Introduction

The use of chemicals in agriculture has caused an imbalance in many agroecosystems (^{Pretty, 2008}; ^{Raza et al., 2019}). Some of the most notable consequences are the increase of affected areas by salt excess and ground water contamination with toxic ions besides the decrease or disappearance of resident microflora in soils and the increase of pathogens that affect cultivations drastically. All these has brought as a consequence harmful effects on human and animal health (^{Emamverdian et al., 2015}; ^{Plá and Cobos-Porras, 2015}; ^{Singh, 2015}; ^{Piculell et al., 2018}).

Thus, the need to apply compatible products with the environment, which at the same time have a positive influence on cultivation yield, emerges as an urgent priority. Currently, the use of biostimulants in agriculture constitutes the center of attention of the international scientific community, which are natural, act in relatively low doses, and accelerate plant metabolism. (^{Duc et al., 2018}; ^{Hastak et al., 2018}; ^{Abd El-Aziz et al., 2019}).

Ecuador is one of the countries where research is developed related to this topic, above all, applied to vegetable products as tomato (Solanum lycopersicum L.) (^{Reyes et al., 2018}). This cultivation has a high demand, not only in the industry where the greatest volumes are distributed but also for high fresh tomato consumption, estimated around 5 kg per capita (^{FAO, 2018}; ^{SINAGAP, 2013}).

Several bioestimulants have been studied, such as nitrogen-fixing bacteria, products that improve nutrition stimulating growth, development and yield (^{Agbodjato et al., 2016}; ^{Capstaff and Miller, 2018}; ^{Wang et al., 2018}). Among other biostimulants, chitosan, a biopolymer derived from chitin, stands out for its versatility in agriculture application stimulating plant growth (^{Barbosa et al., 2017}; ^{Chun and Chandrasekaran, 2018}; ^{Divya et al., 2018a}). Arbuscular mycorrhizal fungi (AMF) are also used in a wide array of cultivations for their capacity to establish symbiosis with plants, facilitating nutrient and water absorption (^{da Silva-Campos et al., 2013}; ^{Ho-Plágaro et al., 2019}). Additionally, the use of humic acids has increased because studies have demonstrated they stimulate plant growth, and intervene in regulatory processes at molecular level that promote plant-soil interface (^{Giachin et al., 2017}; ^{Noroozisharaf and Kaviani, 2018}; ^{Shah et al., 2018}).

Nevertheless, many time biostimulants have been applied jointly, so the contribution of each one is not known in depth and if this contribution changes with different cultivars of one vegetable species. In addition, not much information has been available on how they affect plant nutrient content and if they can correlate with growth indicators. Therefore, the objective of this study was to assess the effect of arbuscular mycorrhizal fungi, chitosan, and humic acid on growth indicators in two tomato cultivars.

Materials and Methods

Results and Discussion

The results showed that no different responses existed among the variables but they did among treatments in variables, such as root and stem length and in stem diameter depending on the treatment used (Table 2).

The best response was made evident with the use of the arbuscular mycorrhizal fungus because both cultivars increased stem length and diameter values, as well as root length when compared with the rest of the treatments (Table 2).

The second best behavior was obtained by using humic acid and chitosan, which produced a similar effect in both cultivars used, except for the stem length variable in the Floradade cultivar where the most discrete response was expressed with respect to the differences found in those with arbuscular mycorrhizal fungi and humic acid. Despite the differences found among them, the application of the bioproducts produced a stimulation in these variables boosting a notable increase in theirs values with respect to the control groups where a significant decrease was observed (Table 3).

The variables related with plant growth showed a similar response when they reacted positively with the three bioproducts used, but the response to the treatment differed among them (Table 3). In this sense, the best results were made evident when HMA was applied to the Pomodoro cultivar, followed by the application in the Foradade cultivar because this bioproduct achieved improving the related variables with biomass accumulation in tomato seedling stem, leaves, and roots with significant differences with respect to the rest of the treatments (Table 3). The second best behavior was obtained when humic acid was applied in both cultivars. However, its results were statistically similar when chitosan was applied although this last one evidenced lower values in Floradade cultivar and in the fresh and dry leaf biomass and fresh root biomass (Table 3).

A similar effect was made evident in total biomass accumulation (Figure 1) where the application of mycorrhizal fungi caused the best effect on the Pomodoro cultivar, followed by Floradade that showed the second best behavior. Nonetheless, Pomodoro responded similarly as Floradade when humic acid was applied (Figure 1b). The use of chitosan was the one that exerted the least positive effect on biomass accumulation of both cultivars used with no significant differences between them. Surprisingly, the best plant biomass values were expressed in the control where no bioproduct was applied (Figure 1b).

HA = humic acid; AMF = arbuscular mycorrhizal fungi; Quitosano = chitosan. Median with different letters on bars indicate significant differences according to Tukey’s (P ≤ 0.05) test

Figure 1: Effect of humic acid, arbuscular mycorrhizal fungi, and chitosan application on tomato plant biomass.  

Nutrient content in plant biomass showed the best results with AMF application (Table 4). In this manner, ions, such as N, K⁺, Ca²⁺, and Mg²⁺ increased significantly in both cultivars with the use of mycorrhizal fungi; however, their application had an opposite effect in the case of phosphorus concentration since a significant biomass decrease was observed. This result constituted a contradictory behavior because a notorious influence of arbuscular mycorrhizal fungi on plants is their role in solubilizing this element in soil, making it easier to be absorbed by the roots (^{Salvioli et al., 2012}; ^{Johri et al., 2015}; ^{Wang et al., 2018}). In this case, the treatment with the most influence was humic acid in both cultivars used, which showed the greatest values. However, no notable differences were observed with respect to chitosan application including the control group, which made evident that the bioproducts exerted very little influence in transporting this element toward the plant; because of their own natural conditions, they were capable of supplying their needs in phosphorus nutrition, probably because of the high availability of this element in the substrate used.

The correlation analysis performed (Table 5) showed a significantly strong and positive lineal relationship in the majority of the variables assessed with the exception of phosphorus content, which expressed the weakest and negative relationship with variables, such as root and stem length, stem diameter, fresh and dry root, stem and leaf biomass. Nonetheless, the rest of the elements, such as N, K⁺, Ca²⁺, and Mg²⁺ had a strong influence on these variables. In the case of N, it had a very high influence on stem length (0.97***) and BMPL (0.94***), as well as in the case of K⁺ on the variables DT (0.97***), MFR (0.97***), and LR (0.95***); in the case of Ca²⁺, it was LT (0.95***), DT (0.92***), MFT (0.95***), MST (0.90***), MSH (0.92***) and BMPL (0.93***), and lastly Mg²⁺ showed a high relationship with LR (0.95***), LT (0.96***), DT (0.96***), MFR (0.93***), MSH (0.94***), and BMPL (0.94***).

In this manner, we could assert that in this research study, phosphorus concentration in tissues did not keep a close relationship with any of the variables mentioned with respect to plant growth and biomass accumulation, contrasting with a very close one with nitrogen, potassium, and magnesium.

Studies have shown the importance of using bioactive products; for example, humic substances may affect plant metabolism directly, exerting an influence in ion transport, facilitating absorption, increasing respiration, speeding enzymatic reaction of Krebs cycle, which results in a greater metabolic energy in the form of adenosine triphosphate (ATP), favoring increase in chlorophyll content, nucleic acid synthesis, selective effect on protein synthesis and increasing or inhibiting different enzymes (^{Khaleda et al., 2017}; ^{Noroozisharaf and Kaviani, 2018}; ^{Shah et al., 2018}; ^{You et al., 2018}). In this manner, the beneficial effect of humic acids may be direct or indirect and related on the one hand, with facilitating nutrient absorption and on the other hand, producing physiological alteration that contributes to tomato plant growth. The experiment in this study showed that humic acids reached as tendency the second best result independently of the cultivar used as expressed in all the variables related with root and stem length, dry stem, leaf, and root biomass, and the increase in leaf concentration of elements, such as nitrogen, phosphorus, and potassium (Table 1, 2, and 3).

With respect to chitosan, it produced benefits on the variables related to growth and development of tomato cultivation, which at the same time kept a close relationship with improving the related variables with yield (^{Terry et al., 2017}; ^{Chun and Chandrasekaran, 2018}). Other authors (^{Paul et al., 2018}) have agreed that it stimulates several physiological processes in plants and has an influence on increasing cell size, maintaining a close relationship with plant nutrient content, which is reflected subsequently on improving the variables related to cultivation growth and yield.

Despite the results obtained in this experiment by chitosan in growth variables, such as stem and root length and plant biomass, as well as in leaf nutrient content, in general, they were surpassed by the use of humic acid and arbuscular mycorrhizal fungi, occasionally including similar to the control groups. This expression could be explained by the fact that in association to the stimulating effect, chitosan showed a stimulating (eliciting) activity of defense reactions that increased enzyme synthesis and defensive metabolites, which bring about an additional energy demand obtained precisely form the accumulated reserves in the plant. Thus, the positive effect for the related variables to growth might not be expressed (^{Giner et al., 2012}; ^{Chun and Chandrasekaran, 2018}; ^{Divya et al., 2018b}; ^{Charitidis et al., 2019}).

Notwithstanding these results, the tomato plants had a better response to the application of arbuscular mycorrhizal fungi related to the use of chitosan and humic acid, independently of the cultivar used, which could have been related to the versatility of these microorganisms (^{Martanto et al., 2018}; ^{Piculell et al., 2018}; ^{Chialva et al., 2019})which can be accompanied by changes in its structure, were already reported during plant interactions with other organisms, such as the mycorrhizal fungi. Arbuscular mycorrhizal (AM. Some of the most notorious effects were related to its influence in mineral nutrition, above all of P and N, as well as with root water absorption, its protective function against pathogenic microorganisms and conferring tolerance to plants to different types of biotic and abiotic stresses. All of these benefits were expressed subsequently with the increase of leaf and radicle biomass, leaf nutrient concentration, and cultivation yield (^{Liao et al., 2018}; ^{Chialva et al., 2019}). The results obtained in this research study agree with those described previously, except for those related to phosphorus content in plant tissues, element that decreased its concentration when mycorrhizal fungi were used (Table 3); in addition, it showed a weak and negative relationship with the other variables assessed (Table 4), which does not agree with the results of the majority of research works related to the topic (^{Salvioli et al., 2012}; ^{Duc et al., 2018}; ^{Chialva et al., 2019}).

Nevertheless, the use of the three bioproducts exerted an important effect on the majority of the variables assessed, which favored their values to go beyond the control groups although better results were reached with the use of arbuscular mycorrhizal fungi.

Conclusions

The arbuscular mycorrhizal fungi exerted the greatest effect on plant growth indicators of the Floradade and Pomodoro cultivars among all the treatments. The leaf phosphorus content was not an adequate indicator to correlate plant growth because it did not keep a direct relationship with biomass accumulation.