**1. Introduction**

The genus *Paecilomyces* includes more than 100 species known for their multiple activities and habitat heterogeneity [1]. Among them, *Byssochlamys spectabilis*(Udagawa and Shoji Suzuki) Houbraken and Samson, formerly known as *Paecilomyces variotii* Bainier, is an ascomycete characterized by its ability to produce secondary metabolites, which belong to different chemical groups with wide biological activity [2–5]. This species has been described as a biological control agent (BCA) against nematodes [6,7], trematode eggs [8] and phytopathogenic fungi, such as *Biscogniauxia mediterranea*, *Fusarium moniliforme* and *Phytophthora cinnamomi* [9], *Pyricularia oryzae* [10], *Fusarium graminearum* [11] and *Magnaphorte oryzae*[12], among others, that function through their raw extracts, secondary bioactive metabolites or their mycelia. *P. variotii* produces metabolites with herbicidal [13] and insecticidal [14] activity and has been reported to control infections caused by pathogenic bacteria in fish [15] and humans [9]. In turn, this fungal species has even been shown to degrade aromatic compounds [16,17], in addition to removing ammonium from synthetic media and reducing ammonia emissions from chicken manure. [18]. However, it is also associated with many types of human infections in immunosuppressed patients [19]. Nevertheless, only studies related to the possible activity of these metabolites as a hormone-like substance or a promoter of phytohormone production by plant hosts have been published to date [20]. Very few references describe the application of *P. variotii* as a plant-growth promoter. The biocontrol agent ZhiNengCong (ZNC), which is an extract of *P. variotii,* is used in China [21]. ZNC is a

highly effective plant elicitor that promotes plant growth by inducing auxin accumulation in root tips with low concentrations [21].

The use of plant probiotic microorganisms (PPMs) is an effective alternative to the use of chemical fertilizers [22–24]. The most studied PPMs are plant growth-promoting bacteria (PGPB), although there are numerous examples of plant growth-promoting fungi (PGPF), which increase crop yield [25–27]. Thus, the most relevant are those that establish endosymbiotic relationships, such as arbuscular mycorrhizal fungi, which solubilize nutrients, such as phosphorous, and micronutrients absorbed by plants [28–30]. *Trichoderma* is one of the most studied genera as PGPF [31], although many others fungi have demonstrated their potential growth-promoting capacity, such as *Penicillium oxalicum* [32], *Penicillium simplicissimum* [33], *Fusarium oxysporum* [34], *Fusarium equiseti* [35], *Alternaria* sp. [36], *Aspergillus* spp. [37] and *Phoma* [38], among others.

In the present study, *P. variotii*, an endophytic fungus isolated from plant roots from the Cabo de Gata Natural Park (Parque Nacional Cabo de Gata—Spain), was tested to evaluate: (a) the effects of seed priming with a fungus suspension on root colonization and tomato and pepper plant vigor; (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 to tomato seedlings and their subsequent transplantation in a greenhouse.

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

#### *2.1. Isolation of P. variotii from Plant Roots*

Twenty roots of different species of autochthonous plants from the Cabo de Gata Natural Park (CGNP; Almería, Spain) were collected for the isolation of fungal organisms in 2017. Collected samples were cleaned under running tap water to remove debris before use, air dried and processed for isolation of endophytic fungi. To remove epiphytic and surface-adhering microbes, the roots were cut into small, 2–3 cm long, pieces, were surface-sterilized with 2% sodium hypochlorite for 3 min and washed three times with sterile distilled water. The surface-sterilized samples were allowed to dry on sterile paper towels. Ten fragments from each root were placed onto potato dextrose agar (PDA, Difco) supplemented with 50 μg mL−<sup>1</sup> chloramphenicol to suppress bacterial growth. After incubation at 25 ◦C for 7 d, individual hyphal tips of the developing fungal colonies were removed, placed on PDA medium and incubated for 5–7 d.

Colony morphology of the pure cultured isolates on PDA and conidiophore morphology were examined and identified by light microscopy, and all selected isolates were stored for further studies. Only one isolate, whose identification under a microscope was consistent with the genus *Paecilomyces* (Figure 1), was selected for this study (*P. variotii* CDG33). Molecular identification of the selected fungi was conducted following the procedure described by Diánez et al. [26]. The sequence was analyzed using a BLAST search in the GenBank database of the National Centre for Biotechnology Information (NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi) and aligned to the nearest neighbors. The sequence has not been deposited in the GenBank database because the isolate is subject to patent.

The culture of *P. variotii* has been deposited in the CECT (Spanish Type Culture Collection, Valencia, Spain) with the collection number CECT 20957. This strain was selected for the experiments based on the results of a preliminary assay (data not shown).

**Figure 1.** Conidiophore of *Paecilomyces variotii* (**A**: 400×; **C**: 200×; **D**: 100×) and an aspect of colony morphology (**B**) in the potato dextrose agar (PDA) medium.

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

Siderophore production was determined on the chrome-azurol S (CAS) medium following the method of Schwyn and Neilands [39] and Louden et al. [40]. Fungal mycelial discs (5 mm) of active culture were transferred to CAS medium and orange halos around the colonies on blue were indicative of siderophore production. The diameter of the orange halo was measured at 24, 48 and 72 h.

Indole-3-acetic acid (IAA) production was estimated according to the procedure described by Diánez et al. [26]. *P. variotii* was grown in 50 mL of glucose peptone broth (GPB) amended with or without L-tryptophan (Sigma-Aldrich) at a concentration of 100 mg L<sup>−</sup>1. The flasks were inoculated and incubated on an orbital shaker at 150 rpm at 25 ◦C in the dark for 7 d. After incubation, the suspension from each flask was centrifuged for 30 min at 12,000× *g*. The supernatant was filtered through sterile Millipore membranes (pore size 0.22 μm) and collected in sterile tubes. The culture supernatants (3 mL) were pipetted into test tubes, and 2 mL Salkowski reagent (2 mL of 0.5 mol L−<sup>1</sup> FeCl3 + 98 mL of 35% HClO4) was added to it. The tubes containing the mixture were left for 30 min for red color development. The intensity of the color was determined by measuring the optical density at 530 nm using a scanning spectrophotometer. The quantity of IAA was determined by comparison with a standard curve for IAA. Five independent replicates of *P. variotii* were analyzed.

The qualitative evaluation of the phosphorus solubilized by *P. variotii* was performed using NBRIP and PVK media supplemented with 2% agar (Difco Laboratories, Detroit, MI, USA). Phosphate solubilization was detected by the formation of transparent zones surrounding fungal colonies in both media [41]. For the quantitative estimation of phosphate solubilization, a modified version of the procedure by Lima-Rivera [42] was followed. Flasks (250 mL capacity) containing 50 mL of NBRIP broth were inoculated with two disks of agar (5 mm diameter) that had been taken from pure cultures of *P. variotii.* Uninoculated flasks were used as a control (three replicates). Incubation was conducted at 26 ◦C at a shaking speed of 100 rpm for 3, 5, 7, 10 and 15 d. Supernatants of each culture were analyzed for pH and phosphate concentration. Phosphates in culture supernatants were estimated using the Fiske and Subbarow method [43] and expressed as equivalent phosphate (μg mL<sup>−</sup>1). The experiments were conducted in triplicate and values were expressed as the mean. The total P in flasks was 10 mg mL<sup>−</sup>1.

#### *2.3. Mass Production of P. variotii on Solid Substrates*

A mixture of two kinds of substrates, buckwheat husk (BH) and oat (O), were tested for the mass multiplication of *P. variotii*. Different percentages (90–10%, 80–20% and 70–30% *v*/*v* BH-O) of both substrates were submerged in different percentages of water (10%, 20% and 30% *v*/*v*) for 24 h. Each mixture was sterilized 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 4 × 106 spores mL−<sup>1</sup> of *P. variotii*. The trays were kept at 25 ◦C in the dark for 10 d. In total, three samples (2 g) of the fungus-colonized 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 hemocytometer. 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 P. variotii on Seed Germination under Laboratory Conditions*

Seeds of tomatoes (*Solanum lycopersicum* 'Red Cherry') and pepper (*Capsicum annuum* 'Largo de Reus') were used in this study. The trial used a random block experimental design with two treatments (control and *P. variotii*) and four repetitions. Each repetition included 50 seeds that were germinated in Petri dishes (150 mm diameter) containing two sheets of Whatman No. 1 filter paper that were moistened with sterile distilled water. The seeds were surface-sterilized sterilized with 1.5% sodium hypochlorite (NaOCl) for 5 min, rinsed twice with sterile distilled water and dried under laminar airflow on sterile paper [26]. Treatments were performed by pipetting 50 μL of *P. variotii* spore suspension (1 × 105 spores mL<sup>−</sup>1) or 50 μL of water (control) on each seed; all boxes were placed in an incubator (25 ± 1 ◦C in the dark). Root length (mm) was measured from the tip of the primary root to the base of the hypocotyl. After 7 and 10 d, for tomatoes and peppers, respectively, percent germination, root length and shoot length were recorded and a seed vigor index (SVI) was calculated as follows: SVI (length) = seed germination% (mean root length + mean shoot length) [44].
