Trichoderma: a multipurpose, plant-beneficial microorganism for eco-sustainable agriculture

Trichoderma is a cosmopolitan and opportunistic ascomycete fungal genus including species that are of interest to agriculture as direct biological control agents of phytopathogens. Trichoderma utilizes direct antagonism and competition, particularly in the rhizosphere, where it modulates the composition of and interactions with other microorganisms. In its colonization of plants, on the roots or as an endophyte, Trichoderma has evolved the capacity to communicate with the plant and produce numerous multifaceted benefits to its host. The intricacy of this plant–microorganism association has stimulated a marked interest in research on Trichoderma, ranging from its capacity as a plant growth promoter to its ability to prime local and systemic defence responses against biotic and abiotic stresses and to activate transcriptional memory affecting plant responses to future stresses. This Review discusses the ecophysiology and diversity of Trichoderma and the complexity of its relationships in the agroecosystem, highlighting its potential as a direct and indirect biological control agent, biostimulant and biofertilizer, which are useful multipurpose properties for agricultural applications. We also highlight how the present legislative framework might accommodate the demonstrated evidence of Trichoderma proficiency as a plant-beneficial microorganism contributing towards eco-sustainable agriculture. This Review discusses the ecophysiology and diversity of Trichoderma and the complexity of its relationships in the agroecosystem environment. Considerations are also presented on how to advance Trichoderma applications in real-world scenarios, contributing towards eco-sustainable agriculture.


Ecophysiology and lifestyle changes
The genus Trichoderma demonstrates enormous morphological uni formity and nutritional diversity and has a high number of species members, greater than that found in other fungal groups with similar lifestyles 1,8 . These traits might be attributed to at least four major shifts over the course of evolution in nutritional habits and ecological roles that could have impacted the Trichoderma lifestyle, in which each transi tion has led to notable bursts in species diversification 9,10 (Fig. 1). These shifts were first, from being a parasite of plantdecomposing fungi to being a feeder on decaying plant matter (saprotroph); second, to living in the soil as a saprotroph, with mycotrophic (obtaining nutrients from live or dead fungi) and phytophagic (obtaining nutrients from plants) abilities; third, to feeding on living fungi (mycoparasite); and fourth, to establishing interactions with living plants. Genetic and comparative genomics studies 5,[9][10][11] have demonstrated that Trichoderma are fungi that have constantly reshaped their genome to improve their ability to rapidly colonize and successfully compete in novel habitats. Myco trophy is a very ancient trait of the Trichoderma genus and is a major lifestyle for many of its species, which has facilitated the evolution of its positive interactions with plants 4,5 .
Phylogenomic analysis has shown that the genus Trichoderma shares at least one common ancestor with entomoparasitic hypoc realean fungi 10 , and the most ancient species of the genus evolved around the time of the Cretaceous-Paleogene extinction event (66 million years ago) 11 . These fungi were mycoparasitic on Basidi omycota hosts, from which they acquired genes by horizontal trans fer that subsequently conferred to Trichoderma the ability to grow on dead wood substrates, a lifestyle typical of their targeted fungal prey 5,9,10 . Trichoderma might have made a successive leap from this habitat as a participant in the strong burst of fungal populations sub sequently found as feeders on the decaying biomass of plants killed by the Cretaceous-Paleogene extinction 12 . Most of the carbohydrate hydrolysing genes required for saprotrophic growth as mycotrophs and phytophages were probably acquired 20-30 million years ago, resulting in the diversification and establishment of noted infrageneric sections and/or clades (such as section Trichoderma (ST), section Longibrachiatum, and clades Harzianum and Virens (HV)) 10,11 .
Many soilliving species of Trichoderma developed a distinctive genus characteristic for their capacity to produce a plethora of hydro lytic enzymes (exochitinase and endochitinase) that enabled them to mycoparasitize Ascomycota fungi or other phylogenetically close spe cies (adelphoparasitism), a trait that was rare or absent in the ancestors of Trichoderma 9,10 . Approximately 40% of the vast number of hydrolytic enzymes secreted by Trichoderma have originated by lateral gene transfer from taxonomically close plantassociated ascomycetes 10 . Trichoderma spp. also expanded their ability to parasitize or hyperpara sitize and to obtain nutrients from diverse soilborne organisms such as Phytophthora, Pythium, Rhizoctonia and nematodes 9 . In addition, they developed mutualistic relationships with insects as demonstrated by the protection of termites from infection by entomopathogenic fungi (Metarhizium) 13 . Species diversity in Trichoderma was favoured by gene gains in the taxonomic groups of HV and ST and losses in section Longibrachiatum, coupled with a rare frequency of sexual reproduction and a high rate of conidiation (asexual reproduction) that increased the adaptive variation input by mutation 9 .
Subsequently, the presence of fungal prey and rootderived nutrients probably attracted the most opportunistic Trichoderma species to colonize the rhizosphere, whereby other species became internal plant colonizers or endophytes as the most recent major Introduction Trichoderma (teleomorph Hypocrea) is a genus of filamentous fungi that is capable of feeding on other fungi (mycotrophism) and is a ubiq uitous colonizer in almost all environments (including agricultural, forestry, mountain, grassland and desert ecosystems, and fresh and marine waters); it prevails in any biotope and has an extensive geo graphical distribution worldwide 1 . Trichoderma species grow rapidly on various substrates and are prolific spore producers, easily recognized by the presence of abundant green conidia.
A review in 2004 by Harman et al. 2 presented Trichoderma species as opportunistic, avirulent plant symbiont fungi, and discussed the diverse mechanisms of action employed by the fungus that contribute to its positive impact on plants. Of particular interest were processes involved in the biological control of plant diseases, with direct action on phytopathogens and indirect mechanisms through induction of local and systemic defences in plants. Also noted was the stimulation of root development and plant growth, producing benefits to both the host plant and the invading fungus that resulted in favourable consequences for agriculture. Harman et al. 2 described the plant-microorganism interaction as intricate, involving multifaceted crosstalk modulated by root colonization and the plethora of compounds produced by Trichoderma that activate biochemical and genetic pathways determining plant defence responses to biotic and abiotic stresses.
The advancement of omics investigations 3,4 has resulted in increased understanding of the ecological events involved in the evo lutionary progression of Trichoderma fungi from common soil dwell ers growing on decaying organic matter (noted for their outstanding saprotrophic action on dead fungi and oomycetes), to mycoparasitism of other fungi (including those of taxonomically close species) and to interactions with plants that involve colonization of the rhizosphere and endophytism 5 . Omics approaches have also served to unveil the processes and regulation dynamics of the beneficial effects of Trichoderma to plants 6,7 that are of agricultural interest. The application of molecular methods for species identification and classification has resulted in the exponential expansion of Trichoderma taxonomystarting 50 years ago with only 9 species aggregates described for the genus to >400 species recognized today 8 (Box 1).
Research interests have since expanded towards an integrated analysis of the multipurpose properties of Trichoderma as fungi ben eficial to plants for applications and improvement of agricultural production. This variation in research objectives can be attributed to ongoing changes in agricultural policies and management over time, with an increasing focus on sustainability for the future. Trichoderma contributes positive effects to the agroecosystem; thus, considerations are being given to the role that this fungus has in innovative agricultural strategies as an established and accepted biotechnological tool.
In this Review, we present the latest advances in Trichoderma research, including ecophysiology and lifestyle changes that result in species diversification; opportunism and competition (discussing the complex relationships between plants and microbiota); use of the fungus as a direct biological control agent (BCA) in crop protection; indirect BCA effects stimulating plant immunity; and the capacity of Trichoderma as a plant biostimulant, both in promoting plant growth and activating defence against abiotic stress. Considerations are also presented on how to advance Trichoderma applications in realworld scenarios, bio formulation improvements and policy deliberations. We conclude by discussing how the use of Trichoderma could maximize opportunities for reducing chemical inputs, thus providing cleaner resources and healthier prospects for a more environmentally sustainable agriculture system.

Review article
evolutionary event of the genus 5,9,11 (Fig. 1). Three key features have contributed to Trichoderma becoming an endophyte 9 as a result of its progressive 'intimacy' in plant-microbiota interactions: as a sapro troph, it initiated the decay-degradation process after plant host death; as a mycoparasite, it was capable of parasitizing the primary fungal decomposers that colonized the vegetative tissues; and as a mutualistic nonpathogenic symbiont, it was able to interact and communicate with the living plant host, providing benefits such as growth promotion and protection against biotic and abiotic stresses. Over time, Trichoderma became an opportunistic plant colonizer, developing mechanisms that enabled it to overcome plant defences and not be recognized as a foe.

Opportunism in the rhizosphere
Trichoderma opportunism is evidenced by the ability of this fungus to colonize a wide range of habitats, employing a combination of traits to compete for space and nutritional resources, resist environmental stresses, repair cell damage, and modify the ecological living conditions to its advantage (for example, by detoxifying noxious compounds or changing the substrate pH). The substantial increase in Trichoderma growth in the presence of pectin, xylan or other mucigelreleased sub stances supports the notion that rootderived nutrients are attractors for Trichoderma, providing incentives for this microorganism to physi cally colonize roots 14,15 . The production of reactive oxygen species (ROS) in Trichoderma has been linked to antagonism against phytopathogens containing cellulose in their cell wall, such as Pythium ultimum 16 , as well as the finetuning of molecular crosstalk communication with plants that establishes beneficial effects 17 . Furthermore, tomato root exudates obtained from plants subjected to various biotic and abiotic stresses (such as pathogen attack, wounding or salt) were enriched with ROS and oxylipins, which were capable of stimulating growth and acting as selec tive chemoattractants to Trichoderma 18 . Trichoderma can also enhance antioxidant defence in plants subjected to abiotic stresses, resulting in a decrease of ROS levels in the plant and thus limiting tissue damage 19,20 . Compared to other filamentous fungi, Trichoderma has its own robust antioxidant system with a potential role in protecting genome stabil ity by elimination of ROS 4 . Evidence suggests that H 2 O 2 and oxylipins produced by Trichoderma atroviride might act as signal molecules in response to injury and cell damage (as in plants and animals) 21 .
The photoreactivation system that repairs DNA damage caused by UV radiation and is involved in the regulation of carbon and nitro gen metabolism in response to light has been studied extensively in Trichoderma; the ENVOY photoreceptor of T. atroviride, a repres sor of blue lightinduced genes, modulates the expression of genes involved in DNA repair, acting as a growth and conidiation check point 22 . The high opportunistic ability of Trichoderma might also be a consequence of the activation of a complete range of heat shock proteins that confers tolerance to cold, heat, oxidative, osmotic or saline stresses 23 . Trichoderma genomes exhibit a high number of genes that encode for ATPbinding cassette (ABC) transporters 4 , which could bestow increased tolerance to toxic compounds present in the rhizo sphere 24 . Trichoderma also secrete siderophores 25,26 , which may help them to compete in the rhizosphere and to solubilize phosphates 27 . The application of selected Trichoderma strains able to solubilize diverse phosphate sources enhanced phosphate uptake by plants, resulting in increased growth promotion 28 . Volatile organic compounds (VOCs), such as 6pentyl2Hpyran2one (6PP), have known antibiotic activity, and, at low doses (similar to those expected to be released by Trichoderma in nature), they may act as signalling molecules to modulate seed germination, plant growth, root architecture and immune responses in the absence of direct physical contact 29,30 .
Trichoderma genomes harbour phytohormone genes 31 involved in the production of auxins, gibberellins, abscisic acid, salicylic acid or cytokinins that, in a straindependent and/or culture medium dependent manner, have been linked to hyphal growth, root coloni zation, activation of the plant antioxidant machinery and promotion of plant performance under abiotic stress 20,32 . The role of auxins produced by Trichoderma has not been established beyond doubt, but these com pounds have been linked to root hair initiation and development effects

Box 1
Trichoderma taxonomic scenario Persoon (1794) proposed the genus name Trichoderma (teleomorph Hypocrea) for a group of wood-decaying filamentous fungi that were producers of green asexual conidia masses at hyphal termini. These fungi are now classified as Division Ascomycota, Order Hypocreales and Family Hypocreaceae. The pleomorphism of this fungus led to the use of a dual nomenclature 1 ; however, the sexual form (Hypocrea) is rarely found in nature, barely growing on plant debris and on specific basidiomycete fungi present in decaying bark, whereas the asexual form (Trichoderma) is abundant. Following the principle of 'one name, one species' 159 , after a vote by the International Subcommission on Trichoderma and Hypocrea taxonomy (www.trichoderma.info), Trichoderma was preferentially chosen by 54 votes to 22 over Hypocrea as the name to be adopted, and this genus name has been officially in use since 2013 (ref. 160 ).
Today, the genus Trichoderma consists of >400 species (375 with valid nomenclature as of 2020) characterized by in vitro cultures, DNA barcoding and integration of publicly available wholegenome sequences 8 . DNA-based analysis has resulted in both the simplification and the complication of species classification and nomenclature for Trichoderma 8 . A species identification system has been elaborated by sequencing the ribosomal RNA locus internal transcribed spacers ITS1 and ITS2, different fragments of the tef1 gene (encoding the translation elongation factor 1α), together with a species confirmation marker based on a fragment of the rpb2 gene (encoding the second largest subunit of RNA polymerase II) 8 . In the present Trichoderma taxonomic framework, some species names have become obsolete, and it might be misleading to assign biological activities, such as the production of certain metabolites, to species described with an old name. For example, DNA barcoding identification of the active substances in four commercial biocontrol products determined that none were Trichoderma harzianum as indicated on the label 63 . Indeed, the worldwide marketed strain T. harzianum T-22 (ref. 2 ) was renamed as Trichoderma afroharzianum after DNA barcoding analysis 63 . Therefore, the systematic reform of Trichoderma taxonomy might produce technical complications as some species names indicated in patents and commercial registration dossiers might need nomenclature revisions.

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in plants 33 . Trichoderma might also stimulate plant auxin transport and signalling, resulting in plant growth promotion 33 . Notably, excessive accumulation of plant auxins can have the opposite effect, as rhizo sphere acidification induced by Trichoderma can lead to inhibition of root growth through an auxindependent mechanism 34 .
Particularly interesting is the role of nonsecreted molecules located in the cell wall of Trichoderma as well as the relative proportions of com ponents in the cell membrane. For instance, the cysteinerich cell wall protein QID74 of Trichoderma harzianum enhances the formation and elongation of root hairs, thus increasing the absorptive surface area plus translocation efficiency of nutrients into the shoots, resulting in more plant biomass 35 . The balance of structural squalene and ergosterol is nec essary to maintain cellular membrane stability in fungi and is important for the ability of Trichoderma to colonize the roots as well as having a criti cal role in the regulation of plant defence mechanisms and biocontrol action 36 . The prolific endophytic colonization by some Trichoderma is not only a direct biocontrol mechanism that prevents pathogen coloni zation in the host but it can also produce plantbeneficial effects, such as increased photosynthetic capacity and growth promotion, accom panied by an increased tolerance to biotic and abiotic stresses [37][38][39] . For example, colonization of the apoplast of olive tree roots by Trichoderma prevents the pathogen Verticillium dahliae from accessing the vascular bundles, thus providing effective disease control of Verticillium wilt 40 . However, endophytic colonization by Trichoderma does not guarantee a corresponding beneficial effect for the plant 20 .

Microbiomes and non-target microorganisms
The evolutionary leap to colonize the rhizosphere and plant roots, cou pled with antagonism or biocontrol action, suggests that Trichoderma is an intrepid conqueror of ecological niches, a determined competi tor and an aggressive antagonist. Therefore, it is important to investi gate and evaluate the impact that Trichoderma strains might have on nontarget organisms and plants as well as the effects in given soil and rhizosphere environments. Trichoderma has been proposed as a marker of healthy soils 41 . A core Trichoderma biome seems to be present in both endemic and cosmopolitan plants from different continents, in which endemic plant populations harbour a substantially higher proportion of antagonistic Trichoderma species 42 . The cultivation systems used and the crops cultivated can have diverse effects on soil properties, which in turn influences fungal diversity (for both pathogenic and beneficial fungi) [43][44][45] . Microbiome diversity is highest in bulk soil and decreases in rhizosphere and endosphere samples 44,45 . However, with inoculations of Trichoderma, prokaryote and eukaryote populations are modified in bulk soil and in the two rootassociated compartments. The use of Trichoderma strains alone or in combination with organic compost in crop plants has been reported to maintain plant growth and cause changes in the structure and function of microbial communities in the rhizosphere in terms of microbial community composition and effect on phosphorus solubilization (resulting in total rhizosphere soil microbial community changes and stimulation of potentially beneficial microbial consortia) 46,47 . Diverse organic amendments added to soil differentially influence the growth and disease suppression capacity of microorgan isms, including beneficial fungi such as Trichoderma, and increase plant root proliferation 48 . Trichoderma can aid in the maintenance of microbiome diversity when growth conditions are compromised as was observed when the application of Trichoderma alone increased the number and diversity of many genera of beneficial plant bacteria in the wheat rhizosphere after the microbiome had been negatively affected by high doses of inorganic nitrogen fertilizers 45 or, similarly, when dual inoculations with Trichoderma and endophytes were found to enrich the microbiome of plants subjected to drought stress 49 .
The compatibility of Trichoderma with mycorrhizal fungi has been frequently questioned for three reasons: first, the high mycoparasitic potential of Trichoderma and its attack on arbuscular mycorrhizal fungi (AMF) in vitro 50 ; second, the distinct and Trichoderma species dependent VOC emission profiles against ectomycorrhizal fungi 51 ; and third, the ability of AMF to compete for nutrients and colonization sites and boost the systemic defences of plants 52 , which might potentially hinder Trichoderma colonization in the rhizosphere. The behaviour of AMF reflects their ability to induce the systemic defences of plants and means that they can be effectively considered as indirect BCA. However, despite assumptions that Trichoderma and mycorrhizal fungi could not be applied simultaneously, their combined use has been noted to increase crop yields 53 , and greenhouse studies have proven their compatibility when applied together to tomato seedlings 54

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nonmycorrhizal Brassica host that improved rapeseed productivity 56 , and a single application of Trichoderma increased the levels of AMF in the wheat rhizosphere 45 . Importantly, compatibility studies need to be conducted for these two beneficial fungi and evaluated on a case bycase basis, bearing in mind that root colonization by Trichoderma is much faster than that of mycorrhizal fungi. Some Trichoderma spp. have been noted to produce harmful effects, including T. aggressivum, T. pleuroti and T. pleuroticola, which have been reported as pathogens of edible mushrooms 8,57 . The produc tion of the trichothecene toxin trichodermin by Trichoderma brevicompactum resulted in phytotoxic effects on tomato plants, thus invalidating the mycotoxinproducing strains as BCAs 58 .
With the ability to grow at high temperatures (37 °C), Trichoderma longibrachiatum has been noted as a human opportunistic pathogen in patients who are immunocompromised 59 . Fortunately, the undesirable activities observed with some Trichoderma do not correspond to those species commonly used in agriculture and commercially marketed. In addition, as a prerequisite for product registration, the microorgan ism must be tested and certified as not having any potential negative health effects.
Interestingly, Trichoderma can have deleterious effects on leaf cutting ants (on the fungal gardens they grow for feeding as well as on their survival) owing to antagonism with their fungal symbiont. How ever, the endophytic colonization by Trichoderma of the plant mate rial transported to the ant nests can act as an effective 'Trojanhorse' strategy that results in beneficial effects to the plant as it provides protection from these damaging agricultural and forestry pests 60 .

Direct biocontrol in crop protection
Trichoderma can be considered as a multipurpose BCA owing to its combined potential actions, with direct antagonism to the target organ ism (Supplementary Table 1) and indirect activities through the plant host to stimulate a defence response to a multitude of biotic stress factors. The direct action of Trichoderma as a BCA (Fig. 2) has been extensively addressed in many reviews, including that by Harman et al. 2 . However, it is important to note that not all Trichoderma species or strains have the same capacity for pathogen or pest control, respond equally to diverse crops or cultivars 61,62 , function effectively in different geographic locations, or are able to maintain a consistent standard level of protection in all field conditions or over extended timeframes. In par ticular, the strains of Trichoderma that are of interest to agriculture 63 are principally distributed among species of the infrageneric groups ST (T. atroviride, T. gamsii, T. viride, T. asperellum and T. asperelloides) and HV (T. harzianum sensu lato, T. afroharzianum, T. guizhouense and T. virens) 8 (Box 1). In general, efficacy as a BCA depends on the biologi cal characteristics of the Trichoderma strain, including rapid growth, prolific sporulation and opportunistic colonization of the environ ment, as well as on the biochemical arsenal of host cell walldegrading enzymes (CWDEs) 2,4,64 , cumulative secondary metabolites 65,66 and released VOCs 67 (all of which affect interactions with the host plant 68 , influence the soil microbiome 51,69 and subsequently affect the biocontrol of plant attackers 70 ).
The antagonism or direct biocontrol activity by Trichoderma of plantdamaging organisms can be attributed to the following five principal mechanisms 2,71 : parasitism, whereby Trichoderma is a preda tor that obtains nutrients from the target prey (that is, if the prey is a fungal phytopathogen, then Trichoderma is a mycoparasite feeding on a fungal disease agent); antibiosis, by production of secondary metabo lites that inhibit competitors, limiting and impeding microorganism proliferation or plant pathogen attack; enzymatic activity (for example, chitinases) and production of secondary metabolites with biological activity against nematodes and insect pests; competition for ecological niches and resources (such as soil and mucigel nutrient uptake, ROS tolerance, growth on roots) that contribute to Trichoderma coloniza tion of the soil, rhizosphere and endosphere (endophytism); and the production and release of VOCs can attract parasitoids and predators of insect pests. Moreover, a sixth mode of action for biocontrol (which is indirect rather than direct) involves the induction of immunity in the plant host, whereby Trichoderma activates plant defence responses and mechanisms that provide protection against biotic and abiotic stress (discussed below).
Trichoderma produces >120 different types of secondary metab olite, with the most relevant chemical structures being terpenes, pyrones, polyketides and nonribosomal peptides. Some of these sec ondary metabolites possess antibiotic activity, inhibiting the growth and multiplication of fungi 66,83 , oomycetes 84,85 and bacteria 66,73 . The application of purified secondary metabolites has biocontrol effects on target pathogens comparable to those obtained by using the living Trichoderma producer 86 . Secondary metabolites that can permeabilize cell membranes might work synergistically with CWDEs to promote cell disruption 87 . Although Trichoderma might be considered a necro trophic mycoparasite that destroys its prey, microscopic evidence 88 demonstrates that the fungus might instead penetrate through open holes in the cell wall and not extensively damage the prey, thus using what might be called a hemibiotrophic parasitic mode of action 75 .
Trichoderma has long been noted to have suppressive effects on Meloidogyne rootknot nematodes (RKN) 89 . Similar findings have been confirmed for other nematodes (Heterodera, Haemonchus, Pratylenchus or Globodera), whereby inhibition occurs via Trichoderma para sitism 90 , egg lysis by proteases 91 and chitinases 92 , or suppression of egg hatching by secondary metabolites 73 . Furthermore, Trichoderma has demonstrated direct biocontrol of insects through enzymatic activity on the midgut peritrophic matrix 93 and inhibition of cuticle formation 94 . In addition, extracts of secondary metabolites can have inhibitory effects on insect larvae 95 .
As competitors in the plant environment, Trichoderma spp. can interfere with or counteract the attack strategies used by phytopatho gens to invade plants. For example, proteases secreted by Trichoderma can inhibit enzymes produced by pathogens to disrupt plant tissues for penetration 96 . Secondary metabolites produced by Trichoderma can downregulate the expression of pathogen genes involved in the pathogenicity process; for example, polyketides released by Trichoderma arundinaceum can modulate the phytotoxic sesquiterpenes

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of Botrytis cinerea that are involved in virulence and growth 97 . A final biocontrol mechanism that is considered a direct effect by Trichoderma and not via the plant is the production of VOCs such as 6PP, which are released in the environment and are able to attract parasitoids and predators of insect pests 98 .

Indirect biocontrol and priming of defences
Trichoderma acts as an indirect BCA by activating plant immune responses (Fig. 3), resulting in a faster and stronger induction of defence mechanisms upon perception of a subsequent triggering stimulus (Box 2); this form of defence is known as priming 99 . By induc ing priming, Trichoderma can provide the plant with longlasting defence through the balance of different phytohormonedependent pathways 6 . Priming is not exclusively associated with indirect biocon trol as reinforcement of plant responses to biotic and abiotic stresses are very similar in their genesis and establishment, although they are activated by stimuli of very different nature. The molecular interaction between Trichoderma and plants, and the manner in which signals are activated and systemically transmitted, have been the subject of several reviews 6,7,100 . Structural components of the Trichoderma cell wall and membrane (for example, chitin, βglucans and sterols) act as microorganismassociated molecular patterns (MAMPs) 36  acidifies the soil and affects the structure and abundance of microbiota in soil and root compartments; in addition, its ability to take up nutrients, tolerate ROS and grow on roots, and its compatibility with other plantbeneficial microorganisms, enable it to compete successfully in the soil (4a), rhizosphere (4b) and endosphere (4c). Trichoderma colonization of the rhizosphere and root tissues (endophytism) inhibits the occupation of these spaces by potentially pathogenic microorganisms and nematodes. (5) VOCs produced by Trichoderma can attract parasitoids and predators of insect pests.

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other microorganisms 104 might function as damageassociated molecu lar patterns, which, once recognized by patternrecognition receptors (PRRs), activate MAMPtriggered immunity, which is stronger than pathogentriggered immunity, thus conferring plant resistance 3 .
The plant reacts to the arrival of Trichoderma at the roots with an increase in levels of salicylic acid (a key phytohormone that controls early root colonization) to limit Trichoderma to the apoplastic space of the epidermis and cortex 40,105 . Next, Trichoderma increases the level of a second layer of plant immunity by means of an array of apoplastic effector proteins and metabolites such as xylanase EIX 106 , LysM protein Tal6 (ref. 107 ), ceratoplatanin Sm1 (ref. 108 ), the peptaibol alamethicin 109 , and the terpenes trichodiene 110 and harzianum A 111 , among others 112 . The apoplastic effectortriggered defence is considered effector triggered immunity regardless of whether it is activated at the PRR level.
The secreted effectors 113 , together with ROS tolerance, might ena ble endophytic colonization and allow Trichoderma to establish an avirulent relationship with the plant and longlasting priming that keeps plant response at or just below the threshold for effective resist ance 3 (Box 2). How the cytoplasmic nucleotidebinding site leucine rich repeat (NLR) receptors interact with the effectors released by Trichoderma is not well understood. In tomato plants, NLR receptors are overrepresented in the leaf proteome of plant roots inoculated with T. atroviride and Rhizoctonia solani 114 , and NLR genes have also been induced by harzianic acid released by T. harzianum 86 . NLRlike proteins are upregulated in the leaf maize proteome after the inoculation of roots with T. afroharzianum 115

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are increasingly being studied and understood, including the potential role of bidirectional crosskingdom small RNA trafficking 7,112 . The salicylic aciddependent defences that limit early growth of Trichoderma can spread throughout the plant, constituting a defence model known as systemicacquired resistance, which has been found to be effective against biotrophic pathogens 116 . Trichoderma suppresses salicylic acid defences and induces jasmonic acid biosynthesis and jas monic acidresponsive genes, which are expressed in the root cells, thus

Box 2
Plant defence layers and the role of Trichoderma in priming defence Plant cells individually auto-defend from invaders and then forward the defence signal to neighbouring cells, resulting in systemic transmission to the entire plant. The first layer of innate defence is regulated by pattern-recognition receptors (PRRs) situated on the plant cell surface that perceive structural components of the invaders known as pathogen-associated molecular patterns, microorganism-associated molecular patterns (MAMPs) and damageassociated molecular patterns (DAMPs; which are small molecules resulting from the hydrolytic action of the attackers on the plant or released by the action of biological control agents on their prey). MAMP-triggered immunity (MTI) or DAMP-triggered immunity is transmitted and amplified through cascades of mitogen-activated protein kinases, which convert external stimuli into intracellular responses, resulting in transcriptional reprogramming that leads to plant cell wall fortification; increases in levels of intracellular calcium; production of reactive oxygen species, antimicrobial secondary metabolites and pathogenesis-related proteins; and accumulation of defence phytohormones such as salicylic acid, jasmonic acid and ethylene. Attackers can overcome and suppress MTI by deploying specific effector proteins into the host cytoplasm. Plants activate a second specific defence layer, known as effector-triggered immunity (ETI), following cytoplasmic effector recognition by nucleotidebinding site leucine-rich repeat protein (NLR) receptors. ETI is quicker and more intense than MTI and is associated with early oxidative burst and hypersensitive response cell death to prevent the invasion of pathogens.
Priming activated in the plant by Trichoderma (see figure) follows a different defence dynamic (green arrows) to the untreated control plant (red arrows). PRRs recognize Trichoderma MAMPs and host plant or Trichoderma prey DAMPs, which increases the level of MTI. Trichoderma induces a stronger and more intense defence than MTI via apoplastic effectors, which are also recognized by PRRs (ETI). The plant enters a state of priming in which defence responses are not activated but remain 'alert' around the threshold for effective resistance. With a stress challenge, there is a faster and stronger induction of plant defence from a level of resistance greater than that of the control plant. When the stress ceases, the Trichoderma-treated plant enters a post-challenge priming state in which the defence level is once again maintained around the threshold for effective resistance. The response events are stored in the 'transcriptional memory' of the plant, which discriminates between single and repeated stresses, and can modulate transcription of response genes to future stress during the current lifetime of the plant (to the dotted line). Subsequently, offspring from the Trichoderma-treated plant acquire an inherited memory, whereby, when exposed to stress, they can activate heritable priming also at a level of effective resistance.

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generating a signal that spreads systemically 102 . This defence model is known as jasmonic acid-ethylenedependent induced systemic resist ance and is especially effective against necrotrophic pathogens and herbivore attack 116 . To colonize the roots, Trichoderma takes advan tage of the antagonism between salicylic acid and jasmonic acid 102 . Pioneering work demonstrated that cucumber roots colonized by T. asperellum accumulated substantial levels of jasmonic acid and ethylene in 24 h (ref. 117 ), supporting the notion that priming activated by beneficial microorganisms conforms to an induced systemic resistance response 116 . However, evidence suggests that Trichodermatriggered defences against pathogenic fungi and even viruses are modulated by both jasmonic acid-ethylene and salicylic acid 111,118,119 . Improved plant fitness, as promoted by Trichoderma, also prevents nematode access to the roots 120 . In tomato roots in which RKN complete their life cycle, Trichoderma reprogrammes plant immunity by adapt ing salicylic aciddependent and jasmonic aciddependent defences according to the nematode infection stage 121,122 . As a general mechanism, Trichoderma also primes defences by different means and at diverse frequencies in leaves and roots via small RNAmediated gene silencing and by inducing the transcription of core components of the RNA dependent DNA methylation machinery that sharpens the expression of both salicylic acid and jasmonic acid-ethylene defencerelated genes 123 .
Another indirect biocontrol mechanism of Trichoderma is the activation of plant systemic defences by VOCs, in which their release results in an oxidative burst that is effective against aphids 124 . Trichoderma can also enhance the expression of genes encoding for protec tive enzymes against moths 125 . Furthermore, Trichoderma is able to alter plant metabolic pathways leading to the induction of various plant systemic defences 126 such as the production of phytocompounds that act as antifeed deterrents 127,128 or negatively affect the insect gut proteome equilibrium 129 , activate the release of plant VOCs with high attractivity to parasitoids and predators of aphids 124,130 , or decrease feeding by herbivorous insects 131 .
The speed and efficiency at which plants adapt to their surround ings might also be facilitated by Trichoderma given that it has a role in balancing defences and growth as well as alleviating the effects of unfavourable environmental conditions. Abiotic stressmediated phyto hormones share common regulators with MAMPs and/or damage associated molecular patterns. However, in natural settings in which plants are exposed to a mix of stimuli, PRR pathways display a substantial divergence in sensitivity to biotic or abiotic perturbations and signal transduction. Given that abiotic stresses modify water fluxes, solute con centrations and ion homeostasis, the Ca 2+ pumps or channels required for PRRdependent defences have an important role in regulating poten tial gradients across membranes and conditioning plant immunity 132 . Abiotic stress sensing induces cytoplasmic Ca 2+ accumulation, leading to an extracellular ROS burst and activation of Ca 2+ dependent protein kinase cascades; these mechanisms enable the plant to cope with the variation in ambient conditions by prioritizing functions involved with plant growth regulation and responses to environmental stresses 132 . A hydrophobin secreted by Trichoderma triggers the plant Ca 2+ signalling pathway 133 , which opens up an interesting line of research on G protein recognition and signalling activation and/or deactivation that is compatible with the effects that Trichoderma has on plants 7 .
Early studies described how Trichoderma favours the production of plant metabolites associated with increased drought tolerance 37,134 and how its enhancement of antioxidant defence delays the onset of a water deficit response 19 . Numerous reports illustrated that Trichoderma could affect ROS scavenging and has 1amino1cyclopropanecarboxylic acid deaminase (ACCD) activity leading, respectively, to activation of plant antioxidant machinery and regulation of plant ethylene levels under drought, waterlogging, and osmotic, salinity, chilling, or heat stress [135][136][137][138] . Trichoderma also increased plant growth and salt tolerance by direct contact 102 or through VOCs 139 . However, the promotion of growth and development triggered by the combination of Trichoderma and inorganic fertilizers to saltstressed plants led to a dysregulation of the phytohormone network as overstimulated plants in suboptimal conditions were unable to adapt to the contradictory signalling 140 . With out a doubt, plant-Trichoderma crosstalk is dynamic and the expres sion of salicylic acid and jasmonic acid-ethylenedependent defence genes might overlap in an undulating pattern that responds to both biotic and abiotic stresses 14,141 . This plant effect disappears over time, becoming imperceptible several weeks after the plant has been in con tact with Trichoderma 142 . Given that the plant-Trichoderma interaction varies with timing and the plant and/or fungal speciesstrain involved, time course studies are needed to determine which transduced signal for each specific plant response is prevalent at a given time.
Once the Trichodermaactivated priming signals gradually dis appear, the plant activates a 'transcriptional memory', in which cells previously primed by a particular stimulus show increased rates of gene expression upon subsequent restimulation 143 . The capacity for defence priming can be inherited, generating a second level of memory known as 'heritable priming', that can be passedon to the offspring (Box 2). These nextgeneration plants express a stronger defence response than offspring of unprimed plants 144 . The beneficial action that Trichoderma has on plants is modulated by molecular networks that condition the immediate and longlasting systemic responses, orchestrating the metabolic tradeoffs between plant growth and defence 7 ; for example, in the Trichoderma-tomato-RKN interaction, the tomato progeny inherit both resistance to RKN as well as growthpromotion effects without compromising the level of defence in the plant offspring in responding to the nematode attack 121 .

Applications in agriculture
Modern agriculture policies have been radically changed by the Sus tainable Development Goals of the 2015 UN General Assembly, which were later focused on food and agriculture as the key factors to address concerns regarding fertilizer handling, pesticide use and management practices 145 . Climatic changes and intensive agricultural practices have created biodiversity loss, changes in the geographic distribution of plantdamaging pests and pathogens, and contamination of soil, air and water resources by chemicals that negatively impact not only the agroecosystem but also human health 146 . Progressive modifications in agricultural policies are aimed at reducing the use of synthetic chemical products; thus, the growing importance of plantbeneficial Trichoderma in this process is apparent from its increased use as a biological alternative to agrochemicals and the intensified research linking the fungus to 'sustainable agriculture' as noted in the recent lit erature searches conducted for this review 38,44,48,71,72 . Trichoderma has become a popular protagonist as the key component of plant biostimu lants, bioprotectants, biofertilizers, soil amendments, soil integrators, biodegraders and bioremediators 147 (Fig. 4; Supplementary Table 2).
Trichoderma has remained as a renowned BCA of phytopathogens and as a mycoparasite that uses direct antagonism and other mecha nisms in the biocontrol of important plant diseases. Therefore, it is of no surprise that Trichoderma is the active BCA substance in many commercial preparations registered as plant protection products (PPPs). In general, authorization as a microbial BCA can only be Review article provided by the appropriate designated institutions after passing a rigorous evaluation process (for efficacy and safety) to support its claims as a PPP, a procedure following that used for chemical phy tosanitary products. The usefulness of Trichoderma is demonstrated by the increase from 21 BCA registrations, with strains of 8 Trichoderma species, worldwide in 2014 (ref. 72 Table 2). This survey indicated that Brazil has the most active market (28% of total registrations), followed by Colombia (18%), then consolidated European Union (15% in 22 countries). The information provided about the manufactured products from India and China, the biggest Asian consumers of Trichodermabased products, as well as the developing markets in Central and South America are not fully complete owing to the diverse registration procedures used by the regulatory authorities in comparison to the European and North American counterparts. For instance, in India, many governmental research institutions are funded to isolate and test 'agriculturally important microbes' for bioefficacy and to then develop the dossier for registration by the Central Insec ticides Board and Registration Committee. Any company can buy the dossier, strain, technology transfer and training, submit for registra tion, and then manufacture their own product. Currently, in India, this process is only possible for the species T. viride and T. harzianum. The product claims for the Trichoderma microbial biofungicide correspond to the control of phytopathogens as previously mentioned for direct BCAs and for use in a large variety of crops, including vegetables, field crops (such as wheat, rice, bean and soya), soft fruits, ornamentals and flowers, herbs and aromatics, golf course turf, arboriculture, coffee, orchards and grapevines (Supplementary Table 2).
Although many Trichoderma species and strains have been reg istered for use as PPPs and have recognized plant growthpromotion effects, by definition, they cannot be registered or commercially dis tributed as plant biostimulants in Europe 147 . The regulatory frame work varies by country and, in some nations, Trichoderma strains are allowed to be marketed with claims as plant inoculants, strengtheners or biostimulants, irrespective of whether the active substances exert direct or indirect biocontrol and without an evaluation process verify ing efficacy. Policymakers are still investigating whether the multipur pose use of Trichoderma species, as both PPPs and biostimulants, is possible and how this can be regulated given that the capacity of many Trichoderma strains to exert indirect biocontrol and to act as biostimu lants is determined by the plant host, depending upon which process the plant exploits (defence versus growth), and by various stimuli in the agroecosystem. This dilemma with Trichoderma is an important issue to be faced for future regulation revisions. Given that scientific evidence supports the validity of Trichoderma applications both for biocontrol and biostimulation effects together (and just singly), it is time to unify the legal framework regarding these nonharmful and agriculturally useful fungi, defining them as 'plantbeneficial microorganisms'.

Eco-sustainable agriculture
To increase the general success and implementation of biological prod ucts in eco-sustainable agriculture, the focus needs to be on improving their shelf life, efficacy and standards to a level similar to that of chemical products used to date 71 . Strategies aimed at improving the yield of conidia and chlamydospores and their stress tolerance are of great importance for the development of costeffective and durable agricultural applica tions of Trichoderma. Technologies to produce an ideal Trichoderma product for agriculture should exploit its multipurpose assets by select ing species or strains that have potential biocontrol, rhizosphere compe tence, endophytic colonization characteristics, and can induce disease resistance and/or promote plant growth (Box 3 and Fig. 4) Table 2). Bioformulations could con tain living Trichoderma plus bioactive compounds from other microbial and/or botanical sources (such as algae or phytoextracts) or with natural carriers to improve application efficacy 72 . For example, Trichoderma, its secondary metabolites (such as 6PP), phytohormones, plant extracts and polymers (such as cellulose, galactomannan or chitosan) have been tested successfully for biocontrol and/or plant growthpromotion effects [148][149][150] . To this end, the search, selection and practical use of synthetic com munities generated from Trichodermabased root microbiomes 20 or synthetic communities from Trichodermafostered microbiomes 47 will be important in developing new generation biofertilizers and agricultural probiotics to aid microbiota recruitment or restoration, particularly where intensively cultivated lands suffer from soil fatigue.
With regard to future agricultural mandates aimed at reducing the use of chemical products in farm management, the selection of Trichoderma strains tolerant to agrochemicals and compatible with inorganic fertilizers will be a useful strategy 151 . Industrial processes can use technologies to improve Trichoderma qualities by inducing the production of bioactive compounds responsible for the benefi cial effects in agricultural production or protecting these qualities during fermentation 152,153 . Other innovations include encapsulation and nanoparticle technologies 154 for the delivery and dissemination of Trichoderma spore inoculum and/or the compounds produced, which could be important during the preparation of fermentation culture supernatants and application over extensive cultivated areas. Trichoderma has a role in the present transitions in agriculture towards a green economy (to reduce environmental impacts and ensure food safety) and a circular economy (to recycle agrifood waste to produce valueadded products, such as substrate sources to cultivate plant beneficial microorganisms, including Trichoderma, or organic matter formulations, with/or without these microorganisms, for direct appli cation as soil amendments). Biotechnological advances might permit the safe and widespread use of Trichoderma gene expression in plants to confer increased resistance to pathogens 155 and tolerance to abiotic stresses 156 , thus reducing the use of agrochemicals and improving the ability of crops to overcome adverse environmental conditions. The ongoing findings in Trichoderma research prompt a reflection on the effective use of this fungus in agriculture and the development of practical uses with selected potential strains (Fig. 4). New appli cations include crop cultivation in marginal lands, improved crop resilience to unfavourable climate changes 157 , bioremediation for the reduction of pollutants in contaminated sites 158 , and a general contri bution to the reduction of methane and carbon dioxide emissions in the atmosphere 38 .

Conclusions
This article has provided an overview of the advances in Trichoderma research that support its applications as a successful BCA and plant biostimulant for improved crop protection and production. Notable changes have occurred in the systematics of the genusspecies complex that will affect the nomenclature and how the fungal group is recognized and nominated, subsequently influencing species or strain selection for use in biotechnological development. Progress is also noted in scientific investigations regarding the multitrophic, interkingdom relationships that Trichoderma establishes in the agroecosystem; the evolutionary events that have given rise to rhizosphere and endophytic colonization of the host plant; and interactions with the plant microbiota and other nontarget organisms that affect the surrounding soil ecology, influence plant growth, and contribute to environmental and human wellbeing. However, the most exciting scientific discoveries -made possible with modern omics techniques -are those offering insights into the plant

Box 3
Ideal characteristics of a Trichoderma-based product for future sustainable agriculture The ideal commercial product based on Trichoderma will be multifunctional and capable of the following diverse beneficial effects for agriculture: 1. Direct biocontrol of plant pathogens and pests, thus reducing the need for chemical pesticides 2. Multiple capabilities for crop protection in a single product, exhibiting a broad spectrum of biocontrol activity against pathogenic microorganisms, nematodes and insects 3. Activation of plant defence mechanisms providing indirect biocontrol of plant pathogens and pests 4. Activation of plant defence mechanisms that increase tolerance to abiotic stress 5. Activation of plant defence priming against biotic and abiotic stresses at the time of attack or damage, which can activate over time and has a long-term duration 6. Provision of heritable beneficial traits in seedbed and nursery plants 7. Stimulation of plant growth to increase crop productivity and yields 8. Improvement of soil nutrient availability and fertilization, leading to increased plant uptake and assimilation 9. Improvement of quality of harvested products by increasing nutritional values and storage attributes 10. Decreased use of chemical products in agriculture, thus reducing risks to the environment and consumer health

Review article
responses to this fungus guest, including induced defence responses that provide indirect biocontrol to a variety of phyto pathogens, the effects on priming and plant memory, and increased tolerance to a diverse range of biotic and abiotic stresses. Trichoderma is a model sys tem for studying and deciphering the beneficial microorganism-plant and microorganism-microorganism interactions that these fungi establish among themselves and their surroundings. Without a doubt, Trichoderma is a fascinating microorganism -an opportunist that is versatile and in continuous evolution, a true survivor of the multitude of ecological changes over the millennia. The main question that arises is, how can we harness Trichoderma diversity to develop longterm efficient strategies to improve agricultural production and protection? Can adverse climatic conditions be counteracted with Trichoderma? Can Trichoderma help assure global food security? Can agriculture truly become autosufficient by using alternative biological solutions such as Trichoderma without the implementation of synthetic chemicals? How can optimal Trichoderma species or strains be selected, formulated and applied to obtain consistent efficacy? At present, the major bottleneck to the development of innovative Trichodermabased products is how to overcome the restrictions imposed by registration and authorization procedures, including inflexible terminology and definitions; inadequate consideration of the variable characteristics of living biological organisms (they are not single purified elements) and the differing effects depending upon interactions between organisms; slow dossier evaluation; and limited communication among researchers, policymakers, stakeholders and end users. These restrictions are in direct contrast to the legislative policies for agriculture of the future, as man dated by many governments globally, aiming to find solutions in the short term that provide alternatives to synthetic chemicals, minimize negative impacts to the environment, develop green and circular economies, and implement a One Health approach (a concept that aims to optimize the health of people, animals and the environment). Multidisciplinary investigations are needed to understand the multipurpose properties of Trichoderma to maximize the benefits from this green fungus, thus leading to improved quality of life and safe, ecosustainable agriculture.