*3.5. E*ff*ects of Dose of Application of T. aggressivum f. europaeum and T. saturnisporum*

Since no significant plant growth-promoting results were found in tomato seedlings, the effects of applying three doses of both species separately, as well as jointly, were determined. The results are outlined in Table 5, wherein values significantly higher than that of the control are highlighted in green, and negative values in red, for better visualisation.

The increase in the dose of both species improved seedling quality, increasing all study parameters in TA D2 and TS D3 treatments, with respect to that of the control. In treatment TA D2, stem length increased 14.37%, plant diameter 9.4%, leaf number 21.58%, shoot dry weight 16.66% and root dry weight 66.66%. In treatment TS D3, stem length increased 39.05%, plant diameter 15.22%, leaf number 11.55%, shoot dry weight 12.5% and root dry weight 33.33%. Although most treatments favoured the development of seedling shoots, no favourable results were found in roots; therefore, the seedling quality was not improved. The combination or mixture of the two species for the three doses tested did not improve the results compared to that of their separate application.

The results of the study parameters after transplantation of the seedlings into the soil are outlined in Table 5 (Figure 3). Three treatments, TA D1, TA D2 and TS D2, led to a good relationship between tomato shoots and roots, with significantly higher plant quality, compared to that of the control, without a new application of *Trichoderma*. Thus, shoot dry weight increased 43.20%, 22.84% and 29.58% and root dry weight increased 29.94%, 39.32% and 31.51% after the TA D1, TA D2 and TS D2 treatment, respectively. The establishment of the endophytic fungus at the root (Figure 4) enabled its effects to persist after transplantation.




 letters indicate significant differences according to the one-way ANOVA test (*<sup>p</sup>* 0.05). DQI: Dickson Quality Index.


**Table 5.** Morphological parameters and DQI of tomato seedlings and plants treated with different doses (105, 106 and 107 spores per plant; D1, D2 and D3, respectively) of *T. aggressivum* f. *europaeum* (TA), *T. saturnisporum* (TS) and mix (M) of two species.


 letters indicate significant differences according to the one-way ANOVA test (*<sup>p</sup>* 0.05). Green: favourable; Red: unfavourable; Orange: no effect compared to control. DQI: Dickson Quality Index.

**Figure 4.** (**A**) Colonization of pepper and tomato roots by *T. aggressivum* f. *europaeum.* (**C**) Mycelium in pepper root (100×). (**B**) Conidiophores and mycelium in tomato root (100×). (**D**) Chlamydospores in pepper root (200×).

#### **4. Discussion**

Numerous *Trichoderma* species have been described as plant-growth promoters, including *T. harzianum, T. longipile*, *T. tomentosum*, *T. viride*, *T. koningii*, *T. asperellum, T. aureoviride* and *T. saturnisporum,* among others [34]. This ability to promote growth depends on several factors, including the existence of isolates of the same species that may or may not promote plant growth, or for example, the crop and/or variety to which the species is applied [34]. Similarly, the use of a mixture of species has been extensively studied and commercialised to increase this activity [10]. In this study, the plant growth-promoting capacity of a new species, *T. aggressivum* f. *europaeum*, which is characterised by its rapid growth and sporulation, was analysed and compared to that of *T. saturnisporum*, a species characterised as a plant-growth promoter by Diánez et al. [16,18]. Although Allaga et al. [35] recommend not using species that produce green mould disease, these species do not create any problems in horticultural crops or pose any danger to mushroom crops, as long as they are applied in different geographical areas. Additionally, mushrooms are produced in closed locations and under completely different conditions. Furthermore, plant remains under horticultural production are not used to prepare substrates for mushroom cultivation, as shown in many commercial species; neither are plant remains that have been studied with plant-growth promoters, which may also cause green mould disease, such as *T. harzianum* [36] or *T. longibrachiatum* [37].

The first objective was to obtain viable spores with high yield on low-cost substrates. This product was used for additional tests, which demonstrated that the nutritional composition of the substrates used did not affect the biostimulant capacity of either *Trichoderma* species. Lane [38] determined that the nutrients provided in the medium could affect the biocontrol or biostimulant capacity of the agent. Different substrates have been used for *Trichoderma* spore production, including barley straw [39], wheat, rice, corn kernels [40] or a mixture of substrates, such as wheat straw, bran, cassava, potato starch and sugar beets [41,42], among others. In general, in our study, high yields, expressed as colony forming unit (CFU) g<sup>−</sup>1, were assessed in all substrate mixtures tested; the yields increased both in 80% buckwheat husk + 20% rice and 70% buckwheat husks + 30% oats. Although in laboratory tests, extraction could be performed without a problem in all mixtures, in the extractor tank, mixtures containing rice adhered to the walls and pipes, complicating the subsequent extraction and filtration processes. For this reason, to develop low-cost production methods for industrial scale-up, rice was rejected as a constituent of the production substrate for TA and TS. A high siderophore and IAA production and P solubilisation by TA and TS compared to other *Trichoderma* species or isolates were demonstrated in our study. These three components play key roles in plant biostimulation by increasing nutrient availability to plants, such as for hormone production [43,44]. However, the direct relationship between IAA production and plant-growth promotion is not yet clear because numerous species can produce IAA, but they do not promote plant growth [45]. Hoyos et al. [45] concluded that IAA production is not a

species-dependent quality of *Trichoderma* and found no direct correlation between biostimulation and IAA and siderophore production or P solubilisation. In turn, Vinale et al. [46] highlighted the effects of siderophore (harzianic acid) production on the germination of tomato seeds and the improved growth of the seedlings even under iron-deficient conditions. Similarly, Qi and Zhao [47] demonstrated that applying *T. asperellum* enhanced cucumber growth by inducing physiological protection under saline stress, and its siderophores played a key role in mitigating the negative effects of salinity.

Many *Trichoderma* species can produce IAA, and high IAA secretion in the presence of tryptophan indicates the importance of tryptophan as a precursor for IAA production [48,49]. Gravel et al. [50] reported that IAA production induced by L-tryptophan increased the fresh weight of tomato shoots and roots. Our results indicate that TA and TS produce much higher amounts of IAA than those assessed by other authors. Accordingly, Saber et al. [48] described IAA production of *T. harzianum* isolates that were 10 times lower than that of *T. aggressivum* f. *europaeum* and *T. saturnisporum* assessed in this study. Bader et al. [51] reported that IAA production ranged from 13.38 to 21.14 μg mL−<sup>1</sup> in *T. brevicompactum, T. gamsii* and *T. harzianum*. Diánez et al. [16] described a highly similar IAA production for *T. saturnisporum*; therefore, the in vitro production capacity of IAA was preserved despite maintaining the isolate in the laboratory for 10 years. Similarly, phosphate solubilisation by *Trichoderma* species has been described both in vitro and in vivo [52–54]. Recently, Tandon et al. [55] evaluated P solubilisation of different *Trichoderma koningiopsis* isolates under abiotic stress conditions and determined a range from 1.6 to 71 μg mL<sup>−</sup>1. Bononi et al. [12] found that *Trichoderma* isolated from soils of the Amazon rainforest demonstrated a high potential for phosphate solubilisation, which ranged from 51.7 to 90.3% 10 days after inoculation. Despite their high P solubilisation capacity, some of these isolates inhibited the germination of soybean seeds. In our study, the P solubilisation range of both isolates was lowest on the tenth day of incubation, at 5.9% and 6.16% for TA and TS, respectively.

Applying PGPMs to seeds makes it possible to use a lower concentration of spores while ensuring that the PGPMs are readily accessible at germination and during early developmental plant stages, stimulating healthy and rapid establishment, and consequently, maximising crop production [43]. However, the direct application of different *Trichoderma* isolates or species to seeds (bioprimming) has not always had beneficial effects. In this study, the seed germination rate was not affected by *T. aggressivum* f. *europaeum* or *T. saturnisporum* application. Similar results were found by Azarmi et al. [18] after applying *T. harzianum* isolates to tomato seeds. Hajieghrari et al. [56] demonstrated that direct exposure of corn seeds to *Trichoderma* spores decreased the percentage of seed germination, as well as radicle and shoot length. However, You et al. [57] demonstrated that *T. harzianum* and *T. koningiopsis* isolates significantly enhanced the tomato seed vigour index when they were used to treat tomato seeds. Our results demonstrated that direct *T. aggressivum* f. *europaeum* and *T. saturnisporum* application decreased seed vigour, significantly so in peppers but not in tomatoes. However, the application of either species under commercial plant nursery conditions, via substrate irrigation, similarly enhanced pepper seedling quality significantly, albeit again non-significantly for tomatoes. Optimising the application dose for each species is a factor that should be considered, among other factors, to enable companies and producers to adopt this technology with higher security [58]. Increasing the dose of *T. aggressivum* f. *europaeum* and *T. saturnisporum* applied to tomato seedlings increased most of the study parameters, as well as the DQI value in treatments TA D2, TA D3 and TS D3. The endophytic establishment of *Trichoderma* in plant nurseries may ensure its colonisation once transplanted. As such, in the TA D2 treatment, tomato plants continued to show better quality, without any additional application of *Trichoderma*, and plant quality improved in other treatments with *Trichoderma* applied separately. The poorest results were obtained for mixtures of both species, with no improvement in study parameters for any dose tested, and even a reduction of 21.62%, 10.63% and 25% in stem length, diameter and shoot dry weight of tomato seedlings in the MD3 treatment, respectively. Similar results were found by Liu et al. [59], who reported that the combination of three species, *T. afroharzianum, T. pseudoharzianum* and *T. asperelloides,* decreased the biocontrol and growth-promoting effects in comparison to the application of each species separately.

Although major reductions in the use of chemical fertilisers without production losses is currently difficult in many farming systems, their gradual decrease accompanied by the use of biostimulants or biofertilizers is a tool that can optimise the use of chemical inputs while reducing environmental pollution and food crop contamination.

#### **5. Conclusions**

The present study demonstrated, for the first time, the biostimulant capacity of *T. aggressivum* f. *europaeum* in pepper and tomato plants under commercial plant nursery and greenhouse conditions, with similar results to those of *T. saturnisporum*.

#### **6. Patents**

This isolate was patented with a Spanish patent number ES2706099: New strain of *T. aggressivum* f. *europaeum*, compositions and applications.

**Author Contributions:** F.D. and M.S. conceived and designed the experiments; B.S.-M., F.J.G., and A.M.-G. performed the experiments; M.S. analysed the data; F.D. wrote the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The present work benefited from the input of the project RTC-2017-6486-2 and was supported by the Spanish Ministry of Science, Innovation and Universities.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


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