**1. Introduction**

The success of applying *Trichoderma* in agriculture results from the multiple benefits that it generates in plants. Thus, the genus *Trichoderma* is characterised by its strong competitive and reproductive potential, presenting high survival rates under unfavourable or abiotic stress conditions, such as salinity [1], water stress [2], or the presence of various toxic chemicals, including fungicides [3], among others. Similarly, *Trichoderma* exhibits high efficiency in the promotion of nutrient uptake [4], the capacity to modify the rhizosphere and root structure in which the fungus is established [5,6], high aggressiveness against plant pathogenic fungi, efficiency in the promotion of plant growth [7–12], and the ability to induce plant defence mechanisms, among many additional benefits [8,9,13]. The properties of *Trichoderma* have generated considerable research interest in these fungi for use in agriculture, and a large number of commercial products have been developed using different *Trichoderma* species [10,14]. Many formulations contain mixtures of different species that provide a wider range of direct and indirect beneficial effects for the plants. Numerous studies have reported the benefits

of *Trichoderma* application for plant growth and even increased production yield. Thus, applying *Trichoderma* species, to both soil and seeds, allows the multiplication of the fungus in conjunction with the developing root system [15]. Its ability to colonise plant roots from the appressorium-like structure directly enhances seed vigour [16] and germination and promotes seedling growth [10,11,17]; thereby, suggesting that these fungi should be applied from the plant nursery stage in the case of horticultural, ornamental, or forest species, which would allow the early colonisation of the roots by *Trichoderma*, before transplanting the seedlings in the field.

It has been reported that plant growth is enhanced in association with *Trichoderma* species similar to that of other plant-growth-promoting microorganisms (PGPMs), but the effects are greater with *Trichoderma* when plants are under biotic, abiotic, or physiological stress conditions [9,18–21]. Recently, *T. aggressivum* f. *europaeum* has been described as a melon seedling growth promoter under saline stress conditions, in addition to its capacity to control *Pythium ultimum*, decreasing the severity of the disease in seedlings [1]. *Trichoderma aggressivum* Samuels & W. Gams is the causal agent of the green mould disease, which causes economic losses in the cultivation of white button mushrooms (*Agaricus bisporus* (J.E. Lange) Imbach) worldwide. There are two subspecies, *T. aggressivum* f. *aggressivum* and *T. aggressivum* f. *europaeum* found in North America and Europe, respectively [22]. *Trichoderma aggressivum*, a fast-growing filamentous fungus, colonises compost and casings used as growth substrates in mushroom cultivation and produces dense white mycelial colonies that change colour to green after sporulation [23]. This aggressive competitor is known to produce metabolites that are toxic to *A. bisporus* [24,25]. In areas colonised by *T. aggressivum*, fruit body formation is retarded, and fruit bodies may be of poor quality because of damage or discolouration [23]. Numerous *Trichoderma* species have been isolated from *Agaricus* compost and *Pleurotus* substrates, such as *T. harzianum, T. longibrachiatum, Trichoderma ghanense, T. asperellu,* and *T. atroviride*, although its aggressiveness has not been determined [26]. Sánchez-Montesinos et al. [1] demonstrated its high mycelial growth and sporulation on roots. Thus, *T. aggressivum* f*. europaeum* is a potential biofertilizer for different crops. In our study, the growth-promoting capacity of this species has been analysed in comparison to that of *T. saturnisporum* Ca1606, which was recently characterised as a biocontrol agent and a seedling growth promoter for different horticultural plants [11,16,27]. Since the effectiveness of microorganisms as growth promoters will depend on the crop, dose and application method, among many other factors, further studies on *T. aggressivum* f. *europaeum* are needed to determine its efficacy.

Consequently, in the present study, *T. aggressivum* f*. europaeum* Tae52481 and *T. saturnisporum* Ca1606, were tested to evaluate: (a) the effects of direct application to seeds of a fungus suspension on root colonisation of tomatoes and peppers and subsequent plant vigour; (b) the promotion of growth and quality of pepper and tomato seedlings under a conventional production system; and (c) the effects of applying different doses and the synergistic effect of both isolates on tomato seedlings and on their subsequent transplantation under greenhouse conditions.

### **2. Materials and Methods**

#### *2.1. Fungal Isolates*

*Trichoderma saturnisporum* Ca1606 (TS), already known for their plant growth promotion properties, were extracted from suppressive soils. TS was cultivated on potato dextrose agar (PDA) for 7 days at 25 ◦C in lightless conditions. The growth results measured were used to establish a comparison value.

For this study *Trichoderma aggressivum* f. *europaeum* Tae52481 (TA) were isolated from samples of substrate used for *Agaricus bisporus* cultivation at mushroom farms. These fungal spore samples were similarly cultivated on potato dextrose agar (PDA) for 7 days at 25 ◦C in dark conditions. The corresponding growth results were recorded. The spore suspensions for both samples were prepared using sterile distilled water. A concentration of 1 × 107 spores/mL was achieved with a Neubauer haemocytometer.

#### *2.2. Analysis of Plant Growth-Promoting Attributes*

In accordance with the method of Louden et al. [28], by the transference of fungal mycelial discs (5 mm) of active culture onto Chrome-Azurol S (CAS) agar medium, siderophore production was determined. At 24, 48 and 72 h the diameter of the siderophore colony indicative orange halos on blue were measured.

Indole-3-acetic acid (IAA) production was estimated according to the procedure described by Diánez et al. [16]. Five independent replicates of TA and TS were analysed. This process is described as follows. A glucose peptone broth (GPB) of 50 mL, amended with or without L-tryptophan (Sigma-Aldrich) at a concentration of 100 mg L−<sup>1</sup> was prepared. Flasks containing this broth inoculated with TA and TS were incubated on an orbital shaker at 150 rpm in dark conditions for 7 days at 25 ◦C. Subsequently the supernatants from each flask, having first being centrifuged for 30 min at 12,000× *g* and filtered through sterile Millipore membranes (pore size 0.22 μm), were collected into sterile test tubes. In order to determine the quantity of IAA, optical density tests were carried out and compared to a standard IAA curve. For both the TA and TS, 3 mL of the culture supernatant and 2 mL (0.5 mol L−<sup>1</sup> FeCl3 + 98 mL of 35% HClO4) Salkowski reagent were combined and left for 30 min. The intensity of the resulting red pigmentation density was measured at 530 nm using a scanning spectrophotometer for each of the samples.

To determine the quantitative estimation of phosphate solubilisation, a modified version of the Lima–Rivera procedure [29] was followed. Then, 250 mL capacity flasks containing 50 mL National Botanical Research Institute's phosphate (NBRIP) broth, inoculated with two 5 mm pure *Trichoderma* isolates agar disks were agitated at 100 rpm and incubated at 26 ◦C for 3, 5, 7, 10 and 15 days. As a control the procedure was carried out on uninoculated flasks containing the same NBRIP broth. The experiments were conducted in triplicate.

Using the Fiske and Subbarow method [30] phosphate concentrations in culture supernatants were estimated as equivalent phosphate (μg mL<sup>−</sup>1), mean values expressed and pH analysed. The total P (phosphate) in the flasks was 10 mg mL<sup>−</sup>1.

#### *2.3. Mass Production of TA and TS on Solid Substrates*

A mixture of two kinds of substrates, one containing buckwheat husk (BH) and oat (O), the other containing BH and rice (R) were tested for the mass multiplication of TA and TS [31]. Different proportions of BH-O (90–10%, 80–20% and 70–30% *v*/*v*) and BH-R (90–10%, 80–20% and 70–30% *v*/*v*) were submerged in 30% *v*/*v* of water for 24 h. Each mixture was sterilised for 1 h at 125 ◦C twice on consecutive days. Each mixture was placed on a tray and aseptically inoculated by spraying with 5 mL of spore suspension containing 1 × 10<sup>7</sup> spores mL−<sup>1</sup> of each isolate. The trays were kept at 25 ◦C in the dark for 15 days. In total, three samples (2 g) of the fungus-colonised substrate were removed from the trays in each treatment. The samples were successively diluted in sterile distilled water + 0.01% Tween 20® and the number of conidia g−<sup>1</sup> of the solid substrate was quantified for each replicate using a Neubauer haemocytometer. There were three replications per treatment. The collected spores were used in the different experiments conducted in this study.

#### *2.4. Analysis of E*ff*ects of TA and TS on Seed Germination under Laboratory Conditions*

Three treatments (control, TA and TS) and four repetitions following a random block experimental design were implemented in this study. For each repetition of the three treatments 50 seeds of tomato (*Solanum lycopersicum* 'Red Cherry') and pepper (*Capsicum annuum* 'Largo de Reus') were germinated on two sheets of sterile distilled water moistened Whatman No. 1 filter paper in (150 mm) Petri dishes. These seeds were first surface sterilized for 5 min with 1.5% sodium hypochlorite (NaOCl), rinsed twice with sterile distilled water and dried under laminar airflow on sterile paper [16]. Germination was achieved by treating the seeds with 50 μL of spore suspension (1 × 105 spores mL<sup>−</sup>1) of TA, TS or 50 μL of sterile water (control). The trays were placed in a lightless incubator at 25 ± 1 ◦C, 7 days

for tomato and 10 days for pepper seeds. For each Petri dish treated with one of the three solutions (control, TA and TS), percent germination, root length and shoot length of tomato and pepper seeds were recorded. A Seed Vigour Index (SVI) was calculated as follows: SVI (length) = seed germination% (mean root length + mean shoot length) [32].

#### *2.5. Analysis of Promoter E*ff*ects of TA and TS on Pepper and Tomato Seedlings: Experiment 1*

The following experiment was conducted in autumn using a completely randomised design at a commercial nursery (Almería, Spain). Pepper (*Capsicum annuum* 'Largo de Reus') and tomato (*Solanum lycopersicum* 'Red Cherry') seeds were sown in 96-cell commercial peat mix filled nursery polystyrene planting trays (70 mL volume) and covered with vermiculite. Trays were relocated to a greenhouse and rinsed with sterile distilled water (control), or a 5 mL (TA or TS) spore suspension per cell at 105 spores per plant, after a 2 day (tomato) or 4 day (pepper) period in a germination room (relative humidity (RH) = 95%; 25 ◦C). Four trays of seedlings for each treatment were cultivated under standard nursery culture conditions (18–28 ◦C; 75.4 ± 6.7% RH). Then, 20 plants per treatment and control were randomly selected from the four replications at 45 days after sowing across the four replications. Different growth parameters: number of leaves, stem length, stem base diameter, total leaf area and root dry weights, as well as leaf area using the WINDIAS 3.1 of the plants, were measured. The formula: DQI = TDW/((LS/D) + SDW/RDW)) where TDW is the total dry weight (g), LS is stem length (cm), D is stem diameter (mm), SDW and RDW are stem and root dry weight (g), respectively; they were employed to determine the Dickson Quality Index (DQI) [33].
