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Microbial-based biostimulants, functioning as biotic and abiotic stress protectants and growth enhancers, are becoming increasingly important in agriculture also in the context of climate change. The search for new products that can help reduce chemical inputs under a variety of field conditions is the new challenge. In this study, we tested whether the combination of two microbial growth enhancers with complementary modes of action, Azotobacter chroococcum 76A and Trichoderma afroharzianum T22, could facilitate tomato adaptation to a 30% reduction of optimal water and nitrogen requirements. The microbial inoculum increased tomato yield (+48.5%) under optimal water and nutrient conditions. In addition, the microbial application improved leaf water potential under stress conditions (+9.5%), decreased the overall leaf temperature (-4.6%), and increased shoot fresh weight (+15%), indicating that this consortium could act as a positive regulator of plant water relations under limited water and nitrogen availability. A significant increase in microbial populations in the rhizosphere with applications of A. chroococcum 76A and T. afroharzianum T22 under stress conditions, suggested that these inoculants could enhance soil microbial abundance, including the abundance of native beneficial microorganisms. Sampling time, limited water and nitrogen regimes and microbial inoculations all affected bacterial and fungal populations in the rhizospheric soil. Overall, these results indicated that the selected microbial consortium could function as plant growth enhancer and stress protectant, possibly by triggering adaptation mechanisms via functional changes in the soil microbial diversity and relative abundance.

Abstract Background Fungi biosynthesize a wide range of chemically diverse secondary metabolites during processes of competition with other micro- and macro-organisms, symbiosis, parasitism, or pathogenesis. Some of these natural compounds have antibiotic properties, which allow the microbe to inhibit and/or kill their microbial competitors. Results In the course of an ongoing search for novel bioactive metabolites from Trichoderma harzianum M10 using OSMAC (One Strain MAny Compounds) strategy, a bioactive chromone derivative has been isolated. The 5-hydroxy-2,3-dimethyl-7-methoxychromone (1), purified for the first time from T. harzianum M10 and induced in specific medium (potato dextrose broth, PDB) and condition (light and shaking), has been obtained as pure crystals and its structure has been fully characterized using X-ray and spectroscopic methods. This metabolite revealed a significant antibiotic activity against Rhizoctonia solani (45% of growth inhibition after 24 h of incubation at a concentration of 100 ng plug−1) and significantly reduced the viability of colorectal human cancer cells in a concentration-dependent manner. Moreover, metabolomic analysis allowed the identification of compounds modulated by the cultivating conditions. Among the statistically different molecules detected it was possible to identify siderophores, such as ferricrocin and coprogen B, harzianic acid (and its derivatives), and butenolides. Conclusion OSMAC strategy represents a valuable approach to overcome the limitations of experiments performed under laboratory conditions. Indeed, it is possible to modulate metabolites production by changing medium and conditions applied to the cultures. A specific set of conditions induced the production of a secondary metabolite never isolated from T. harzianum M10. The compound, a methoxychromone derivative, characterized by X-ray diffraction, mass spectrometry, IR, and NMR, displayed antimicrobial and antitumor activities. Graphical Abstract

Intensive use of copper-based pesticides in agriculture can impact human health and biodiversity: the accumulation of this metal in soil negatively affects crop yields. There is growing interest in developing new tools that are capable of monitoring copper occurrence in agricultural contexts through the use of quick and user-friendly approaches for non-specialists, e.g., farmers. This work focuses on the development of a glove-based electrochemical sensor, enhanced with gold nanoparticles, for the detection of copper ions on leaves. The developed device proved was capable of detecting copper ions contained in a copper-based pesticide commonly used in agriculture (Cupravit Bio Advanced). The on-glove analytical device was characterized using garden leaf as the model system and subsequently applied to on-site detection of copper ions on vine leaf treated with Cupravit Bio Advanced. The procedure was very facile: sampling was carried out by touching the leaf with the strip and the stripping-voltammetric measurement was performed by adding a few microliters of an acidic solution to the strip. The on-glove approach allowed evaluation of the level of copper-based pesticide used, avoiding complex and time-consuming tasks. Such operation opens up a wide range of possibilities for improving precision agriculture and sustainable development at the point-of-need for the use of non-specialists.

The use of biostimulants has gained popularity in recent years as a sustainable approach to increase the yield and quality of horticultural crops. However, information is missing concerning their ability to enhance the concentration of some beneficial elements (macro- and microelements) in the edible tissues of plants, which, in turn, are useful for human health. For this reason, we investigated the effects of different microbial and non-microbial biostimulants on the content of essential macro- and micro-nutrients (P, K, Ca, Mg, Cu, Fe, Mn, Zn, Se) in San Marzano and Datterino tomatoes (Solanum lycopersicum L.) and spinach (Spinacia oleracea L.) by atomic adsorption spectrometry, also estimating the Daily Intake (EDI) and the Nutrient Contribution (NC) of fresh produce. All the biostimulants were able to increase the content of macro- and micro-nutrients in the studied horticultural products. Specifically, compared with control, application of Trichoderma harzianum T22 on the Pixel tomato increased Fe, Zn, Cu, Mn and Se contents by 49.66, 38.68, 129.79, 64.03 and 72.72%. In the San Marzano tomato, higher values of Fe (55.16 µg 100 g−1 fw), Mn (30.63 µg 100 g−1 fw), Zn (20.89 µg 100 g−1 fw), Cu (1.91 µg 100 g−1 fw) and Se (0.266 µg 100 g−1 fw) were obtained after application of a tropical plant extract (TPE) biostimulant. Similarly, compared with control, application of a vegetal-derived protein hydrolysate (VPDH) on spinach increased EDI-Fe, EDI-Zn, EDI-Mn, EDI-Cu and EDI-Se by 98.98, 127.09, 125.93, 68.52 and 230.76%, respectively. Therefore, biostimulants, regardless of their origin and nature, could be an ecological tool for biofortification programs for both fruit and leafy vegetables.

Eugenol and linalool are often the most abundant volatile compounds found in basil (Ocimum basilicum L., Lamiaceae) leaves, and they are interesting for the aroma they provide and for their numerous beneficial bioactivities. Their determination is thus needed for several purposes. In the present study, to avoid the previous isolation of essential oil, the direct solvent extraction is proposed coupled with a transmethylation to convert acyl lipids into fatty acids methyl esters (FAMEs), thus assessing the possible simultaneous analysis of eugenol and linalool with FAMEs by gas chromatography coupled to flame ionization detector (GC-FID). The method has been validated and applied to ten basil leaves samples in which eugenol and linalool were found in mean concentrations of 2.80 ± 0.15 and 1.01 ± 0.04 g kg−1 (dry weight), respectively. FAMEs composition was dominated by linolenic acid (52.1–56.1%) followed by palmitic acid (19.3–22.4%) and linoleic acid (9.6–11.3%). The ratio of n6-polyunsaturated fatty acids (PUFAs)/n3-PUFAs was in the range of 0.17–0.20 in the investigated samples. The proposed method exploits a rapid procedure requiring 40 min, making use of a small amount of solvent and allowing the simultaneous determination of molecules contributing to assess the quality of this worldwide appreciated herb.

The cultivation of different species of Eucalyptus has recently expanded in Liguria (Italy) due to the growing demand of the North European floricultural market. Eucalyptus tree branches are cut and selected for their quality, resulting in large amounts of waste biomass to be disposed of. The aim of our study was to evaluate the phytotoxic and antimicrobial activities of essential oils (EOs) from pruning wastes of E. cinerea (EC) and E. nicholii (EN), for potential applications in agriculture. Phytochemical analyses showed eucalyptol (1,8-cineole) as the major component in both EOs, but the EO yield of EN was higher than that of EC, in agreement with a significantly higher oil gland density on EN leaves. EOs from both species showed phytotoxicity on both weeds tested, but no significant inhibition on horticultural crop seed germination, except for Raphanus sativus. The EO from EC showed the strongest antibacterial activity, while the EO from EN showed the strongest antifungal activity. Concluding, EOs from Eucalyptus pruning may be used as possible alternatives to synthetic herbicides and pesticides, acting as antimicrobial and antifungal agents, thus representing a safe strategy for crop management programs.

The contamination of agricultural products with mycotoxins causes risks to animal and human health and severe economic losses. Mycotoxicoses can be reduced by preventing fungal infection using chemical and biological approaches. The chemical strategies can release toxic molecules; therefore, strategies for biological control are being evaluated, such as using nontoxic fungi and their metabolites. This work evaluated the effect of exoenzymes produced by the beneficial fungus Trichoderma afroharzianum strain T22 in degrading Aflatoxin B1 (AFB1) and Ochratoxin A (OTA). The ability of Trichoderma to produce hydrolases was stimulated by using different inducing substrates. The highest AFB1 and OTA degradation activity was obtained using a medium containing lyophilized mushrooms and crude fiber. The T. afroharzianum T22’s ability to reduce mycotoxins may be attributed to peroxidase enzymes. This study showed that T.afroharzianum strain T22 or its peroxidase supplementation could represent a sustainable strategy for the degradation of AFB1 and OTA in feed and food products.

Abstract Background The study describes the Salmonella Rissen phage ϕ1 isolated from the ϕ1-sensitive Salmonella Rissen strain RW. The same phage was then used to select the resistant strain RRϕ1+, which can harbour or not ϕ1. Results Following this approach, we found that ϕ1, upon excision from RW cells with mitomycin, behaves as a temperate phage: lyses host cells and generates phage particles; instead, upon spontaneous excision from RRϕ1+ cells, it does not generate phage particles; causes loss of phage resistance; switches the O-antigen from the smooth to the rough phenotype, and favors the transition of Salmonella Rissen from the planktonic to the biofilm growth. The RW and RRϕ1+ strains differ by 10 genes; of these, only two (phosphomannomutase_1 and phosphomannomutase_2; both involved in the mannose synthesis pathway) display significant differences at the expression levels. This result suggests that phage resistance is associated with these two genes. Conclusions Phage ϕ1 displays the unusual property of behaving as template as well as lytic phage. This feature was used by the phage to modulate several phases of Salmonella Rissen lifestyle.

Kunitz-type (PKPI) and Potato type I (Pin1) protease inhibitors (PIs) are two families of serine proteinase inhibitors often associated to plant storage organs and with well known insecticidal and nematicidal activities. Noteworthy, their ability to limit fungal and bacterial pathogenesis in vivo or to influence plant physiology has not been investigated in detail. To this aim, we generated a set of PVX-based viral constructs to transiently and heterologously express two potato PKPI (PKI1, PKI2) and three potato Pin1 (PPI3A2, PPI3B2, PPI2C4) genes in Nicotiana benthamiana plants, a widely used model for plant-pathogen interaction studies. Interestingly, transgenic plants expressing most of the tested PIs showed to be highly resistant against two economically important necrotrophic fungal pathogens, Botrytis cinerea and Alternaria alternata. Unexpectedly, overexpression of the PKI2 Kunitz-type or of the PPI2C4 and PPI3A2 Potato type I inhibitor genes also lead to a dramatic reduction in the propagation and symptom development produced by the bacterial pathogen Pseudomonas syringae. We further found that localized expression of PPI2C4 and PKI2 in N. benthamiana leaves caused an increase in cell expansion and proliferation which lead to tissue hypertrophy and trichome accumulation. In line with this, the systemic expression of these proteins resulted in plants with enhanced shoot and root biomass. Collectively, our results indicate that PKPI and Pin1 PIs might represent valuable tools to simultaneously increase plant fitness and broad-spectrum resistance toward phytopathogens.

Periodic epidemics of black rot disease occur worldwide causing substantial yield losses. Xanthomonas campestris pv. campestris (Xcc) represents one of the most common bacteria able to cause the above disease in cruciferous plants such as broccoli, cabbage, cauliflower, and Arabidopsis thaliana. In agriculture, several strategies are being developed to contain the Xanthomonas infection. The use of bacteriophages could represent a valid and efficient approach to overcome this widespread phenomenon. Several studies have highlighted the potential usefulness of implementing phage therapy to control plant diseases as well as Xcc infection. In the present study, we characterized the effect of a lytic phage on the plant Brassica oleracea var. gongylodes infected with Xcc and, for the first time, the correlated plant metabolic response. The results highlighted the potential benefits of bacteriophages: reduction of bacterium proliferation, alteration of the biofilm structure and/or modulation of the plant metabolism and defense response.

Seed quality is an important aspect of the modern cultivation strategies since uniform germination and high seedling vigor contribute to successful establishment and crop performance. To enhance germination, beneficial microbes belonging to arbuscular mycorrhizal fungi, Trichoderma spp., rhizobia and other bacteria can be applied to seeds before sowing via coating or priming treatments. Their presence establishes early relationships with plants, leading to biostimulant effects such as plant-growth enhancement, increased nutrient uptake, and improved plant resilience to abiotic stress. This review aims to highlight the most significant results obtained for wheat, maize, rice, soybean, canola, sunflower, tomato, and other horticultural species. Beneficial microorganism treatments increased plant germination, seedling vigor, and biomass, as well as overcoming seed-related limitations (such as abiotic stress), both during and after emergence. The results are generally positive, but variable, so more scientific information needs to be acquired for different crops and cultivation techniques, with considerations to different beneficial microbes (species and strains) and under variable climate conditions to understand the effects of seed treatments.

The growing demand for safer foods reflects the public perception of the adverse consequences of chemicals used in agriculture. This research aimed at developing new biological formulations based on the combination of small microbial consortia containing two Trichoderma spp., with a medium–long chain fatty acids mixture (FAm). The bioactivity of these formulations was investigated on different vegetable crops in terms of biocontrol, growth promotion, yield and quality improvements. FAm application reduced Botrytis cinerea necrosis by up to 90% compared to the infected control plants and some of the assayed Trichoderma spp. + FAm combinations contained Rhizoctonia disease, reaching more than 90% reduction of tomato and lettuce plant mortality. An increasing yield, ranging to 25 and 90%, was recorded on treated tomato, lettuce and kohlrabi compared to untreated plants. A significant enrichment in carotenoids (+60%) and glucosinolates (+39%) was measured on biotreated plants compared to controls. Untargeted LC-MS analysis revealed a higher content of tomatine and dehydro-phytosphingosine, glycoalkaloids involved in defense responses, in Trichoderma spp. + FAm combination-treated plants. The combined biostimulant applications of Trichoderma spp. with FAm were able to improve the performances of different horticultural plant species, producing a premium quality marketable vegetable with higher antioxidant content.

Increasing attention is being given to the development of innovative formulations to substitute the use of synthetic chemicals to improve agricultural production and resource use efficiency. Alternatives can include biological products containing beneficial microorganisms and bioactive metabolites able to inhibit plant pathogens, induce systemic resistance and promote plant growth. The efficacy of such bioformulations can be increased by the addition of polymers as adjuvants or carriers. Trichoderma afroharzianum T22, Azotobacter chroococcum 76A and 6-pentyl-α-pyrone (6PP; a Trichoderma secondary metabolite) were administrated singularly or in a consortium, with or without a carboxymethyl cellulose-based biopolymer (BP), and tested on sweet basil (Ocimum basilicum L.) grown in a protected greenhouse. The effect of the treatments on basil yield, photosynthetic activity and secondary metabolites production was assessed. Photosynthetic efficiency was augmented by the applications of the bioformulations. The applications to the rhizosphere with BP + 6PP and BP + T22 + 76A increased the total fresh weight of basil by 26.3% and 23.6%, respectively. Untargeted LC-MS qTOF analysis demonstrated that the plant metabolome was significantly modified by the treatments. Quantification of the profiles for the major phenolic acids indicated that the treatment with the T22 + 76A consortium increased rosmarinic acid content by 110%. The use of innovative bioformulations containing microbes, their metabolites and a biopolymer was found to modulate the cultivation of fresh basil by improving yield and quality, thus providing the opportunity to develop farming systems with minimal impact on the environmental footprint from the agricultural production process.

Biocontrol fungal strains of the genus Trichoderma can antagonize numerous plant pathogens and promote plant growth using different mechanisms of action, including the production of secondary metabolites (SMs). In this work we analyzed the effects of repeated applications of selected Trichoderma strains or SMs on young olive trees on the stimulation of plant growth and on the development of olive leaf spot disease caused by Fusicladium oleagineum. In addition, metabolomic analyses and gene expression profiles of olive leaves were carried out by LC–MS Q-TOF and real-time RT-PCR, respectively. A total of 104 phenolic compounds were detected from olive leave extracts and 20 were putatively identified. Targeted and untargeted approaches revealed significant differences in both the number and type of phenolic compounds accumulated in olive leaves after Trichoderma applications, as compared to water-treated plants. Different secoiridoids were less abundant in treated plants than in controls, while the accumulation of flavonoids (including luteolin and apigenin derivatives) increased following the application of specific Trichoderma strain. The induction of defense-related genes, and of genes involved in the synthesis of the secoiridoid oleuropein, was also analyzed and revealed a significant variation of gene expression according to the strain or metabolite applied.

Xanthomonas campestris pv. campestris is known as the causative agent of black rot disease, which attacks mainly crucifers, severely lowering their global productivity. One of the main virulence factors of this pathogen is its capability to penetrate and form biofilm structures in the xylem vessels. The discovery of novel approaches to crop disease management is urgent and a possible treatment could be aimed at the eradication of biofilm, although anti-biofilm approaches in agricultural microbiology are still rare. Considering the multifactorial nature of biofilm, an effective approach against Xanthomonas campestris implies the use of a multi-targeted or combinatorial strategy. In this paper, an anti-biofilm strategy based on the use of fatty acids and the bacteriophage (Xccφ1)-hydroxyapatite complex was optimized against Xanthomonas campestris mature biofilm. The synergic action of these elements was demonstrated and the efficient removal of Xanthomonas campestris mature biofilm was also proven in a flow cell system, making the proposed approach an effective solution to enhance plant survival in Xanthomonas campestris infections. Moreover, the molecular mechanisms responsible for the efficacy of the proposed treatment were explored.

Many Trichoderma are successfully used to improve agriculture productivity due to their capacity for biocontrol and to stimulate plant growth and tolerance to abiotic stress. This research elucidates the effect of applications with Trichoderma harzianum strain T22 (T22), or biopolymer (BP) alone or in combination (BP + T22 or BP + 6-pentyl-α-pyrone (6PP); a Trichoderma secondary metabolite) on the crop performance, nutritional and functional quality of greenhouse tomato (Solanum lycopersicum L. cultivar Pixel). T22 elicited significant increases in total yield (+40.1%) compared to untreated tomato. The content of lycopene, an important antioxidant compound in tomatoes, significantly increased upon treatment with T22 (+ 49%), BP + T22 (+ 40%) and BP + 6PP (+ 52%) compared to the control. T22 treatments significantly increased the content of asparagine (+37%), GABA (+87%) and MEA (+102%) over the control; whereas BP alone strongly increased GABA (+105%) and MEA (+85%). The synthesis of these compounds implies that tomato plants are able to reuse the photorespiratory amino acids and ammonium for producing useful metabolites and reduce the pressure of photorespiration on plant metabolism, thus optimizing photosynthesis and growth. Finally, these metabolites exert many beneficial effects for human health, thus enhancing the premium quality of plum tomatoes.

Biofilm protects bacteria against the host’s immune system and adverse environmental conditions. Several studies highlight the efficacy of lytic phages in the prevention and eradication of bacterial biofilms. In this study, the lytic activity of Xccφ1 (Xanthomonas campestris pv. campestris-specific phage) was evaluated in combination with 6-pentyl-α-pyrone (a secondary metabolite produced by Trichoderma atroviride P1) and the mineral hydroxyapatite. Then, the antibiofilm activity of this interaction, called a φHA6PP complex, was investigated using confocal laser microscopy under static and dynamic conditions. Additionally, the mechanism used by the complex to modulate the genes (rpf, gumB, clp and manA) involved in the biofilm formation and stability was also studied. Our results demonstrated that Xccφ1, alone or in combination with 6PP and HA, interfered with the gene pathways involved in the formation of biofilm. This approach can be used as a model for other biofilm-producing bacteria.

The current research elucidated the agronomical, physiological, qualitative characteristics and mineral composition of lettuce (Lactuca sativa L. var. longifolia) after treatments with a beneficial fungus Trichoderma virens (TG41) alone or in combination with a vegetal biopolymer-based biostimulant (VBP; ‘Quik-link’). The experiment consisted of lettuce plants grown in three N conditions: sub-optimal (0N kg ha−1), optimal (70N kg ha−1), and supra-optimal (140N kg ha−1) N levels. Lettuce grown under 0N fertilization showed a significant increase in fresh yield when inoculated with TG41 alone (45%) and a greater increase with TG41 + VBP biostimulant (67%). At 48 days after transplanting, both the TG41 alone or TG41+VBP biostimulant induced higher values of CO2 assimilation in comparison to the control. The mineral concentrations in leaf tissues were greater by 10% for K and 12% for Mg with the TG41+VBP treatments compared to the untreated lettuce. The lettuce plants receiving either TG41 alone or TG41+VBP biostimulants had a significantly lower nitrate content than any of the untreated controls. In non-fertilized conditions, plants treated with TG41+VBP biostimulants produced lettuce of higher premium quality as indicated by the higher antioxidant activity, total ascorbic acid (+61%−91%), total phenols (+14%) and lower nitrate content when compared to the untreated lettuce.

The present study investigated the transcriptomic and metabolomic changes elicited in tomato plants (Solanum lycopersicum cv. Micro-Tom) following treatments with the biocontrol agent Trichoderma harzianum strain M10 or its purified secondary metabolite harzianic acid (HA), in the presence or the absence of the soil-borne pathogen Rhizoctonia solani. Transcriptomic analysis allowed the identification of differentially expressed genes (DEGs) that play a pivotal role in resistance to biotic stress. Overall, the results support the ability of T. harzianum M10 to activate defense responses in infected tomato plants. An induction of hormone-mediated signaling was observed, as shown by the up-regulation of genes involved in the ethylene and jasmonate (ET/JA) and salicylic acid (SA)-mediated signaling pathways. Further, the protective action of T. harzianum on the host was revealed by the over-expression of genes able to detoxify cells from reactive oxygen species (ROS). On the other hand, HA treatment also stimulated tomato response to the pathogen by inducing the expression of several genes involved in defense response (including protease inhibitors, resistance proteins like CC-NBS-LRR) and hormone interplay. The accumulation of steroidal glycoalkaloids in the plant after treatments with either T. harzianum or HA, as determined by metabolomic analysis, confirmed the complexity of the plant response to beneficial microbes, demonstrating that these microorganisms are also capable of activating the chemical defenses.

Microbial inoculants such as Trichoderma-based products are receiving great interest among researchers and agricultural producers for their potential to improve crop productivity, nutritional quality as well as resistance to plant pathogens/pests and numerous environmental stresses. Two greenhouse experiments were conducted to assess the effects of Trichoderma-based biostimulants under suboptimal, optimal and supraoptimal levels of nitrogen (N) fertilization in two leafy vegetables: Iceberg lettuce (Lactuca sativa L.) and rocket (Eruca sativa Mill.). The yield, nutritional characteristics, N uptake and mineral composition were analyzed for each vegetable crop after inoculation with Trichoderma strains T. virens (GV41) or T. harzianum (T22), and results were compared to non-inoculated plants. In addition, the effect of the Trichoderma-based biostimulants on microbes associated with the rhizosphere in terms of prokaryotic and eukaryotic composition and concentration using DGGE was also evaluated. Trichoderma-based biostimulants, in particular GV41, positively increased lettuce and rocket yield in the unfertilized plots. The highest marketable lettuce fresh yield was recorded with either of the biostimulant inoculations when plants were supplied with optimal levels of N. The inoculation of rocket with GV41, and to a lesser degree with T22, elicited an increase in total ascorbic acid under both optimal and high N conditions. T. virens GV41 increased N-use efficiency of lettuce, and favored the uptake of native N present in the soil of both lettuce and rocket. The positive effect of biostimulants on nutrient uptake and crop growth was species-dependent, being more marked with lettuce. The best biostimulation effects from the Trichoderma treatments were observed in both crops when grown under low N availability. The Trichoderma inoculation strongly influenced the composition of eukaryotic populations in the rhizosphere, in particularly exerting different effects with low N levels in comparison to the N fertilized plots. Overall, inoculations with Trichoderma may be considered as a viable strategy to manage the nutrient content of leafy horticulture crops cultivated in low fertility soils, and assist vegetable growers in reducing the use of synthetic fertilizers, developing sustainable management practices to optimize N use efficiency.

Fungi belonging to the genus Trichoderma are among the most active and ecologically successful microbes found in natural environments, because they are able to use a variety of substrates and affect the growth of other microbes and virtually any plant species. We isolated and characterized a novel type II hydrophobin secreted by the biocontrol strain MK1 of Trichoderma longibrachiatum. The corresponding gene (Hytlo1) has a multiple role in the Trichoderma–plant–pathogen three-way interaction, while the purified protein displayed a direct antifungal as well as a microbe-associated molecular pattern and a plant growth promotion (PGP) activity. Leaf infiltration with the hydrophobin systemically increased resistance to pathogens and activated defense-related responses involving reactive oxygen species, superoxide dismutase, oxylipin, phytoalexin, and pathogenesis-related protein formation or activity. The hydrophobin was found to enhance development of a variety of plants when applied at very low doses. It particularly stimulated root formation and growth, as demonstrated also by transient expression of the encoding gene in tobacco and tomato. Targeted knock-out of Hytlo1 significantly reduced both antagonistic and PGP effect of the wild-type strain. We conclude that this protein represents a clear example of a molecular factor developed by Trichoderma spp. to establish a mutually beneficial interaction with the colonized plant.

Trichoderma are ubiquitous soil fungi that include species widely used as biocontrol agents in agriculture. Many isolates are known to secrete several secondary metabolites with different biological activities towards plants and other microbes. Harzianic acid (HA) is a T. harzianum metabolite able to promote plant growth and strongly bind iron. In this work, we isolated from the culture filtrate of a T. harzianum strain a new metabolite, named isoharzianic acid (iso-HA), a stereoisomer of HA. The structure and absolute configuration of this compound has been determined by spectroscopic methods, including UV-Vis, MS, 1D and 2D NMR analyses. In vitro applications of iso-HA inhibited the mycelium radial growth of Sclerotinia sclerotiorum and Rhizoctonia solani. Moreover, iso HA improved the germination of tomato seeds and induced disease resistance. HPLC-DAD experiments showed that the production of HA and iso HA was affected by the presence of plant tissue in the liquid medium. In particular, tomato tissue elicited the production of HA but negatively modulated the biosynthesis of its analogue iso-HA, suggesting that different forms of the same Trichoderma secondary metabolite have specific roles in the molecular mechanism regulating the Trichoderma plant interaction.

Metabolites from a collection of selected fungal isolates have been screened for insecticidal activity against the aphid Acyrthosiphon pisum. Crude organic extracts of culture filtrates from six fungal isolates (Paecilomyces lilacinus, Pochonia chlamydosporia, Penicillium griseofulvum, Beauveria bassiana, Metarhizium anisopliae and Talaromyces pinophilus) caused mortality of aphids within 72 h after treatment. In this work, bioassay-guided fractionation has been used to characterize the main bioactive metabolites accumulated in fungal extracts. Leucinostatins A, B and D represent the bioactive compounds produced by P. lilacinus. From P. griseofulvum and B. bassiana extracts, griseofulvin and beauvericin have been isolated, respectively; 3-O-Methylfunicone and a mixture of destruxins have been found in the active fractions of T. pinophilum and M. anisopliae, respectively. A novel azaphilone compound, we named chlamyphilone, with significant insecticidal activity, has been isolated from the culture filtrate of P. chlamydosporia. Its structure has been determined using extensive spectroscopic methods and chemical derivatization.

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.