*3.1. Mass Production of P. variotii on Solid Substrates*

The fungus multiplied well in all mixtures tested. Among the different treatments, whose composition varied in water content and the percentage of oats or buckwheat husk that were tested for mass multiplication of *P. variotii*, the proportion of 70% + 30% for buckwheat husk and oats, respectively, with 20% water content, resulted in significantly higher spore production (Figure 3), followed by 90% + 10% with 10% water content (Table 1). The lowest spore production rate was observed at a percentage of 80% + 20% with 10% water (Figure 3).

**Figure 3.** Mass production of *P. variotii* spores at a proportion of 70% + 30% buckwheat husk and oats, respectively, +20% water (**A,B**) and at 80% + 20% with 10% water (**C**).



BH: buckwheat husk; O: oats. Data were analyzed by ANOVA and treatment means were compared according to Fisher's least significant difference (LSD) statistical procedure (*F*-test at *p* < 0.05).

#### *3.2. Plant Growth-Promoting Characteristics of P. variotii: Siderophore Production, IAA and P Solubilization*

The formation of an orange-colored zone around the fungal colonies was observed, which indicated siderophore production by *P. variotii* (Figure 4A). The diameter of the halo (mm) was estimated at 3.88 ± 0.33, 5.55 ± 0.22 and 8.83 ± 1.29 for 24, 48 and 72 h, respectively.

IAA was produced by *P. variotii* in medium supplemented with 100 mg L−<sup>1</sup> tryptophan during a 7 d period, and the final concentration was 0.049 ± 0.001 mg mL<sup>−</sup>1. The final concentration of IAA was 0.03 ± 0.001 mg mL−<sup>1</sup> in medium supplemented without tryptophan.

No halo of P solubilization by *P. variotii* was detected in any of the media used (NBRIP and PVK media supplemented with 2% agar). The effect of *P. variotii* on the soluble phosphate concentration is shown in Figure 4. The initial concentration of P in the medium was used to quantify the concentration of P solubilized by *P. variotii.* As shown in Figure 4B, no P solubilization was detected during up to 15 d of incubation, assessing a soluble P of 2.01 ± 0.68 g L−<sup>1</sup> versus 0.74 ± 0.25 in the control (*p* = 0.0243). In turn, no change in the pH of the medium was detected, which remained at approximately 7.

**Figure 4.** Formation of orange-colored halos owing to production of siderophores by *P. variotii* (**A**). Effects of *P. variotii* on phosphate solubilization (**B**) in NBRIP broth containing tribasic calcium phosphate (10 g). Mean standard deviation is expressed in error bar. The results are shown as the average of the three replicates, in g L<sup>−</sup>1.

#### *3.3. P. variotii Inoculation E*ff*ects on Tomato and Pepper Seed Germination*

Table 2 outlines the results of the in vitro application of *P. variotii* to tomato and pepper seeds. Direct inoculation of seeds by *P. variotii* isolate spores had a significant effect (*p* < 0.05) on the percent seed germination, root and shoot length, and SVI in tomatoes. The increase in root and shoot length was 18.23% and 17.85%, respectively. However, pepper seeds treated with *P. variotii* showed no improvement in SVI (*p* > 0.05).


**Table 2.** Effects of *P. variotii* isolate on tomato and pepper seed germination 7 and 10 d after treatment, respectively. pp

T0: control without *P. variotii.* Data were analyzed using Student's *t*-test (*p* < 0.05).

#### *3.4. Promoter E*ff*ects of P. variotii Isolates on Tomato and Pepper Seedlings: Experiment 1*

The effects of *P. variotii* application on morphological parameters and DQI are shown in Table 3. Most of the growth parameters evaluated in tomato and pepper were improved by *P. variotii*, when compared with those of the experimental control, with the best results in the development of pepper seedlings.

These increases were statistically significant for most parameters. The increases assessed in pepper and tomato seedlings (P/T) were 9.7%/6.9% for stem length, 4.9%/0.8% for stem diameter, 10.6%/6.0% for leaf number, 18.2–6.7% for root dry weight, 16.7%/10.7% for aerial dry weight and 10.1%/7.5% for leaf area. In tomatoes, *P. variotii* applications resulted in a decrease in root dry weight, albeit without significant differences from that of the control. *P. variotii* application improved plant quality, albeit without significant differences for tomato plants (*p* = 0.2059). In all tests (experiments 1 and 2), *P. variotii* was observed in tomato and pepper roots analyzed in PDA medium.


**Table 3.** Morphological parameters and quality index of pepper and tomato seedlings treated with *P. variotii* isolate at 45 d after sowing.

T0: control without *P. variotii.* Data were analyzed using Student's *t*-test (*p* < 0.05).

### *3.5. E*ff*ects of Dose of Application of P. variotii Isolates on Tomato Seedlings and Transplanted Plants: Experiment 2*

The effects of applying three doses on the development of tomato seedlings under commercial plant nursery conditions and subsequent transplantation into soil under greenhouse conditions are shown in Table 4. As shown in Table 4, applying different doses of *P. variotii* promoted seedling shoot development, significantly decreasing, in some cases, root development. The best results were assessed for dose 3, which resulted in a 30.2% increase in stem length, 15.7% in stem diameter, 19.5% in leaf number and 46.2% in aerial dry weight. However, root dry weight decreased 25.0%. The decrease in root length can cause stress when transplanting tomato seedlings, thereby decreasing plant quality. The DQI [45] expresses the global aptitude of a plant to successfully overcome the transplantation phase, based on overall plant development, while considering the balance between plant shoots and roots. Higher values of this index indicate higher seeding quality. In this case, despite the decrease in root length, a higher value of this index was observed for dose 3, with a significant difference for all doses tested, but not with the control treatment.

**Table 4.** Morphological parameters and Dickson quality index (DQI) of tomato seedlings (30 d after sowing) and plants (90 days after transplanting) treated with different doses (104, 105 and 106 spores per plant, (D1, D2 and D3, respectively) of *P. variotii.*


T0: control without *P. variotii.* Different letters indicate significant differences according to the one-way ANOVA test (*p* = 0.05). Treatment means were compared according to the Fisher's LSD statistical procedure (*F*-test at *p* < 0.05).

Once transplanted in soil, the findings showed that the three doses favored several morphological parameters studied in the development of tomato plants in relation to those of control plants, with higher values for dry weights and DQI but without significant differences for the three doses tested. The establishment of the endophytic fungus at the root (Figure 5), therefore, enabled its effects to persist after transplantation.

**Figure 5.** *P. variotii* mycelium and conidiophores colonizing tomato (**A**: 100×; **B**: 200×) and pepper roots (**C**: 400×).

#### **4. Discussion and Conclusions**

In this study, fungal isolates from surface-sterilized root segments of native plants and soil were collected from the CGNP. This park is located in an arid-to-semiarid Mediterranean region where the predominant fungi genera are *Penicillium* and *Aspergillus*, as well as isolates from the group termed Dark Septate Endophytic (DSE) fungi, which colonize plant roots. The DSE fungi isolated herein formed dark brownish microsclerotia-like structures. The role of DSE fungi in nature has been considered to be similar to that of mycorrhizal fungi [46,47]. These isolates will be analyzed in future studies. Among all isolates, the plant growth-promoting capacity of the only isolate from *P. variotii* was analyzed in this study.

Nevertheless, *P. variotii* was selected given the sparse literature on this species as a plant-growth promoter. The ability of *P. variotii* to stimulate plant growth is poorly studied, while for other species of the same genus, the growth-promoting effect is generally associated with an improvement in the plant status for its nematicide effect for the control of diseases caused by different nematode species [48].

A key requirement for selling microorganisms with a biostimulant capacity is that they have a high-spore production capacity in substrates with a low-production cost. Nevertheless, grains whose agitation leads to a rapid release of spores must be selected to obtain the formulation. For this reason, buckwheat husk and oats were selected for high performance in the spore production of *P. variotii.* This production occurred in the entire substrate, not only on the surface. Generally, commercial spore production methods often only use cereal grains, rice or other starch-based substrates [49]. The nutritional composition of the medium in which the growth promotor multiplies can affect its biocontrol or biostimulant capacity [50]. Based on our results, *P. variotii* can stimulate plant development under the production conditions described herein.

The *P. variotii* isolate has shown the ability to produce siderophores and IAA. Biostimulation is generally associated with increased nutrient availability, similar to biofertilization [51], but it is also caused by multiple other factors, such as mechanisms including enhancement of plant systemic resistance [52]. Thus, siderophore production plays a key role by enhancing the Fe uptake of plants and can be considered an ecofriendly alternative to the use of chemical fertilizers and pesticides in the agricultural sector [53]. Vala et al. [54] describe the production of both hydroxamate and carboxylate-type siderophores by *P. variotii* isolated from the surface of mangrove plants. Terrestrial *P. variotii* has previously been shown to secrete the trihydroxamate siderophore ferrirubin [55]. The *P. variotii* isolate has shown a relatively high IAA production in relation to its genus, and tryptophan incorporation slightly increased in vitro IAA biosynthesis by *P. variotii*. Other studies also reported that Trp considerably stimulated microbial IAA yield in vitro [56–58]. Ali [59] described IAA production by *P. variotii* lower than 2 ng mL−<sup>1</sup> produced in Czapek medium supplemented with tryptophan. In our study, we described a much higher production for the same species. Waqas [60] described IAA production by *P. formosus* at 34.07 ± 3.92 μg mL<sup>−</sup>1, a result very similar to the findings of this study, and consequently, a plant growth-promoting effect. Nevertheless, Waqas [61] analyzed the variability of IAA production as a function of the culture medium used and conversely found that *P. variotii* had virtually undetectable phosphatase activity. These results differed from the high phosphatase production by a *P. variotii* isolate from the medicinal plant *Carallum*a *acutangula* [59]. In soil, P-solubilizing fungi constitute approximately 0.1–0.5% of the total fungal population [62]. The use of microbial inoculants (biofertilizers) possessing P-solubilizing activities in crop productivity is considered as an alternative to further application of mineral P fertilizers [63]. Endophyte co-inoculation of plants exponentially improved the phosphatase activity of soil compared to that of the non-inoculated plants under stress conditions [64]. Etesami et al. [65] showed the relationship among ACC deaminase activity, IAA production, siderophore production and phosphate solubilization of bacterial strains and their effect on root elongation of rice seedlings. In this study, this correlation could not be determined since an isolate of *P. variotii* was analyzed.

The application of beneficial microorganisms (biopriming) may not only help to improve germination and vigor parameters but also relieve a wide range of physiological, abiotic and biotic stresses in both seeds and seedlings [66]. Nevertheless, biopriming can potentially lead to a more resistant plant after transplanting. This can depend on numerous factors, such as plant species, microorganisms, applied dose and substrates, among others. Thus, in this study, the *P. variotii* isolate enhanced germination and root vigor in tomato seeds. However, the results with pepper plants were different, with no significant differences from that of the controls and even with a 19.27% decrease in shoot length (*p* < 0.05). This may be because germination of pepper seeds is often more heterogeneous. Cochran [67] determined that the germination percentage and the accumulation of dry matter in the large seeds of bell pepper were higher in relation to the small seeds. *P. variotii* enhanced several tomato and pepper seedling parameters. The application doses for different crops must be studied for optimal outcomes. In this study, the application of a higher dose (106 spores mL−<sup>1</sup> versus 10<sup>5</sup> spores mL<sup>−</sup>1) improved the different morphological parameters value in tomato seedlings under commercial plant nursery conditions. After transplanting in the greenhouse, the biostimulant effects persisted 4 months after applying the three doses without any additional application. Similar benefits have been observed as a result of the application of other species of the genus *Paecilomyces*. Waqas [68] described improved soybean seedling germination and SVI when applying the endophyte *Paecilomyces* sp. The application of *P. variotii* extracts significantly increased cherry radish yield, dry matter accumulation, the root–shoot ratio and quality. This extract has a very high biological activity, with a low cost, which has a great application prospect [69]. Similar studies conducted by Anis [70] showed increased sunflower seedling vigor under biotic stress conditions when applying *P. variotii* and *Macrophomina phaseolina* spores, in vitro, with no favorable results when conducting the tests in pots. In turn, Maitlo [71] assessed chickpea plant biostimulation when inoculating them with *P. lilacinus* and *F. oxysporum f.* sp. *ciceris*, which also reduced chickpea wilt.

In intensive horticulture under plastic, the benefits of the application of biostimulants or biofungicides based on microorganisms, are in question, because farmers perceive low efficacy of these products as disease controllers when compared with the rapid response presented by chemical fertilizers or fungicide. The current changes in legislation regarding the reduction of active ingredients and the commercialization of biostimulants and biopesticides, together with the need to increase the sustainability of agriculture in terms of public health and the environment, require the use of PPMs as a key element in horticulture.

To the best of our knowledge, the present study demonstrated for the first time the biostimulant capacity of *P. variotii* in pepper and tomato plants under commercial plant nursery and greenhouse conditions.

#### **5. Patents**

This isolate was patented with a Spanish patent number ES2684858A1: New strain of *Paecilomyces variotii*, compositions and applications.

**Author Contributions:** F.D. and M.S. conceived and designed the experiments; A.M.-G. and B.S.-M. performed the experiments; F.D. analyzed the data; M.S. 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 was supported by the Spanish Ministry of Science, Innovation and Universities.

**Conflicts of Interest:** The authors declare that there is no conflict of interests regarding the publication of this manuscript.

#### **References**


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