaches. Differential gene expression (IAA 11, IAA 14, IAA 17, TIR 1, and AFB3) analysis during the induction phase was also carried out. The results showed that the endogenous IAA content is considerably higher in embryogenic than in non-embryogenic calli, with a tendency to increase as the dedifferentiation of the original explant (leaf segments) evolves. Furthermore, the degradation rates of IAA seem to be related to these levels, as non-embryogenic tissue presents a higher degradation rate. The immunohistochemical results support the quantifications made, with higher observable labeling on embryogenic tissue that tends to increase along the induction phase. Differential gene expression also suggests a distinct molecular response between EC and NEC.

Isolation and identification of Arbutus unedo L. fungi endophytes and biological control of Phytophthora cinnamomi in vitro.

Strawberry tree (Arbutus unedo, Ericaceae) is an evergreen tree with a circum-Mediterranean distribution. It has a great ecological and economic importance as a source of bioactive compounds with industrial applications and for fruit production. This study aims to characterize the fungi microbiome of this forestry species in order to develop biological control strategies in the increasing orchard production area. For this purpose, fungi endophytes were isolated from wild strawberry tree plants, and a molecular identification was carried out. In vitro assays were carried out to evaluate and characterize the antagonism of some endophytes. Among the several fungi endophytes isolated from strawberry tree (a total of 53 from 20 genera), a Trichoderma atroviride strain proved to have antagonism effect against several phytopathogens, including Alternaria alternata, Botrytis cinerea, Glomerella cingulata, and Mycosphaerella aurantia. This antagonism was particularly effective against Phytophthora cinnamomi, causing a reduction in growth of about 80% on this invasive oomycete. An enzymatic assay revealed the production of several enzymes by T. atroviride, such as cellulases, chitinases, glucosidases, alkaline phosphatases, and proteases, which is one of the several mechanisms known to be involved on Trichoderma biological control ability. The enzymatic activity, in particular that of cell wall-degrading enzymes, was accentuated when in a dual culture with P. cinnamomi. The production of serine proteases, aspartyl proteases, metalloproteases, and cysteine proteases was also detected in an experiment carried out in liquid medium, suggesting the involvement of these proteases on Trichoderma mycoparasitism mechanisms. Finally, in a three-way interaction with in vitro strawberry tree plants, the T. atroviride strain identified on this study (Au50) was able to protect the plants against P. cinnamomi, thus proving its potential as a biological control agent.