A complete understanding of the taxonomical identity, molecular polymorphisms, antagonism level, and potential as a plant growth promoter of a collection of Trichoderma spp strains is essential for any biological control program. Here, we provided key information that could strengthen current strategies at the same time that optimizes inputs use in tomato cultivation.
4.1 There are two different Trichoderma species with polymorphisms among strains
Most previous efforts have been focused on describing genetic and phylogenetic characteristics of novel Trichoderma isolates from different origins (Błaszczyk et al., 2011; Druzhinina et al., 2006; Druzhinina et al., 2012; Feitosa et al., 2019; Hermosa et al., 2004; Jiang et al., 2016; Kamala et al., 2014; Zhang et al., 2005) and their potential as biocontrol agent in a broad number of pathogen species such as Alternaria spp., Botryodiplodia theobromae, Curvularia sp., Macrophomina spp., Mucor racemosus, Candida albicans, Plasmopara viticola, Pseuperonospo cubensis, Rhizopus spp., Sphaerotheca fusca (Ghazanfar et al., 2018), Rhizoctonia spp., (Filizola et al., 2019), Aspergillus spp., Botrytis spp., Colletotrichum spp., Pseudomonas syringae, Sclerotinia spp., Xanthomonas campestris; (Singh et al., 2018), Fusarium spp., (Martínez-Medina et al., 2011), Pythium spp., (Harman, 2011), Phytophthora spp., Sclerotium rolfsii (Roberts et al., 2010) and Rhizoctonia (Wu et al., 2017). But, there are no references of studies that describe the genetic diversity and polymorphisms of strains under production and promotion in tomato crop.
According to DNA sequence analyses, strains were classified into two species T. asperellum and T. harzianum, correcting previous perception of having only one species. Indeed, some reports indicate that several isolates of genus Trichoderma have been initially misclassified because of their similarity, scarcity of morphological characters, and lacking molecular tools needed to confirm taxonomic identity (Druzhinina et al., 2005; Kullnig et al., 2001; Schuster and Schmoll, 2010). Because T. harzianun is the most abundant species in diverse niches (Błaszczyk et al., 2011), it is very common to attribute any novel isolate to that species.
Usually, DNA sequences are compared to reference databases in order to achieve taxonomic identification, a study of the assessment of the main public repositories of DNA Barcode sequences, BOLD and GeneBank, accuracy and reliability outlined that both databases performed comparably for fungi identification, and described BOLD as a curation tool that also stores sequences and GenBank just as a sequence repository with basic quality checks (Meiklejohn et al., 2019). With the purpose of having a high level species identification confiability, we used a third database repository, TrichOkey, which is a method for molecular identification of Trichoderma at the genus and species levels, using a combination of several genus specific hallmarks and species clades specifically allocated within the internal transcribed spacer 1 and 2 (ITS1 and 2) sequences of rDNA repeat (Druzhinina et al., 2005). All top hits from blasts in GenBank and BOLD provided an E-value of 0.0, with expected identity scores higher than 99.9% and were an exact match with TrichOkey results. Hence, DNA sequences obtained using ITS1‒F and ITS4 primers were capable of disaggregating strains into two species with high level of confidence (> 99.9%) without the need of sampling more genome regions for this purpose. However, it would probably be necessary to analyze at least two additional gene regions to detect more nucleotide polymorphisms for each strain, for traceability purposes (Meiklejohn et al., 2019) or to explore more secondary fungal DNA barcodes such as the stated optimal for secondary DNA barcoding in Ascomycota, DNA topoisomerase I (TOPI) and phosphoglycerate kinase (PGK) (Stielow et al., 2015). All these options are promising for fingerprinting all the Trichoderma strains.
Phylogenetic analyses suggested a consensus tree (considering both approaches, Bayesian and Parsimony) desegregating strains into two main clusters according to species. This result was expected considering the reduced number of taxa and species. There was no apparent correlation between origin and phylogenetic structure as reported in other studies where geographic origin and host crop influence the genetic structure of Trichoderma populations (Jiang et al., 2016; Kamala et al., 2014; Zhang et al., 2005). On the other hand, it was interesting that our results confirmed that strain TZ01 was clearly different from strains TC01 and TC02, something that has been suggested by researchers at Zamorano University based on morphological information (personal observation).
Within cluster I, T. asperellum strains were divided into two sub-clusters, differences are due to a T deletion at 180 nt and a transversion (T‒G) at 438 nt. As these two nucleotide changes were consistent in two groups of T. asperellum strains, phylogenetic algorithms conducted in both approaches divided strains into two sub-clusters. On the contrary, in cluster III, strains TN1C and MMR1 showed two separate changes in nucleotide sequences, a T deletion at 144 nt and a transversion (G‒C) at 576 nt, respectively. Although, these variations were not significant for sub-branching those taxa during phylogenetic analyses. However, these polymorphisms are useful for strain barcoding. These kind of mutations are reported as the main cause of genetic differentiation within Trichoderma species, affecting in some cases gene functionality and exposing sub-clustering in phylogenetic analyses under diverse approaches (Feitosa et al., 2019; Jiang et al., 2016; Rai et al., 2016; Skoneczny et al., 2015).
It was interesting that during the growth of the strains in PDA media, we observed variations in morphology that suggest that more than one strain could be present in the pure culture. Although it was not confirmed in the present study, it is something to consider in future research. This would make a lot of sense if we consider the complex relationships between microorganisms and plants, as studied in sugarcane (Armanhi et al., 2016; Armanhi et al., 2018) where the community plays a key role in the success of the symbiosis. This could change the perception of the “genetic purity of the strains” in the future and adopt the concept of “community-based collections” instead.
4.2 Trichoderma spp reduces incidence and severity of F. solani on tomato plants
The fungus F. solani, causative agent of the wilt disease, is one of the most harmful plant pathogens around the world, acting in some cases in complex with other pathogens, for instance, Pythium spp and Rhizoctonia spp in the damping-off disease in vegetable seedlings. In tomato, wilt disease causes important losses that could be estimated between 20 and 50% (Ramyabharathi et al., 2012; Singh and Dwivedi 2014) depending on environmental conditions, variety and pathogen’s strain. The identification of biological alternatives that control F. solani is of great importance for the improvement of tomato productivity, its safety and the reduction of production costs, something important under current global context.
In this study, the results of incidence and severity of F. solani, considering the positive control, demonstrated that Trichoderma spp is an effective alternative for reducing the damage caused by this pathogen. The endogenous presence of F. solani (37.50%) in the positive control is not surprising since it is a seed-borne pathogen frequently found in seeds (Al-Askar et al., 2014; Mehedi et al., 2016). However, despite of this, the supply of Trichoderma spp improves the health of plants expose to high concentration of inoculum. If we consider another scenario where there is no F. solani inoculum on the field, only the one present in the seed, even so, strain TC01 (T. asperellum) seems to suppress the incidence of the disease by 9.37% and its severity by 5%, improving the performance of infected seeds in nurseries.
Indeed, Trichoderma antagonistic effects, such as substrate competition, mycoparasitism, and antifungal antibiotic production, are useful to inhibit the mycelial growth of pathogens (Li et al, 2016). Some hydrolytic enzymes and metabolites could play a key role in controlling Fusarium wilt. Sundaramoorthy and Balabaskar (2013) confirmed that under in vitro conditions the isolate ANR-1 (T. harzianum) was found to effectively inhibit the radial mycelial growth of this pathogen. In the meantime, under greenhouse conditions, this isolate exhibited the least disease incidence (by 15.33%) in the experiment. It is suggested that Trichoderma is able to trigger a long-lasting up‐regulation of the salicylic acid pathway even without any pathogen infection, probably stimulating a priming mechanism in the plant (Tucci et al. 2011). Also, it has been observed the Trichoderma can induce genes involved in the jasmonic and ethylene transduction pathways confirmed by microarray and qRT-PCR analyses, suggesting a transitory increment of plant defense (Moreno et al. 2009).
4.3 The use of Trichoderma spp improves the seed germination and growth of tomato plants
Although the physiological quality of the seed lot used in this study was very low, the use of these seeds in these experiments was an opportunity to test the benefits of using Trichoderma spp. It was impressive how Trichoderma spp, regardless of the strain, almost doubled the germination of tomato seeds compared with the control. This demonstrates the potential of these strains used as biological seed protectants, which could provide added value to seed inputs that are often very scarce for agriculture based on less use of inputs. Some studies suggest that Trichoderma spp could modify mRNA levels of 45 genes, 41 in roots and 4 in leaves (Moreno et al. 2009) promoting more growth in tomato seedlings expressed as an increasing in plant height, stem diameter and number of leaves, root length, dry matter parameters (Chowdappa et al. 2013; Nzanza et al. 2011, 2012).
In our experiment, even at 27 days after sowing, the variables plant height, stem diameter and number of leaves was still higher in plants inoculated with Trichoderma spp compared to the control, showing that plant growth is effectively promoted beyond germination period. This same trend was observed when analyzing the same variables at 41 days after sowing. In addition of creating a biological protection against pathogens during seed germination, Trichoderma spp establishes an effective symbiosis with tomato plants, steadily promoting plant growth due to the stimulation of the secretion of growth hormones. In this respect, Chowdappa et al. (2013) found that the use of T. harzianum in tomato seedlings increased the levels of indoleacetic acid by 54.34% and gibberellic acid by 67.59% in the root tissue. That study reported increases in shoot length (32.04%), leaf area (62.68%) and fresh weight of shoots (28.87%) in tomato plants. Similar results were obtained by Nzanza et al., (2011) who evaluated the performance of tomato seedlings supplied with T. harzianum at sowing stage or two weeks after sowing during two years. In that study, the application of T. harzianum at both times increased plant height of tomato in 63.41% and 74.29% respectively in 2008 and by 33.67% and 35.65% in 2009, respectively. Other authors report increases in the growth rate of tomato, maize, tobacco, radish (Windham et al., 1986), beans (Mayo et al., 2015; Pereira et al., 2014), cucumber (Li et al., 2019; Yedidia et al., 2001), peper and certain ornamental species (Chang et al,. 1986) as a result of the application of Trichoderma.
Although not addressed in depth in this study, it is known that Trichoderma spp can induce an improvement in photosynthetic capacities in plants (Harman et al. 2019), promoting greater generation of carbohydrates and therefore increased yields of many crops. These benefits can be further enhanced when added to other management actions that improve the availability of nutrients in the soil and the use of sunlight.
The supply of Trichoderma spp influenced root growth only in plants 28 days after sowing. The improvement expressed as root length and root volume was similar for both strains, but significantly superior to the control. In average, Trichoderma spp strains improve root length by 41.15% and root volume by 34.43% compared with the control. Nzanza et al. (2011) reported that tomato seedlings inoculated with T. harzianum increased plant root length by 23.20% when T. harzianum was inoculated at planting time and 39.46% when it was applied two weeks after planting obtained similar results. In another experiment, Chowdappa et al. (2013) reported increases of 38.53% in root length and 36.21% in fresh weight of tomato root due to the use of T. harzianum compared to the control. Other authors report average increases of 66% in shoots and roots of sweet corn (Björkman, et al. 1998) as well as increases of 75% in root length (Yedidia et al., 2001) and 100% in root volume in cucumber (Li et al., 2019).
The application of Trichoderma as a seed treatment colonizes the roots of the seedlings and induces their growth and development, which allows them to reach greater depths of soil, improving the dynamics with the microbiota, tolerance to drought, the performance in compacted soils and the yields (Bailey and Lumsden, 1998; Harman, 2000; Harman et al., 2004). However, the host plant, pH, temperature and other microorganisms present in the rhizosphere (Ahmad and Baker, 1988; Harman, 1992) could influence the growth and development of these populations. For example, Nzanza et al. (2011) reported 90% colonization by T. harzianum in tomato seedlings at nursery and 85% in open field conditions (Nzanza et al. 2012). Thus, it is important together with Trichoderma to provide a proper crop management that improve the performance of the binomial Trichoderma – plant, such as incorporation of organic matter to the soil, crop/varieties rotations, and minimum soil tillage.
The response of tomato plants to the application of Trichoderma was different between the seedling stage and the stages after seedling transplanting. In the early stages of seedlings, Trichoderma directly influenced the increase in root length and volume, an effect that did not occur during the productive stages. This behavior agrees with the results obtained by Nzanza et al. (2012) who evaluated the effect of T. harzianum on the dry weight of tomato roots in the open field without presenting significant differences with respect to the control in two productive cycles. However, Nzanza et al. (2011) previously found an average increase of 53.44% of the dry weight of seedlings roots under greenhouse conditions. This behavior is because plants at early stages of development that is during seed germination and seedling growth, they experience the highest growth rates in their life cycle, then Trichoderma is able to influence significantly the hormone machinery producing the results expressed here and in other studies.
The efficiency of the reproductive structure was different depending on the experiments. In the plants that were transplanted, the conidia stood out statistically over the microsclerotia in the interaction over time, as the independent factor. This is contrary to what happened in tomato seedlings at 28 days in which the microsclerotia had a better performance with respect to conidia at plant height. This performance could be due to the effect of environmental factors, in time and space. The temperature and relative humidity values recorded during both experiments (not shown) presented values above those registered in the antagonism experiment (in vivo) under greenhouse conditions. This would be reflected in an increase in the evaporation rate. Then, the soil moisture would be affected, creating stress conditions for both the plant and the Trichoderma strains, influencing the performance of the reproductive structures. In this case, the microsclerotia showed a better behavior under conditions of water stress compared to the conidia, which agrees with the description by Coley-Smith and Cooke (1971).