In agriculture, especially in low-input or organic cultivation, plants can benefit from interactions with microorganisms, such as arbuscular mycorrhizal fungi (AMF) (He and Nara, 2007). In this case, their extraradicular mycelium provides a wide nutrient interception area and a high nutrient uptake efficiency, decreasing demands on applied fertilizers and reducing soil nutrient loss, in addition to improving plant nutrition and growth (Hogberg, 1989; Finlay and Söderström, 1992; He and Nara, 2007; Cavagnaro et al., 2015). Furthermore, evidence exists that AMF can acquire nutrients from organic matter, probably after its mineralization, and transfer them to the host plant (Hodge et al., 2001; Hodge and Fitter, 2010). However, fundamental studies and production of a high quality inoculum (free of impurities) of AMF have been limited by the fact that such fungi are obligate biotrophs, requiring establishment of effective associations with metabolically active roots for their development and multiplication (Sylvia and Jarstfer, 1994).

Dark septate endophytic (DSE) fungi are quite diverse facultative biotrophic ascomycetes characterized by dark pigmentation, melanized septate hyphae, and formation of microsclerotia inside the plant roots. Such fungi are capable of colonizing root tissues intra and intercellularly of more than 600 plant species [including Solanum lycopersicum (L.) and those non-mycorrhizal ones] without causing pathologies. They can also act as plant growth promoters (Jumpponen and Trappe, 1998; Upson et al., 2009; Andrade-Linares et al., 2011a,b; Yuan et al., 2011; Mahmoud and Narisawa, 2013).

Tomato plants belongs to the family Solanaceae and have been determined to be the second most economically important vegetable worldwide after the potato (Foolad, 2004; Reis Filgueira, 2008). Tomato fruits are an important source of lycopene, which exhibits antioxidant, hypolipidemic, and anti-carcinogenic properties (Viuda-Martos et al., 2014). In addition, tomatoes are cholesterol free and rich in mineral salts, vitamins A and C, and fibers (Block et al., 1992; Gerster, 1997; Rao and Agarwal, 2000; Viuda-Martos et al., 2014).

Studies have suggested that DSE fungi are capable of accessing complex carbon (C), nitrogen (N), and phosphorus (P) compounds within the soil when associated with plants (Mandyam and Jumpponen, 2005), making nutrients available to their host plant (Mandyam, 2008). This is probably because they produce hydrolytic enzymes, which induce release of nutrients absorbed by plants. Additionally, organic compounds taken up by DSE fungi, including amino acids and small peptides, are transferred directly to host plants (Jumpponen and Trappe, 1998), resulting in better use of organic nutrient sources (Reeve et al., 2008).

In previous studies, three isolates of DSE fungi, A101, A104, and A105 obtained from wild rice (Oryza glumaepatula [Steud.]) were determined by ITS phylogeny to belong to the order Pleosporales (suborder Massarineae) (A104 and A105) and to an unknown taxon (A101) (Ribeiro, 2011; Vergara et al., 2017). These fungi colonize wild [Oryza glumaepatula (Steud.)] and commercial [Oryza sativa (L.)] rice and tomato (cv. Santa Clara I-5300) plants, with no apparent symptoms of disease (Ribeiro, 2011; Vergara et al., 2017). Furthermore, a neutral effect was observed in the N content of tomato plants inoculated with these fungi when NH₄NO₃ (an inorganic source) was supplied to the plants.

In the present study, the effects of inoculation with three DSE fungi on tomato plants (cv. Santa Clara I-5300) was evaluated in non-sterile sand soil in greenhouse conditions. Plant growth, nutrient accumulation (N, P, K, Ca, Mg, Fe, Mn, and Zn), the amount of N in the plants derived from ammonium sulfate-¹⁵N or from a finely ground legume Canavalia ensiformis-¹⁵N, and recovery efficiency of ¹⁵N, P, and K by plants were determined. Thus, the present study investigated the effects of DSE inoculation on nutrient recovery efficiency, which is the proportion of a given fertilizer nutrient taken up by a plant, nutrient accumulation, and growth of tomato plants fertilized with organic and inorganic N sources.

Canavalia ensiformis (L.), a plant that is extensively used in tropical agriculture as a green manure for nutritional enrichment of soils (Rodrigues et al., 2004), and ammonium sulfate were used as N source for tomato plants inoculated with DSE fungi. To study the differences between these N sources, two experiments were conducted in parallel: (i) an experiment with the inorganic N source, ammonium sulfate; and (ii) an experiment with the organic N source, C. ensiformis. Ammonium sulfate is an important source of N, but also provides sulfur for plants. On the other hand, green manure C. ensiformis (a legume) contributes considerable amounts of N to the soil-plant system due to its association with N₂ fixing bacteria (Perin et al., 2003). C. ensiformis also maintains soil moisture (Tejada et al., 2008) and mobilizes nutrients such as P and some micronutrients (Lambers et al., 2015).

The DSE fungi tested in this study were obtained from wild rice (O. glumaepatula) collected from the Amazon region and identified by the ITS phylogeny to belong to order Pleosporales (suborder Massarineae) (A104 and A105) and an unknown taxon (A101) (Ribeiro, 2011; Vergara et al., 2017). DSE fungi were able to colonize the roots of tomato plants with no negative symptoms, even when the hyphae colonized the epidermis and the cortex of root plants and formed microsclerotia in the roots cells. Other studies have also reported colonization by DSE fungi in epidermis and cortex cells of tomato plants with no symptoms of pathologies (Andrade-Linares et al., 2011a; Mahmoud and Narisawa, 2013).

Similar to previous works on plant responses to inoculation with DSE fungi (Usuki and Narisawa, 2007; Diene et al., 2013; Mahmoud and Narisawa, 2013; Qin et al., 2017), the present study showed that inoculation with DSE fungi led to an increase in aboveground dry biomass of tomato plants supplied with an organic N source (finely ground C. ensiformis-¹⁵N). The same effect was not observed in plants treated with an inorganic N source (ammonium sulfate-¹⁵N). Conversely, Andrade-Linares et al. (2011b) reported that the DSE fungus Leptodontidium orchidicola was capable of increasing the shoot dry matter of tomato plants, as well as the fruit mass and glucose content, even in plants grown with an inorganic N source (NH₄NO3-).

A meta-analysis was performed from 18 studies carried out independently and confirmed that plants inoculated with carefully-selected DSE fungi responded positively when supplied with organic N sources, showing increases in dry matter and contents of N and P ranging from 26 to 106% (Newsham, 2011). Similarly, in the present study, which was carried out under greenhouse and non-sterile soil conditions, the DSE-tomato interaction was more efficient with use of N from an organic source. Increases were observed in the amount of nitrogen in the plant derived from finely ground C. ensiformis-¹⁵N, in the recovery efficiency of ¹⁵N, P, and K, in the contents of N, P, K, Ca, and Mg, plant height, leaf number, and total leaf area (A104), leading to increases in aboveground dry biomass ranging from 25 to 41% in comparison with control plants. Such results indicate that the DSE fungi A101, A104, and A105 can enhance tomato plant nutrition and growth when fertilized with an organic N source. Conversely, plants supplied with an inorganic N source only showed a positive inoculation effect for fertilizer-K recovery efficiency by plants, K content, and leaf area (A104), while other evaluated variables showed no effect. This suggested that the cultivar Santa Clara I-5300 does not need the fungus to absorb readily assimilable N as N-NH4+, since breeding programs have frequently selected for plants more responsive to mineral fertilization (Borlaug and Dowswell, 1997). In a previous study performed under controlled hydroponic conditions, significant increases were not also observed in shoot N content using inorganic N sources (Torres-Júnior, 2014), although experimental conditions were different from those of the present study.

The ability of DSE fungi to promote growth and increase nutrients contents in tomato plants fertilized with finely ground C. ensiformis-¹⁵N suggests that these fungi are capable of degrading organic C, N, and P compounds, increasing macro and micronutrient availabilities to plants. Indeed, studies have reported that DSE fungi degrade organic compounds including cellulose, starch, proteins, lipids, amino acids, gelatin, urea, and pectin under in vitro conditions (Caldwell et al., 2000; Menkis et al., 2004; Mandyam et al., 2010; Surono and Narisawa, 2017). Additionally, DSE fungi promote the growth of several plant species supplied only with an organic N source (amino acids) or organic P source (phytate) under in vitro conditions (Usuki and Narisawa, 2007; Diene et al., 2013; Mahmoud and Narisawa, 2013; Surono and Narisawa, 2017). Recently, three isolates of Phialocephala fortinii led to the growth of Asparagus officinalis (L.) in agar medium supplied only with corn steep liquor (0.1%) or with corn steep liquor amended with inorganic nutrients (Surono and Narisawa, 2017). Although the preference in degrading a specific organic nutrient source can differ among species of DSE fungi (Diene et al., 2013; Surono and Narisawa, 2017), studies have suggested that DSE fungi are capable of mineralizing organic compounds containing N and P, making them available to plants. A recent study demonstrated that DSE fungi are capable of mineralizing organic P compounds under in vitro conditions (Della Monica et al., 2015). However, the mechanisms leading to an increase in the growth of plants inoculated with DSE fungi still need further research.

The highest K content and fertilized-K apparent recovery efficiency was observed for fertilization with ammonium sulfate-¹⁵N (for the fungus A104) and with finely ground C. ensiformis-¹⁵N (in all inoculation treatments), indicating that the DSE-tomato interaction was more responsive to K compare to other evaluated nutrients. This may be because K is the most required cation for plants and the most abundant cation in cytosol and plant dry matter (Marschner, 1995; Meurer, 2006). K acts in several physiological processes in plants, including photosynthesis, activation of more than 60 enzymatic systems, regulation of stomatal opening and closure, cell growth and elongation by generating cell turgor, and protein synthesis (Marschner, 1995).

During uptake K is transported from the soil solution to the root surface mainly by diffusion. Mass flow can significantly contribute to transport when the K concentration is high in the soil solution (Barber, 1995; Ruiz et al., 1999). However, diffusion is limited to very short distances from the root surface, usually around 1–4 mm (Meurer, 2006; Zeiger et al., 2017). Thus, the high K content and fertilizer-K apparent recovery efficiency observed under inoculation treatments strongly indicated that these fungi helped tomato plants absorb K through transposition of the K depletion zone and K interception in remote locations unattainable by the root surface.

Higher K and N contents are directly related to a greater total leaf area, since K acts on cell extension and N acts on leaf elongation, leading to a greater leaf area (Meiri et al., 1992; Chapman and Lemaire, 1993; Neto et al., 2007) and thus to greater plant exposition to sunlight (Neto et al., 2007). Contrary to previous studies (Melin, 1922; Wilcox and Wang, 1987; Stoyke and Currah, 1993; Mandyam et al., 2010; Diene et al., 2013), negative effects of inoculation with DSE fungi on plant growth were not observed.

Dark septate endophytic (DSE) fungi can also facilitate the uptake of micronutrients, including Fe, present in the soil (Bartholdy et al., 2001; Haselwandter, 2009). In this study, inoculation led to an increase in Fe content from 72 to 178%, and in Mn and Zn contents from 17–25% to 41–46%, respectively. These increases in Fe, Mn, and Zn contents under inoculation are related to a better use of these micronutrients in their source (soil, FTE BR12, and C. ensifomis-¹⁵N). Bartholdy et al. (2001) observed that DSE Phialocephala fortinii synthesized siderophore hydroxamate which led to an increase in Fe (III) uptake by host plants. When absorbed by plants, Fe acts on redox reactions in hemoproteins, such as cytochromes, and in non-heme proteins, such as ferredoxin (Dechen and Nachtigall, 2006). Ferredoxin and cytochromes are carriers of electrons during photosynthesis (Dechen and Nachtigall, 2006). Zn and Mn also act on enzymes involved in C metabolism. For instance, Zn is a structural component of carbonic anhydrase, which catalyzes CO₂ dissolution (previous to its assimilation), and activates triphosphate dehydrogenase, an essential enzyme for glycolysis (Dechen and Nachtigall, 2006). Mn acts on photosystem II (in the Mn complex), which is responsible for water photolysis (Broadley et al., 2012). Currently, Mn has been recognized as an indicator of P acquisition efficiency by plants (Lambers et al., 2015). Thus, an increase in Fe, Mn, and Zn contents corroborates previous studies (Zhang et al., 2012, 2017; Ban et al., 2017) which suggested involvement of DSE fungi in photosynthetic activity by increasing chlorophyll levels, photochemical efficiency of photosystem II, net photosynthetic rate, stomatal conductance, and transpiration rate, leading to higher glucose (Andrade-Linares et al., 2011b) and soluble sugar contents (Vergara et al., 2017).

In general, tomato plants fertilized with finely ground C. ensiformis-¹⁵N showed significant differences among inoculation treatments, especially fungus A104, which had greater leaf number and area, and fungus A105, which had a higher Fe content. On the other hand, plants treated with ammonium sulfate-¹⁵N showed significant differences for K content and fertilizer-K apparent recovery efficiency, with an emphasis on fungus A104. These differences suggest that plant responses to inoculation with DSE depend on each fungus isolate, as it has already been observed in AMF inoculation (Mensah et al., 2015).

Our findings indicated that tomato plants inoculated with DSE fungi acquired macro and micronutrients more efficiently, especially when an organic N source is used, resulting in increased plant growth. This finding was demonstrated using finely ground ¹⁵N-labeled C. ensiformis or ammonium sulfate-¹⁵N applied to soil. The A104 isolate seems to be the best option for inoculation of tomato cv. Santa Clara I-5300. However, a more detailed understanding of biochemical and molecular DSE-tomato interactions is still needed.

CV, KA, SU, NS, FB, PM, LS, and JZ designed, performed experiments and analyzed data. CV, KA, FB, GX, and JZ conceived the experiments and wrote the paper. All authors read, edited, and approved the final manuscript.