By 2050, water consumption for agricultural practices will increase by 60% to support a growing world population (Boretti and Rosa, 2019). Concurrently, drought is becoming a dominant environmental stressor for crop production globally, limiting food security with precarious economic and sociological impacts (Trenberth et al., 2014). Due to climate change, water deficit is spreading to regions where drought was infrequent in the past (Somerville and Briscoe, 2001). Globally, the reduction in yield due to drought is likely to exceed the combined loss of all other possible causes of yield decline (Blum, 1996; Foolad, 2007). Therefore, there is a need to identify solutions to mitigate water deficit stress and its impact on food security.

Tomato (Solanum lycopersicum L.) is the world’s second most important vegetable crop in per capita consumption (Singh et al., 2018). On a global scale, tomatoes are grown on 5 million hectares of land with an annual production of about 180 million tons, and a production value of more than 90 billion USD (FAO, 2022). Currently, drought is a significant environmental stressor challenging tomato productivity in areas of inadequate or erratic rainfall that lack irrigation options (Petrozza et al., 2014; Pal et al., 2016). Drought decreases crop production on 25% of arable land throughout the world. Thus, where tomatoes are produced under rainfed agricultural conditions, there is a need to identify multi-faceted, low-cost, environmentally friendly, and sustainable solutions to water deficit that can be implemented on a short-term basis. Symbiotic associations with stress-mitigating soil microorganisms may serve as one of those solutions.

Naturally occurring beneficial soil microorganisms, including members of the genus Trichoderma (Family – Hypocreaceae), are studied as accessible and sustainable tools for enhancing plant growth and mitigating biotic and abiotic stressors in both research and commercial production settings (Van Wees et al., 2008; Grover et al., 2011; Kim et al., 2012; Meena et al., 2017; Khadka and Miller, 2021). As such, specific commercial Trichoderma isolates are widely used as effective biocontrol agents, biofertilizers, and phyto-stimulators (Harman, 2011; Hermosa et al., 2012; Waghunde et al., 2016; Kashyap et al., 2017; Khan et al., 2017), but evidence of contributions to abiotic stress amelioration in crop species is sparse. Several recent studies suggest that Trichoderma inoculation of water-stressed plants enhanced growth by improving root biomass, enhancing water holding capacity, and mobilizing nutrients (Mastouri et al., 2010; Harman, 2011; Bakhshandeh et al., 2020). Further, Trichoderma inoculated plants exhibit delayed wilting, increased stomatal conductance, enhanced leaf chlorophyll content, and greater net photosynthesis levels under water deficit stress conditions (Bae et al., 2009; Shukla et al., 2012; Alwhibi et al., 2017; Harman et al., 2019).

Plant genotype influences the degree of colonization and crop stress-mitigating potential of Trichoderma even for the established commercial isolates (Harman et al., 2004; Prashar and Vandenberg, 2017). The beneficial effects of Trichoderma, however, are more pronounced when plants are under stress compared to when grown under optimal conditions (Mastouri et al., 2010; Lombardi et al., 2018). As it has been demonstrated for other fungi, there is also potential that changes in climate patterns can influence Trichoderma distribution and abundance, as well as the outcome of the plant–microbial interaction (Compant et al., 2010; Tyagi et al., 2014; Delgado-Baquerizo et al., 2020). However, whether different species or isolates of Trichoderma have adapted as symbionts in stress environments remains unclear.

The overall goal of this research was to evaluate the ability of novel isolates from several species of Trichoderma, primarily native to different agroecological regions in Nepal, to improve growth in different tomato genotypes exposed to water deficit stress. Our study involved a series of experiments focused on: (1) assessing these novel isolates for their desiccation tolerance and their efficacy for alleviating water deficit stress symptoms (e.g., reduced biomass accumulation and diminished physiological function) in tomato genotypes differing in water deficit tolerance; (2) delineating the physiological (e.g., relative water content, electrolyte leakage, and photosynthetic efficiency) and biochemical (e.g., total and individual phenolic, proline and hormonal level) changes in leaves of a Trichoderma-inoculated, susceptible tomato genotype subjected to water stress; and (3) identifying and mapping genes differentially expressed in leaves of the inoculated tomato genotype under water stress and irrigated conditions (Rawal, 2021). Herein, we report our assessment of the novel Trichoderma isolates as plant growth promoters with the potential to alleviate growth-related and physiological stress symptoms across tomato genotypes that differ in water deficit stress tolerance. We hypothesized that (a) Trichoderma isolates will differ in their efficacy to diminish the effects of water deficit stress on tomato plant growth; (b) that isolates collected from the drier regions will tolerate desiccation conditions and have a greater impact on water deficit stress amelioration than those from wetter regions, and (c) that the effect of selected Trichoderma isolates on growth and water deficit stress will depend on tomato genotype. To ascertain the validity of the latter, we pursued a supporting goal of surveying heirloom and land races within The Ohio State University tomato germplasm collection for water deficit susceptibility or tolerance with the aim of including one or more of these genotypes in our Trichoderma trials.

Nepalese Trichoderma isolates used in this study were imported under the US Department of Agriculture Animal and Plant Health Inspection Service (APHIS) permit #P526P-19-03121. All experimental procedures involving fungal isolates were approved by The Ohio State University Institutional Biosafety Committee as described in Protocol 2019R00000043. These experiments were performed in BSL-2 rated laboratories and greenhouses. A general overview of the experiments reported in this work is shown in Figure 1.

The application of biological agents like water deficit-tolerant Trichoderma could be a low-cost, environmentally friendly, and sustainable near-term solution to mitigate drought stress (Shukla et al., 2012; Brotman et al., 2013). However, specific fungal isolate-plant cultivar relationships are complex, and the mechanisms of drought tolerance induced in the plant by Trichoderma colonization are not well-understood. Herein, we report the evaluation of novel Trichoderma isolates collected from different agroecological regions of Nepal for their ability to proliferate under osmotic stress and then to mitigate stress responses in tomato genotypes during and after exposure to water deficit conditions. We had a unique opportunity to test isolates from highly diverse ecological niches ranging in mean annual precipitation from <200 to 5,000 mm, in elevation above sea level from 58 to 8,848 m, and temperature extremes from –12°C to 43.3°C (Karki et al., 2016; Khadka et al., 2022).

Supplementation of growth medium with PEG is designed to measure the effect of water potential on the growth of fungi (Jin et al., 1991; Aujla and Paulitz, 2017). We screened 44 isolates and identified some isolates which exhibited the ability to survive and sporulate under different osmotic stress conditions (Figures 2A–C and Supplementary Table 2). The potential efficacy of these isolates to mitigate water deficit stress in planta was ascertained in greenhouse experiments, in which tomatoes inoculated with NT16, NT3, NT33 and subjected to water deficit stress amassed significantly greater growth parameters than the non-inoculated water deficit stressed control plants in different tomato genotypes (Figure 3A and Table 1). Similarly, other researchers have successfully employed various in vitro culture techniques to identify isolates of Trichoderma (Shukla et al., 2012; Poosapati et al., 2014; Khoshmanzar et al., 2020) or other soil microbiota (Mhadhbi et al., 2011; Subramanian et al., 2016) that conferred tolerance to abiotic stressors, such as osmotic (salt, drought) or temperature (heat, cold) stress.

Microorganisms collected from environmentally diverse sites (arid, saline, alpine regions) are reservoirs of potential traits that may benefit plant hosts when grown under the abiotic stress conditions from where they were isolated (Subramanian et al., 2016; Giauque et al., 2019). Among the six Trichoderma isolates from Nepal that consistently performed in the top third of the collection in the in vitro assay, NT3 was isolated in Jumla, the driest site sampled (766 mm/year precipitation; Supplementary Tables 1, 2 and Supplementary Figure 1). Isolates NT31, NT33, and NT37 were obtained from Banke, another dry site (1175 mm annual precipitation with 85% of the rainfall accumulated during a three-month monsoon season). Conversely, isolates NT10, NT19, NT23, and NT27 ranking in the lower third of the collection for growth under water deficit conditions were collected at sites receiving 1993–2329 mm annual precipitation, from 160 to 200% greater than that received at Jumla. To our knowledge, site-specific Trichoderma adaptation to stress environments, either as free-living organisms or in mutualistic relationships, has yet to be reported. Adaptation to mutualism as a means of survival under harsh environmental conditions may be predicated upon co-evolution involving heritable genetic variation in both plants and microorganisms that promote colonization as well as abiotic stress responses (Hoeksema, 2010). Kubicek et al. (2019) characterized Trichoderma as an “environmental opportunist and generalist” that can rapidly evolve to thrive in new or stressful environments as a free-living organism or a plant symbiont. The fungi can produce the asexual conidiospores and chlamydospores that can survive under severe environmental conditions. Therefore, it is plausible that specific Trichoderma isolates in this study, most notably NT33, are effective at ameliorating water deficit stress in tomatoes because they evolved in drier regions of Nepal. The evolution of these and other effective isolates may also have been influenced by micro-climatic conditions not examined during the collection process.

Water deficit stress significantly inhibits crop productivity and quality. Trichoderma inoculation could ameliorate the negative effect of water deficit stress and facilitate growth promotion in plants (De Palma et al., 2016; Bashyal et al., 2021). Mastouri et al. (2010, 2012) demonstrated the effects of Trichoderma inoculation on plant performance to be apparent only in stressful environments. In their studies, inoculation of tomato seed with T22 improved germination and other growth parameters when grown at water deficit conditions. Other researchers have uncovered similar trends among treatments and controls, suggesting significant Trichoderma effects under different biotic or abiotic stress conditions (Donoso et al., 2008; Lombardi et al., 2018). Our results support these findings in the first greenhouse experiment where shoot weights of ‘Roma Organic’ tomato genotype inoculated with five Trichoderma isolates belonging to two different species T. asperellum and T. asperelloides were higher than those of controls when subjected to the water deficit regimen (Figure 3A). However, in the Trichoderma × Tomato Genotype experiment, there was a significant effect of Trichoderma inoculation on growth parameters of tomato plants under both irrigated and water deficit stress conditions (Tables 1, 2), supporting Trichoderma’s role as growth promoting agent.

Genotypes of the plant and symbiont are essential factors determining the level of the beneficial effect of Trichoderma inoculation, even for established and commercial isolates of Trichoderma like T22 and the T. virens strain G-41 (Harman et al., 2004; Prashar and Vandenberg, 2017). Trichoderma-cultivar specificity has been studied most extensively upon exposure to biotic stresses, but our study documents this relationship under the abiotic stress condition (water deficit stress) as well. We conducted the greenhouse trial with three genotypes of tomato differing in their drought tolerance capacity. Since plant- microbe interaction is host specific, it is important to identify the isolates that are effective across wide range of crops. In our study, NT33 generally outperformed the other Nepalese Trichoderma isolates and the commercial Trichoderma T22 when inoculated in the different tomato genotypes under both water deficit stress and non-stress conditions. We also found that the overall performance of the selected Trichoderma isolates was better in drought susceptible genotypes ‘Jaune Flamme’ and ‘Roma Organic’ as compared to drought tolerant ‘Punta Banda’ (Tables 1–4). Prashar and Vandenberg (2017) exposed 23 wild and cultivated lentil genotypes colonized with either commercial Trichoderma strain T22 or G41 to the root rot pathogen Aphanomyces euteiches. Responses to the pathogen and overall plant performance were strongly governed by the origin and genetic makeup of the lentil genotype. Similarly, using Trichoderma commercial strains P1 (T. atroviride) and T22 and five tomato genotypes, Tucci et al. (2011) demonstrated significant variability in performance (negative, neutral, positive) among inoculated pairs vs. controls. These authors introduced the feasibility of breeding plant genotypes to be specifically responsive to common commercial Trichoderma isolates.

Water deficit stress stimulates different physiological responses such as stomatal closure, repression of photosynthesis, activation of respiration, and reduction of cell growth in the plants. The plants inoculated with Trichoderma isolates can enhance water-deficit tolerance by improving root development, regulation of drought-induced changes in the stomatal opening, photosynthesis, and chlorophyll content in leaf (Contreras-Cornejo et al., 2009; Brotman et al., 2013; Guler et al., 2016). Our study also found that the net photosynthetic rate and stomatal conductance in the tomato leaves were less affected by water deficit stress in the NT33 inoculated tomato plants compared to non-inoculated control (Table 3). In addition, NT33 and NT3 increased the chlorophyll content in the drought susceptible genotypes as compared to non-inoculated controls, which is an important pigment essential for absorbing light during photosynthetic process (Table 3). Bashyal et al. (2021) reported that genes related to the photosynthesis process, such as those coding for photosystem I subunits, photosystem II core complex proteins, osmotin like proteins, and stomatal development were upregulated in the Trichoderma inoculated plants as compared to non-inoculated controls. Water deficit exposed plants inoculated with Trichoderma exhibit improved physiological health (Shukla et al., 2012; Harman et al., 2019; Khoshmanzar et al., 2020), similar to our observation in NT33-colonized tomato plants. It has been reported that the plants inoculated with Trichoderma species increase the accumulation of different metabolites like phenolics, flavonoids, and proline which increase antioxidant activity, protect photosynthetic pigments, and photosynthetic pathways from photooxidative damage (Mona et al., 2017; Scudeletti et al., 2021; Tripathi et al., 2021). The earliest response of plants to drought is considered to be due to an increase in abscisic acid content and regulates stomatal movements and helps to maintain water balance. Martinez-Medina et al. (2013) reported that Trichoderma harzianum colonized plants displayed higher expression levels of the ABA-responsive marker gene involve in different defense-related pathways compared to non-inoculated control plants.

Relative water content is an important indicator of the physiological water status of the leaf. It indicates the appropriate water status of plant in terms of cellular hydration and is used as a selection criterion for drought tolerance in different crops (Teulat et al., 1997). Under water deficit stress conditions, the RWC% in the plants is significantly reduced; however, the Trichoderma colonization augmented the relative water status in the leaves (Bae et al., 2009; Guler et al., 2016). In our study NT33 and NT3 inoculation increased the relative water content in the leaf of Jaune Flamme’ and ‘Roma Organic’ plants and allowed them to defend against the abiotic stress (Table 3). Trichoderma spp. can improve the drought avoidance mechanism in plants through morphological adaptation by increasing root growth and also changing root architecture in many crops, thereby increasing the plant yield under the water deficit stress condition (Contreras-Cornejo et al., 2009; Meng et al., 2019). Some Trichoderma species secrete auxin (indole acetic acid) and other indole derivatives like nitrilase, that induce the lateral root growth and increase the root surface area (Hermosa et al., 2012). In this study we found that the ‘Jaune Flamme’ plants inoculated with NT33 had higher root biomass compared to T₀ (Table 1) under water deficit stress condition and also increases the lateral root number (Rawal, 2021) that could have increased the water status in the plant.

The salient results of this study are as follows: (1) We demonstrated the ability of Trichoderma isolates to reduce water deficit stress in tomato, which may provide a grower-friendly strategy for crop improvement and crop protection. (2) We identified superior strains from among a novel collection of 41 Trichoderma isolates collected from diverse ecological niches in Nepal with the ability to thrive under osmotic stress and to improve the horticultural performance of inoculated tomatoes when cultured in the greenhouse under water deficit stress conditions. (3) We ascertained that the best performing isolates were collected in dry to relatively dry regions of the country, suggesting adaptations to mutualism predisposing them to confer water deficit tolerance to the plants they colonized. This potential adaptation response to environmental conditions may have significant implications for the acquisition of Trichoderma strains capable of enhancing crop performance under specific biotic and abiotic stress conditions. (4) We validated plant cultivar-Trichoderma isolate specificity in greenhouse experiments with inoculated tomato plants differing in water stress tolerance. The improvement in horticultural performance was most evident in inoculated, water stress-susceptible tomato genotypes. (5) We distinguished NT33 (T. asperelloides) isolate for its ability to deliver consistent performance advantages to its tomato partners suggesting its potential as an element in multi-faceted environmentally sound approaches to manage crop water deficit stress (Figure 5). It is noteworthy that NT33 also consistently outperformed the commercially available T22 (T. harzianum) strain suggesting that limits to the potential benefits of Trichoderma inoculation under abiotic stress conditions have yet to be reached. Different biochemical and genetic determinants underlying the induction of resistance in the plants are unknown and are currently under investigation. In the future, research should also focus on the mechanisms adopted by these isolates like solubilization and mobilization of nutrients, production of plant growth-promoting hormones, and other secondary metabolites for plant growth promotion and adoption to abiotic stress.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

RR, JS, and M-SB conceived the project. RR performed most of the experiments, analyzed the data, and wrote the manuscript. SF and DF conceived the experiments on water deficit susceptible tomato genotypes. SF performed those experiments and wrote the corresponding sections. SM provided Trichoderma isolates. JS, M-SB, SM, and DF edited the manuscript. All the authors contributed to the article 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.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.