Numerous strains of Trichoderma species are widely used in agriculture and commercialized as biocontrol agents (BCA) of plant pathogens (Harman et al., 2004), biostimulants, and biofertilizers (Woo et al., 2014).

Their ability to control plant pathogens is mediated by different mechanisms, including competition for nutrients (Chet et al., 1997), the ability to modify the rhizosphere (Benítez et al., 2004), the production of useful secondary metabolites (Vey et al., 2001; Vinale et al., 2014), and direct antagonism of disease agents (mycoparasitism) (Harman et al., 2004). It is also well known that root colonization by these and other non-pathogenic microorganisms may modulate plant defense reactions to challenge the pathogen attack, based on a localized or systemic response, namely, Localized Acquired Resistance (LAR) (Anees et al., 2010), associated with the induction of hormone pathways that involve the activation of Salicylic Acid in Systemic Acquired Resistance (SAR) and of Jasmonic Acid (JA) in Ethylene Induced Systemic Resistance (ISR) (Pieterse et al., 2009). In fact, Trichoderma colonization may stimulate plant defense, resulting in the establishment of ISR, which represents an effective response to pathogens, particularly active at the root level, as it has been shown with nematodes (Degenkolb and Vilcinskas, 2016). Moreover, it has been indicated that Trichoderma colonization induces a state of priming in the plant that leads to a faster defense response to pathogens (Conrath et al., 2015; Martìnez-Medina et al., 2017), achieved by increasing the intensity of the plant immune response to microbial elicitors by means of microbe-associated molecular patterns (MAMPs); this is mediated by Trichoderma, which reduces the effector-triggered susceptibility and concurrently enhances the effector-triggered immunity, thus resulting in a higher level of plant resistance due to a faster and more effective defense response to a future pathogen attack (Lorito et al., 2010).

These findings indicate that these beneficial fungi have a well-documented capacity to manipulate plant defense barriers against pathogens. Their use is completely in line with the mandate of EU Directive 2009/128/EC to achieve a sustainable (reduced) use of chemical plant protection products (PPPs) in agriculture, by promoting the implementation of integrated pest management (IPM) practices and the application of alternative non-synthetic approaches/tools to reduce the negative impact on human health and on the environment. However, it would be highly desirable if the plant metabolic changes induced by these beneficial microorganisms could be active also against insect pests.

To date, only limited studies have determined the insect control activity by plants exposed to Trichoderma colonization. Namely, the available information is limited to aphids (Coppola et al., 2019), thrips (Muvea et al., 2014), and caterpillars (Contreras-Cornejo et al., 2018). Battaglia et al. (2013) registered a growth-stimulation effect of Trichoderma longibrachiatum MK1 treatments, both on tomato plants and on aphids feeding on them. Maag et al. (2013) observed a positive impact on oilseed rape development following T. atroviride LU132 treatment, which was not associated with any change in the defense reaction against Plutella xylostella, and in the titer of defense-related hormones, such as JA and SA. Therefore, it is evident that there is a remarkable variability of plant metabolic changes induced by a specific Trichoderma strain that may influence feeding and development of different pests.

To unravel the functional basis of these interactions and to identify the crucial plant metabolic changes associated with defense responses relevant from a crop protection perspective, we used an experimental approach based on the integration of accurate bioassays and concurrent transcriptional analyses of plants, to determine and analyze the effect of root colonization by the symbiont, in order to identify the key regulatory genes underlying Trichoderma-induced plant defense response.

Three interacting organisms were considered in this study: (1) the tomato plant, Solanum lycopersicum L., a staple crop in many areas of the world¹ ; (2) two major pests of tomato that adopt different strategies of plant attack: the noctuid moth Spodoptera littoralis (Boisduval), a chewing herbivore, and the aphid Macrosiphum euphorbiae (Thomas), a piercing–sucking feeder. For this latter, we also considered an effective biological control agent, the parasitic wasp Aphidius ervi (Haliday), which is effectively recruited by attractive volatile organic compounds (VOCs) emitted by the plant in response to pest attack, as reported for many other insect natural enemies (Rasmann et al., 2017 and references therein); and (3) the rhizosphere fungus T. atroviride strain P1, a laboratory strain known for its antagonistic activity against numerous phytopathogens, including the soil-borne plant pathogenic fungus Rhizoctonia solani (Tronsmo, 1989).

The colonization of tomato plants by T. atroviride strain P1 triggered plant metabolic changes that limited the survival and development of two pests, the moth S. littoralis and the aphid M. euphorbiae, which are characterized by different feeding habits, i.e., chewing and piercing-sucking, respectively, eliciting different defense response pathways (Erb and Reymond, 2019).

The Spodoptera caterpillars feeding on P1 plants, compared to controls, consumed the same amount of foliar tissue, but showed (1) a reduced larval survival rate, with a lower number of individuals attaining the pupal stage, and (2) a reduced weight gain over time, which resulted in a lower final weight. These developmental alterations are typically associated with the activity of digestive enzyme inhibitors, often aggravated by the compensative hyper-production of unaffected enzymes, which further enhances the overall nutritional impairment (Broadway, 1996; Brito et al., 2001; Chen et al., 2005; Brioschi et al., 2007). Plant inoculation with Trichoderma P1 is able to enhance the production of PIs in the plant tissues, likely as a consequence of the jasmonate pathway activation. This is further corroborated by the induction of transcripts coding for different classes of PIs and other insect “anti-nutritional” proteins, such as Threonine deaminase, Polyphenol oxidase, and Leucine aminopeptidases, that hinder development. Similarly, maize plants colonized by the same Trichoderma species showed reduced leaf herbivory by S. frugiperda (Contreras-Cornejo et al., 2018), as a consequence of the octadecanoid pathway induction, leading to JA accumulation in the shoots. These plant changes were associated with an altered feeding behavior and symptoms of midgut damage (presented as a ventral dark area extending over 1/5 of the larva), induced by the exposure to Trichoderma-produced VOCs, 6-pentyl-2H-pyran-2-one, and 1-octen-3-ol (Contreras-Cornejo et al., 2018). Moreover, the fine-tuning of defense reactions is associated with arginine catabolic process (VanEtten et al., 1963; Winter et al., 2015), and the up-regulation of genes involved in such process confirms that P1-treated plants are in a “defense state” that is tightly regulated.

The aphid M. euphorbiae showed a significantly reduced survival rate when reared on P1-treated plants compared to controls. This effect cannot be related to the release of volatile compounds that affect aphid fixation and behavior (Digilio et al., 2012), as in the first days of the assay, the number of aphids fixed and feeding on plant was similar between P1-treated and control plants. The difference in aphid development can be attributed to the impact of Trichoderma on plant direct defenses, which include the production of anti-feedant or inhibitory compounds, such as oryzacystatin, which has inhibitory effect when administered to Myzus persicae (Rahbé et al., 2003) and A. pisum (Carrillo et al., 2011). The activation of tomato defense response, indicated by the up-regulation of genes active early and late in the plant defense reaction, may be responsible for the impaired aphid performance on P1-treated plants. Starting from early events, P1 effects on tomato transcriptome indicate the activation of the oxidative defense compartment, known for its effect on aphid survival (Coppola et al., 2013; Enders et al., 2014). In addition, the overexpression of PI genes in the attacked plant may reduce the activity of aphid salivary proteases that appear to degrade defense proteins present in the sieve-tube sap (Furch et al., 2015). Intriguingly, it was proposed that the plant protects sap protein degradation by glycosylation that prevents proteolysis (Taoka et al., 2007; Russel et al., 2009). The concerted up-regulation of PIs and glycosyltransferases in Trichoderma-treated tomato plants may therefore reduce the aphid ability to degrade sap proteins involved in the defense response. Among the 1247 up-regulated genes, 112 could be grouped as “kinases,” indicating a strong impact of the beneficial fungus on the activation of defense signaling pathways. Much evidence shows the essential role of protein phosphorylation in the regulation of plant immunity (Park et al., 2012 and references therein). For instance, in Arabidopsis, SNF1/AMPK/SnRK1 protein kinases play a role in detecting the damages caused by insect feeding (Crozet et al., 2014), while in tobacco plants, kinases were found to be involved in the induction of responses to herbivores and wounds (Seo et al., 2007; Wu et al., 2007). Interestingly, it has been demonstrated that protein kinases play a key role in Arabidopsis responses against aphids (Shoala et al., 2018).

One of the peculiar aspects of Trichoderma–tomato interaction is the positive effect exerted by the fungus on TFs regulating defense gene expression (Segarra et al., 2009; Pieterse et al., 2014; Conrath et al., 2015; Rubio et al., 2017). In our data, genes coding for several families of defense-related TFs (i.e., ERF, WRKY, MYB, and bZIP) are all up-regulated, similarly to what was observed following the interaction of tomato plants with T. harzianum T22 (Coppola et al., 2019, this issue). These TFs are involved in innate immunity. For example, in Arabidopsis, AP2/ERF proteins are involved in JA inducible gene expression and known as octadecanoid-responsive elements that positively regulate the expression of JA- and ET-mediated defense-related genes (Pré et al., 2008). Specifically, in rice, OsERF3 plays positive roles in resistance against the chewing herbivores, influencing the expression of genes involved in the MAPK cascades and hormone biosynthesis (Lu et al., 2011). Similarly, MYB factors are implicated in JA signaling pathways, playing a role in the defense response against aphids and lepidoptera (AtMYB44 regulates resistance to the green peach aphid and diamondback moth by activating EIN2-affected defenses in Arabidopsis). Taken together, these data demonstrate that P1 treatment of tomato plants promoted the expression of a gene network underlying plant defense responses.

The fungus colonization promotes a plant transcriptome reprogramming in which both SA and JA pathways are potentiated, independently from the reported antagonism between these plant hormones (Walling, 2000, 2009; Coppola et al., 2013; Zhang et al., 2015; Li et al., 2016). Recently, higher constitutive levels of ABA and JA, and basal expression of ABA- and JA-related transcripts were found in soybean tolerant genotype (Chapman et al., 2018). In our dataset, the induction of ABA-related (i.e., Abscisic acid inducible protein) and the above-cited JA-related transcripts can be retrieved. In addition, our data are consistent with the recent finding of JA predominance over SA signaling occurring in cotton plants infested by aphids (Eisenring et al., 2018). The potentiation of physical barriers is also a reasonable hypothesis, since the induction of genes involved in cellulose biosynthesis and associated molecules is observed, as well as the up-regulation of several genes involved in phenylpropanoid pathway, which underlies the biosynthesis of lignin precursors and anti-microbial compounds (Naoumkina et al., 2010).

Tomato plants treated with Trichoderma P1 showed enhanced attractiveness toward the aphid parasitoid A. ervi compared to controls. Such behavioral observations are supported by differential VOC profiles, explaining the parasitoid altered behavior. The terpenoid biosynthesis, evidenced by the KEGG analysis, showed the enhanced expression of several transcripts coding for seven enzymes involved in this VOC-generating pathway. Parasitoid attraction to odor source may result from the release of a “blend” of compounds, rather than by single compounds (Bruce and Pickett, 2011). Among the six compounds whose release was significantly enhanced by the treatment with P1, two can have an important role in parasitoid response: z-3-hexenol and methyl salicylate. These two compounds are both released at a higher rate by tomato plants following aphid attack (Sasso et al., 2007) and have been shown to be detected by A. ervi antennae at a concentration as low as 0.1 and 0.01 mg/mL, respectively (Sasso et al., 2009). The same compounds are associated with a significantly higher attraction toward tomato, when the plant was challenged at the same time by T. harzianum T22 and M. euphorbiae in respect to plants challenged by either of the two (Coppola et al., 2017).

The significant differences in VOC release between treated and untreated plants are further corroborated by the up-regulation of genes involved in both the octadecanoid and salicylic acid pathways. In addition, several enzymes involved in early and late steps of phenylpropanoid biosynthesis, as well as in phenylalanine (PAL) metabolism, are coded by DEGs induced by P1 treatment in tomato. This result is consistent with many of the described effects on the enhancement of tomato defenses against insects described in this work. Phenylpropanoid metabolism generates a wealth of secondary metabolites, based on the few intermediates of the shikimate pathway as the core unit, which are molecules with antimicrobial activity (Didry et al., 1999; Naoumkina et al., 2010) and showing direct repellent activity (Vogt, 2010). PAL and the shikimate are channels for SA biosynthesis in plant (Chen et al., 2009). Thus, our transcriptomic results not only suggest a likely enhancement of physical barriers but also support an antixenotic/antibiotic effect on insects and are consistent with the registered increased emissions of MeSA.

In conclusion, P1-treated tomato plants exert a negative impact on the development of S. littoralis caterpillars and on M. euphorbiae. This direct defense barrier against aphids is nicely complemented by more intense attraction of A. ervi, an aphid parasitoid widely used in biocontrol plans and IPM strategies. This makes our results appealing from an applied perspective.

The datasets generated for this study can be found in dd, PRJNA533559.

Animal subjects involved in the study are insect pests (aphids and caterpillars) that we manage on agricultural crops by the use of biocontrol agents (BCA).

MCD, FP, RR, SW, and ML contributed to the study design. MC, PC, and IDL performed the experiments and analyzed the results. MC and MCD wrote the first draft of the manuscript. MC, SW, RR, FP, EG, and MCD wrote sections of the manuscript. All authors contributed to revise the manuscript, read, and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.