Trichoderma carraovejensis: a new species from vineyard ecosystem with biocontrol abilities against grapevine trunk disease pathogens and ecological adaptation.

Trichoderma strains used in vineyards for the control of grapevine trunk diseases (GTDs) present a promising alternative to chemical products. Therefore, the isolation and characterization of new indigenous Trichoderma strains for these purposes is a valuable strategy to favor the adaptation of these strains to the environment, thus improving their efficacy in the field. In this research, a new Trichoderma species, Trichoderma carraovejensis, isolated from vineyards in Ribera de Duero (Spain) area, has been identified and phylogenetically analyzed using 20 housekeeping genes isolated from the genome of 24 Trichoderma species. A morphological description and comparison of the new species has also been carried out. In order to corroborate the potential of T. carraovejensis as a biological control agent (BCA), confrontation tests against pathogenic fungi, causing various GTDs, have been performed in the laboratory. The compatibility of T. carraovejensis with different pesticides and biostimulants has also been assessed. This new Trichoderma species demonstrates the ability to control pathogens such as Diplodia seriata, as well as high compatibility with powdered sulfur-based pesticides. In conclusion, the autochthonous species T. carraovejensis can be an effective alternative to complement the currently used strategies for the control of wood diseases in its region of origin.

Vineyard Management and Physicochemical Parameters of Soil Affect Native Trichoderma Populations, Sources of Biocontrol Agents against Phaeoacremonium minimum.

Native strains of Trichoderma in vineyard soil represent an opportunity for reducing the incidence of grapevine trunk diseases (GTDs) in vineyards. Moreover, its relationship with the environment (physicochemical soil characteristics and farming management practices) remains unclear. In the current study, a survey was carried out on farming management used by viticulturists, and soil samples were studied to analyze their physicochemical properties and to isolate Trichoderma strains. Later, statistical analyses were performed to identify possible correlations between Trichoderma populations, soil management and soil characteristics. In addition, in vitro tests, including antibiosis and mycoparasitism, were performed to select those Trichoderma strains able to antagonize Phaeoacremonium minimum. In this study a positive correlation was found between the iron content and pH in the soil, and a lower pH increases Trichoderma populations in soils. Vineyard management also affects Trichoderma populations in the soil, negatively in the case of fertilization and tillage and positively in the case of herbicide spraying. Two Trichoderma native strains were selected as potential biocontrol agents (Trichoderma gamsii T065 and Trichoderma harzianum T087) using antibiosis and mycoparasitism as mechanisms of action. These results led to the conclusion that native Trichoderma strains hold great potential as biological control agents and as producers of secondary metabolites.

Effects of trichothecene production by Trichoderma arundinaceum isolates from bean-field soils on the defense response, growth and development of bean plants (Phaseolus vulgaris).

The trichothecene toxin-producing fungus Trichoderma arundinaceum has potential as a biological control agent. However, most biocontrol studies have focused only on one strain, IBT 40837. In the current study, three Trichoderma isolates recovered from bean-field soils produced the trichothecene harzianum A (HA) and trichodermol, the latter being an intermediate in the HA biosynthesis. Based on phylogenetic analysis, the three isolates were assigned to the species T. arundinaceum. Their genome sequences had a high degree of similarity to the reference IBT 40837 strain, in terms of total genome size, number of predicted genes, and diversity of putative secondary metabolite biosynthetic gene clusters. HA production by these bean-field isolates conferred significant in vitro antifungal activity against Rhizoctonia solani and Sclerotinia sclerotiorum, which are some of the most important bean pathogens. Furthermore, the bean-field isolates stimulated germination of bean seeds and subsequent growth of above ground parts of the bean plant. Transcriptomic analysis of bean plants inoculated with these T. arundinaceum bean-field soil isolates indicated that HA production significantly affected expression of plant defense-related genes; this effect was particularly significant in the expression of chitinase-encoding genes. Together, these results indicate that Trichoderma species producing non-phytotoxic trichothecenes can induce defenses in plants without negatively affecting germination and development.

Effect of Farnesol in Trichoderma Physiology and in Fungal-Plant Interaction.

Farnesol is an isoprenoid intermediate in the mevalonate (MVA) pathway and is produced by the dephosphorylation of farnesyl diphosphate. Farnesol plays a central role in cell growth and differentiation, controls production of ubiquinone and ergosterol, and participates in the regulation of filamentation and biofilm formation. Despite these important functions, studies of farnesol in filamentous fungi are limited, and information on its effects on antifungal and/or biocontrol activity is scarce. In the present article, we identified the Trichoderma harzianum gene dpp1, encoding a diacylglycerol pyrophosphatase that catalyzes production of farnesol from farnesol diphosphate. We analyzed the function of dpp1 to address the importance of farnesol in Trichoderma physiology and ecology. Overexpression of dpp1 in T. harzianum caused an expected increase in farnesol production as well as a marked change in squalene and ergosterol levels, but overexpression did not affect antifungal activity. In interaction with plants, a dpp1-overexpressing transformant acted as a sensitizing agent in that it up-regulated expression of plant defense salicylate-related genes in the presence of a fungal plant pathogen. In addition, toxicity of farnesol on Trichoderma and plants was examined. Finally, a phylogenetic study of dpp1 was performed to understand its evolutionary history as a primary metabolite gene. This article represents a step forward in the acquisition of knowledge on the role of farnesol in fungal physiology and in fungus-environment interactions.

Novel culture chamber to evaluate in vitro plant-microbe volatile interactions: Effects of Trichoderma harzianum volatiles on wheat plantlets.

The field of plant-microbe interactions mediated by Biogenic Volatile Organic Compounds (BVOCs) still faces several limitations due to the lack of reliable equipment. We present a novel device designed to evaluate in vitro plant-microbe volatile interactions, the plant-microbe VOC Chamber. It was tested by evaluating the effects exerted on wheat development by volatiles from three Trichoderma harzianum strains, a wild type and two genetically modified strains; one expressing the tri5 gene, which leads to the synthesis and emission of the volatile trichodiene, and the other by silencing the erg1 gene, impairing ergosterol production. The wild type and the erg1-silenced strain enhanced fresh weight and length of the aerial part, but reduced root dry weight. Interestingly, no differences were found between them. Conversely, the tri5-transformant strain reduced root and aerial growth compared to the control and the other strains. No differences were observed regarding chlorophyll fluorescence quantum yield and leaf chlorophyll content, suggesting that the released BVOCs do not interfere with photosynthesis. The plant-microbe VOC Chamber proved to be a simple and reliable method to evaluate the in vitro effects of microbial BVOCs on plant development, perfect for the screening of microorganisms with interesting volatile traits.

Use of VOC Chambers to evaluate the impact of microbial volatile compounds on dry grain insect pests.

Although increasing focus is being given to the control of insect pests with natural products, the study of volatile mediated microbe-insect interactions has been largely neglected so far. Currently, no standardized method is available for the in vitro evaluation of the effects produced on insects by Volatile Organic Compounds (VOCs) directly emitted by actively growing microbial strains. This manuscript presents a reliable protocol using VOC Chambers (Álvarez-García et al., 2021) to assess these effects and specifically those exerted on dry grain insect pests. The methodology also serves to evaluate the impact of these volatile interactions on the grain damage produced by the insects. It allows to vary the level of gas exchange with the environment, thus providing different experimental conditions to emulate diverse natural ones in which these interactions may occur. Our method adapts the use of VOC Chambers for the evaluation of microbe-insect volatile interactions and the in vitro screening of microbial strains that produce potentially bioactive VOCs against insects. It allows assessing the effects of these volatile interactions on the damage produced by insect pests on stored dry grains. Sealed and unsealed setups can be used to determine the impact of ventilation and gas exchange on these interactions.

Distribution, Function, and Evolution of a Gene Essential for Trichothecene Toxin Biosynthesis in Trichoderma.

Trichothecenes are terpenoid toxins produced by species in 10 fungal genera, including species of Trichoderma. The trichothecene biosynthetic gene (tri) cluster typically includes the tri5 gene, which encodes a terpene synthase that catalyzes formation of trichodiene, the parent compound of all trichothecenes. The two Trichoderma species, Trichoderma arundinaceum and T. brevicompactum, that have been examined are unique in that tri5 is located outside the tri cluster in a genomic region that does not include other known tri genes. In the current study, analysis of 35 species representing a wide range of the phylogenetic diversity of Trichoderma revealed that 22 species had tri5, but only 13 species had both tri5 and the tri cluster. tri5 was not located in the cluster in any species. Using complementation analysis of a T. arundinaceum tri5 deletion mutant, we demonstrated that some tri5 homologs from species that lack a tri cluster are functional, but others are not. Phylogenetic analyses suggest that Trichoderma tri5 was under positive selection following its divergence from homologs in other fungi but before Trichoderma species began diverging from one another. We propose two models to explain these diverse observations. One model proposes that the location of tri5 outside the tri cluster resulted from loss of tri5 from the cluster in an ancestral species followed by reacquisition via horizontal transfer. The other model proposes that in species that have a functional tri5 but lack the tri cluster, trichodiene production provides a competitive advantage.

Green Strategies of Powdery Mildew Control in Hop: From Organic Products to Nanoscale Carriers.

Humulus lupulus L. is a long-lived, perennial, herbaceous, and dioecious climbing plant. The foremost producers in the European Union are Germany, the Czech Republic, Poland, Slovenia, and Spain. The Spanish cultivated area is concentrated in the province of León. Powdery mildew, caused by Podosphaera macularis, menaces hop production and quality in all hop growing regions located in the Northern hemisphere, colonizing leaves, petioles, inflorescences, and finally cones. In this work, powdery mildew control was monitored, comparing nine fungicide strategies: five organics, two integrated disease management (IDM)-based, with and without Nutragreen® nanoscale carrier, and two conventional treatments (CON) with and without Nutragreen® nanoscale carrier. The organic treatments were able to diminish P. macularis on leaves, but no effect was observed in cones. CON treatments reduced the infection on leaves and cones and increased the cone quantity and quality. Likewise, IDM-based treatments provided satisfactory results as they diminished powdery mildew on leaves and cones. Finally, dose reduction using a Nutragreen® nanoscale carrier showed beneficial effects in the control of powdery mildew compared to the commercial dose. Hence, the use of nanoscale carries permits a 30% reduction in pesticide dose, which optimizes yield and hop quality, reduces risks linked to pesticides, and aids in compliance with public and international policy demands.

Volatile Organic Compound Chamber: A Novel Technology for Microbiological Volatile Interaction Assays.

The interest in the study of microbiological interactions mediated by volatile organic compounds (VOCs) has steadily increased in the last few years. Nevertheless, most assays still rely on the use of non-specific materials. We present a new tool, the volatile organic compound chamber (VOC chamber), specifically designed to perform these experiments. The novel devices were tested using four Trichoderma strains against Fusarium oxysporum and Rhizoctonia solani. We demonstrate that VOC chambers provide higher sensitivity and selectivity between treatments and higher homogeneity of results than the traditional method. VOC chambers are also able to test both vented and non-vented conditions. We prove that ventilation plays a very important role regarding volatile interactions, up to the point that some growth-inhibitory effects observed in closed environments switch to promoting ones when tested in vented conditions. This promoting activity seems to be related to the accumulation of squalene by T. harzianum. The VOC chambers proved to be an easy, homogeneous, flexible, and repeatable method, able to better select microorganisms with high biocontrol activity and to guide the future identification of new bioactive VOCs and their role in microbial interactions.

Effect of Cry Toxins on Xylotrechus arvicola (Coleoptera: Cerambycidae) Larvae.

The beetle Xylotrechus arvicola is a destructive pest in vineyards (Vitis vinifera) in the main wine-producing areas of the Iberian Peninsula. X. arvicola larvae bore into the grapevine wood-making galleries, thus damaging the plant both directly and indirectly; the latter through the proliferation of wood fungi, which can invade the inside of the plant, decreasing the quality and quantity of its production. The susceptibility of X. arvicola larvae to five coleopteran toxic Cry proteins (Cry1B, Cry1I, Cry3A, Cry7A, and Cry23/37) was evaluated under laboratory conditions in order to deepen the knowledge of the effect of these proteins on this insect throughout its biological development. Cry7Ab and Cry1Ba were the most effective in controlling X. arvicola larvae due to the significant reduction in larvae survival (32.9 and 25.9 days, respectively), and by causing serious alterations in the larvae during the remaining months of their development. The developmental stage of the prepupal and pupal stages was not affected by the previous ingestion of Cry proteins. The Cry proteins tested could be applied to control X. arvicola larvae since they were able to kill them and cause serious alterations in the larvae during the remaining months of development that followed. The data presented suggest that these Cry proteins can be used as bioinsecticides against the larvae of this insect, applying them only at the moment when the larvae hatch from the egg outside the grapevine wood (this would only be useful and justified if the economic threshold is exceeded) in order to avoid the rapid evolution of resistance against these toxins since not all of the larvae were killed and thus increase vine wood protection.

Genetic Response of Common Bean to the Inoculation with Indigenous Fusarium Isolates.

Fungal species from the genus Fusarium are important soil-borne pathogens worldwide, causing significant economic losses in diverse crops. The need to find sustainable solutions against this disease has led to the development of new strategies-for instance, the use of biocontrol agents. In this regard, non-pathogenic Fusarium isolates have demonstrated their ability to help other plants withstand subsequent pathogen attacks. In the present work, several Fusarium isolates were evaluated in climatic chambers to identify those presenting low or non-pathogenic behavior. The inoculation with a low-pathogenic isolate of the fungus did not affect the development of the plant, contrary to the results observed in plants inoculated with pathogenic isolates. The expression of defense-related genes was evaluated and compared between plants inoculated with pathogenic and low-pathogenic Fusarium isolates. Low-pathogenic isolates caused a general downregulation of several plant defense-related genes, while pathogenic ones produced an upregulation of these genes. This kind of response to low-pathogenic fungal isolates has been already described for other plant species and fungal pathogens, being related to enhanced tolerance to later pathogen attacks. The results here presented suggest that low-pathogenic F. oxysporum and F. solani isolates may have potential biocontrol activity against bean pathogens via induced and systemic responses in the plant.

Colonization of Vitis vinifera L. by the Endophyte Trichoderma sp. Strain T154: Biocontrol Activity Against Phaeoacremonium minimum.

Trichoderma strains used in biological control products usually exhibit high efficiency in the control of plant diseases. However, their behavior under field conditions is difficult to predict. In addition, the potential of indigenous strains has been poorly assayed as well as their possible behavior as endophytes. Hence, niche colonization is a key feature for an effective protection. In this study, we aimed to: (i) explore the possibility of using a new Trichoderma strain isolated from vine to control pathogens, (ii) study the in planta interaction with the pathogen Phaeoacremonium minimum W. Gams, Crous, M.J. Wingf. & L. Mugnai (formerly Phaeoacremonium aleophilum), a pioneer fungus involved in Grapevine Trunk Diseases (GTDs) such as esca. For this purpose, fluorescently tagged Trichoderma sp. T154 and a P. minimum strain were used for scanning electron microscopy and confocal scanning laser microscopy analyses. Data showed that the Trichoderma strain is able to colonize plants up to 12 weeks post inoculation and is located in xylem, fibers, as well as in parenchymatic tissues inside the wood. The beneficial fungus reduced colonization of the esca-related pathogen colonizing the same niches. The main observed mechanism involved in biocontrol of Trichoderma against the esca pathogen was spore adhesion, niche exclusion and only few typical hypha coiling was found between Trichoderma and the pathogen. These results suggest that the Trichoderma strain has potential for reducing the colonization of Phaeoacremonium minimum and thus, an inoculation of this biological control agent can protect the plant by limiting the development of GTD, and the strain can behave as an endophyte.

Identification of plant genes putatively involved in the perception of fungal ergosterol-squalene.

Trichoderma biocontrol strains establish a complex network of interactions with plants, in which diverse fungal molecules are involved in the recognition of these fungi as nonpathogenic organisms. These molecules act as microbial-associated molecular patterns that trigger plant responses. Previous studies have reported the importance of ergosterol produced by Trichoderma spp. for the ability of these fungi to induce plant growth and defenses. In addition, squalene, a sterol biosynthetic intermediate, seems to play an important role in these interactions. Here, we analyzed the effect of different concentrations of ergosterol and squalene on tomato (Solanum lycopersicum) growth and on the transcription level of defense- and growth-related genes. We used an RNA-seq strategy to identify several tomato genes encoding predicted pattern recognition receptor proteins or WRKY transcription factors, both of which are putatively involved in the perception and response to ergosterol and squalene. Finally, an analysis of Arabidopsis thaliana mutants lacking the genes homologous to these tomato candidates led to the identification of a WRKY40 transcription factor that negatively regulates salicylic acid-related genes and positively regulates ethylene- and jasmonate-related genes in the presence of ergosterol and squalene.

Toxicity of five Cry proteins against the insect pest Acanthoscelides obtectus (Coleoptera: Chrisomelidae: Bruchinae).

The beetle Acanthoscelides obtectus (Say) causes severe post-harvest losses in the common bean (Phaseolus vulgaris). Under laboratory conditions, the susceptibility of A. obtectus to five coleopteran-specific Cry toxic proteins from Bacillus thuringiensis (Cry1Ba, Cry1Ia, Cry3Aa, Cry7Ab, and Cry23/37) was evaluated. After 30 days exposure, Cry proteins demonstrated high activity against A. obtectus adults (100% mortality). Proteins showed statistical differences in toxicity parameters compared to the control treatment, but the parameters were similar among them, and indicated that the final toxic effects can be observed after the 24th day. The toxic effects on A. obtectus larvae were evaluated indirectly by allowing adults to oviposit on treated beans and recording the emergence of F1 adults. All treatments resulted in a lower rate of successful emergence compared to the control treatment, ranging from 60% (Cry23/37) to 10% (Cry1Ia) reduction in eclosion. Finally, to evaluate the ability of Cry proteins to protect the beans against A. obtectus; the number of beans infested, the number of holes in each bean and bean weight loss were determined 45 days after the treatment. The parameters showed significant bean protection by all Cry proteins analyzed compared to control treatment. Cry23/37 showed the best results, however, results for the other proteins were similar. The proteins belong to different Cry protein families, which suggest that they could be used in combination to increase plant protection without compromising resistance management. Moreover, adult emergence and bean protection results indicate differences among the proteins, which may suggest different modes of action. Our results indicate that the studied Cry proteins can be applied for the control of A. obtectus larvae and adults.

Insecticidal Properties of Ocimum basilicum and Cymbopogon winterianus against Acanthoscelides obtectus, Insect Pest of the Common Bean (Phaseolus vulgaris, L.).

The bean weevil, Acanthoscelides obtectus Say (Coleoptera: Chrysomelidae: Bruchinae), causes severe post-harvest losses in the common bean, Phaseolus vulgaris L. The control of this insect is still poor and involves the use of conventional insecticides. There is an increasing demand in the search for new active substances and products for pest control towards reduction of adverse effects on human health and the environment. The protection of grains with alternative products, such as essential oils, is a possible alternative to meet the needs described above. Therefore, this investigation evaluated the applications of basil, Ocimum basilicum, and citronella, Cymbopogon winterianus, essential oils for A. obtectus control. These essential oils significantly reduced the bean weight losses and the number of beans damaged by A. obtectus at higher doses than 60 or 120 µL/sample. The number of holes per bean did not differ between the doses of basil essential oil, not even at the dose of 60 µL, while it was higher at 120 µL, probably due to a lower capacity of movement of the insects treated with this dose and/or the oil's direct or indirect effects on the insects. Basil and citronella oils exhibited similar patterns of insecticidal activity over the insect, both directly in adult insects or indirectly over bean seeds. These essential oils affected the development of A. obtectus since the greatest doses applied on beans decreased the emergence of the bean weevil. The results prove the insecticidal capacity of the tested essential oils and hence their potential as active substances against A. obtectus in environmentally low risk pest control strategies. Supplementary trials should be conducted under real storage conditions.

Effect of Trichoderma velutinum and Rhizoctonia solani on the Metabolome of Bean Plants (Phaseolus vulgaris L.).

The common bean (Phaseolus vulgaris L.) is one of the most important food legume crops worldwide that is affected by phytopathogenic fungi such as Rhizoctonia solani. Biological control represents an effective alternative method for the use of conventional synthetic chemical pesticides for crop protection. Trichoderma spp. have been successfully used in agriculture both to control fungal diseases and to promote plant growth. The response of the plant to the invasion of fungi activates defensive resistance responses by inducing the expression of genes and producing secondary metabolites. The purpose of this work was to analyze the changes in the bean metabolome that occur during its interaction with pathogenic (R. solani) and antagonistic (T. velutinum) fungi. In this work, 216 compounds were characterized by liquid chromatography mass spectrometry (LC-MS) analysis but only 36 were noted as significantly different in the interaction in comparison to control plants and they were tentatively characterized. These compounds were classified as: two amino acids, three peptides, one carbohydrate, one glycoside, one fatty acid, two lipids, 17 flavonoids, four phenols and four terpenes. This work is the first attempt to determine how the presence of T. velutinum and/or R. solani affect the defense response of bean plants using untargeted metabolomics analysis.

Investigations of Trichoderma spp. and Beauveria bassiana as biological control agent for Xylotrechus arvicola, a major insect pest in Spanish vineyards.

Xylotrechus arvicola (Olivier) (Coleoptera: Cerambycidae) is an important pest in vineyards (Vitis vinifera) in the main wine-producing regions of Spain. Effective control of this pest is difficult due to the biology of this pest. Biological control agents (BCAs) have proven to be an effective tool in controlling and preventing the spread of a variety of plant pests and diseases. Consequently, the aim of the present study was to assess the capacity of different Trichodema spp., isolated from various vineyards and one commercial isolate of Beauveria bassiana Vuillemin (Hypocreales: Cordycipitaceae), as BCAs of X. arvicola. Isolates of Trichoderma spp. and one isolate of B. bassiana were evaluated against X. arvicola eggs, larvae and adults. Trichoderma harzianum and Trichoderma gamsii demonstrated a good ovicidal control, 100.0% with T. harzianum and over 92.0% with T. gamsii. These Trichoderma strains achieved an over 65.0% larval mortality and 87.5% adult mortality. B. bassiana was the most effective treatment against X. arvicola larvae. These results confirm that Trichoderma spp. can be used to inhibit egg development. In addition, Trichoderma spp. and B. bassiana can help to prevent larvae boring into vines and to kill adults. Therefore, Trichoderma spp., especially T. harzianum and T. gamsii, and B. bassiana can be considered as highly effective BCAs of X. arvicola in vineyards.

Field evaluation of 3-hydroxy-2-hexanone and ethanol as attractants for the cerambycid beetle pest of vineyards, Xylotrechus arvicola.

BACKGROUND: The beetle Xylotrechus arvicola (Coleoptera: Cerambycidae) is a serious pest of vineyards in the Iberian Peninsula. In previous work, the male beetles, but not females, were shown to produce (R)-3-hydroxy-2-hexanone, and female beetles were attracted to this compound in a laboratory bioassay. In this study, release rates of 3-hydroxy-2-hexanone from different dispensers were measured in the laboratory, and the attractiveness of these to X. arvicola adults was determined in trapping tests in three traditional wine-growing regions in Spain. RESULTS: As a result of laboratory experiments, for field experiments 3-hydroxy-2-hexanone was formulated as 100 µL in a polyethylene sachet (50 mm × 50 mm × 250 µm), and ethanol was formulated as 1 mL in a polyethylene press-seal bag (76 mm × 57 mm ×50 µm). Field catches were similar at all three study sites. Catches in traps baited with 3-hydroxy-2-hexanone alone were not significantly different from those in unbaited control traps, but catches in traps baited with 3-hydroxy-2-hexanone and ethanol in separate sachets, with 3-hydroxy-2-hexanone and ethanol in the same sachet or with ethanol alone were significantly greater than those in control traps. These results confirm that the beetles are attracted to ethanol, and the addition of 3-hydroxy-2-hexanone does not seem to make any difference. CONCLUSIONS: Attraction of females for the male-produced compound (R)-3-hydroxy-2-hexanone has been observed in laboratory but not in field experiments. Traps baited with ethanol are highly attractive to both sexes of adults of X. arvicola, and these can be used for improved monitoring of the adult emergence and for population control by mass trapping. © 2016 Society of Chemical Industry.

Development of a qPCR Strategy to Select Bean Genes Involved in Plant Defense Response and Regulated by the Trichoderma velutinum - Rhizoctonia solani Interaction.

Bean production is affected by a wide diversity of fungal pathogens, among them Rhizoctonia solani is one of the most important. A strategy to control bean infectious diseases, mainly those caused by fungi, is based on the use of biocontrol agents (BCAs) that can reduce the negative effects of plant pathogens and also can promote positive responses in the plant. Trichoderma is a fungal genus that is able to induce the expression of genes involved in plant defense response and also to promote plant growth, root development and nutrient uptake. In this article, a strategy that combines in silico analysis and real time PCR to detect additional bean defense-related genes, regulated by the presence of Trichoderma velutinum and/or R. solani has been applied. Based in this strategy, from the 48 bean genes initially analyzed, 14 were selected, and only WRKY33, CH5b and hGS showed an up-regulatory response in the presence of T. velutinum. The other genes were or not affected (OSM34) or down-regulated by the presence of this fungus. R. solani infection resulted in a down-regulation of most of the genes analyzed, except PR1, OSM34 and CNGC2 that were not affected, and the presence of both, T. velutinum and R. solani, up-regulates hGS and down-regulates all the other genes analyzed, except CH5b which was not significantly affected. As conclusion, the strategy described in the present work has been shown to be effective to detect genes involved in plant defense, which respond to the presence of a BCA or to a pathogen and also to the presence of both. The selected genes show significant homology with previously described plant defense genes and they are expressed in bean leaves of plants treated with T. velutinum and/or infected with R. solani.

Trichothecenes and aspinolides produced by Trichoderma arundinaceum regulate expression of Botrytis cinerea genes involved in virulence and growth.

Trichoderma arundinaceum (Ta37) and Botrytis cinerea (B05.10) produce the sesquiterpenoids harzianum A (HA) and botrydial (BOT), respectively. TaΔTri5, an HA non-producer mutant, produces high levels of the polyketide compounds aspinolides (Asp) B and C. We analyzed the role of HA and Asp in the B. cinerea-T. arundinaceum interaction, including changes in BOT production as well as transcriptomic changes of BcBOT genes involved in BOT biosynthesis, and also of genes associated with virulence and ergosterol biosynthesis. We found that exogenously added HA up-regulated the expression of the BcBOT and all the virulence genes analyzed when B. cinerea was grown alone. However, a decrease in the amount of BOT and a down-regulation of BcBOT gene expression was observed in the interaction zone of B05.10-Ta37 dual cultures, compared to TaΔTri5. Thus, the confrontation with T. arundinaceum results in an up-regulation of most of the B. cinerea genes involved in virulence yet the presence of T. arundinaceum secondary metabolites, HA and AspC, act separately and together to down-regulate the B. cinerea genes analyzed. The present work emphasizes the existence of a chemical cross-regulation between B. cinerea and T. arundinaceum and contributes to understanding how a biocontrol fungus and its prey interact with each other.