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Introduction
In Mexico, the tomato (Solanum lycopersicum L.) has been a very valued vegetable since pre-Hispanic times (Escofet, 2013; Medina et al., 2017), since it represents a food with beneficial properties for human health due to its content of bio-active compounds like lycopene, ascorbic acid, tocopherol, β-carotene, phenolic acids, flavonoids, folates, fiber, coumarin heterosides, phytoene and phytofluene, vitamin E, fatty acids, carbohydrates, potassium, calcium, magnesium and other micro-elements like zinc, iron and manganese, among others (Ahmed et al., 2011; Perveen et al., 2015). Nowadays, its global production and economic importance has only been compared with the cultivation of potatoes, which highlights its enormous value in the market as well as in international food matters (ASERCA, 1995; Atubi-Yeboah, 2013).
In 2013, even though 52 % of the surface destined to the cultivation of this Solanaceae was concentrated in four countries: China (20.9 %), India (18.8 %), Turkey (6.6 %), and Nigeria (5.8 %) (FIRA, 2016), continuous efforts have been made in various countries in order to enhance its quality and yield with different purposes, even implementing efficient technological developments so that its sowing and harvesting can be brought about in adverse climatic conditions.
Despite the inconveniences that might present when handling this crop, Mexico has continued to be an efficient producer and main provider of tomato in the international market (SAGARPA, 2017) where the value of exportations in 2012-2017 increased from 15.6 % to 58.1 % equivalent to 1,001 to 1,583 million dollars (SAGARPA/SIAP, 2018).
On the other hand, the demand of this crop places the United States as the main destination market of Mexican exportations with 90.1 %, which positions our country as the tenth tomato producer in the world with a production of 3,469,707 t per year-1 (SAGARPA/SIAP, 2018).
70 % of the land surface destined for the cultivation of tomato in Mexico is found under the agricultural protection system (Juarez-Maldonado et al., 2015), which makes this technology more efficient in terms of substantial increases in the yield and quality of the fruits (Marquez-Hernandez et al., 2013) if compared to the production of this vegetable in open field. However, this high productivity implicates an intensive use of agrochemicals (Cih-Dzul et al., 2011;Preciado et al., 2011).
Fortunately, nowadays, there are new alternatives of production that allow to increase the yield and quality of the agro-products under protected agriculture, highlighting the application of bio-molecules (Xu et al., 2009), compost (Cesaro et al., 2015), biological control with natural enemies (Bueno & van Lentren, 2010), microbial antagonists (Shahid et al., 2017), plant growth promoting bacteria (BPCV) (Ahmed et al., 2017), arbuscular mycorrhizal fungi (AMF) (Babaj et al., 2014; Hart et al., 2015), among others.
Within the most important genera of BPCV, Bacillus subtilis stands out due to its capacity to directly or indirectly promote plant growth (Choudhary & Johri, 2009) by means of the phytohormone synthesis (Fahad et al., 2015) and/or the increase in the availability of nutrients in the soil (Barriuso et al., 2008; Xie et al., 2014). Research studies with siderophores have also been realized in the antagonist activity against phytopathogen microorganisms (Sanchez et al., 2014; Fernandez-Herrera et al., 2018), solubilization of phosphates (Sharma et al, 2013), antibiotics, lithic enzymes and nitrogen fixation (Tejera-Hernandez et al., 2011), or else, where the formation of bacterial tridimensional biofilms could have an effect on the choosing of strategies tending to guarantee food safety (Morris & Monier, 2003; Nihorimbere et al. 2010), most importantly when the agrochemical options have failed.
Some research studies have referred to the importance symbiosis AMF-plant has had on the agro-productive systems due to the accumulation of evidence that has allow to confirm the benefits on the host species (Field et al., 2015) such as conferring tolerance to drought (Ortiz et al., 2015), salinity (Kumar et al., 2015; Dar et al., 2018) and toxicity to heavy metals (Singh et al., 2011); favoring the discrete absorption of nutrients of low mobility in soil (Jain & Chahar, 2016), the bidirectional interaction with herbivores (Zamioudis & Pieterse, 2012), its natural enemies and pollinators (Pineda et al., 2010), inducing systemic resistance (Pieterse et al., 2014; Chen et al., 2018) or decreasing the attack of pathogenic agents from the soil (Vilvert et al., 2017). Therefore, they broaden plants’ potentials to survive in adverse environmental conditions (Kim et al., 2017).
The use of beneficial and/or extremophile microorganisms constitutes a vital component of the biotechnology focused on the sustainable production of biomass with agricultural value (Pereg & McMillan, 2015; Moreno, 2016; Glick, 2018) or on applications of scientific interest by diversifying with industrial or commercial vision (Oliart-Ros et al., 2016; Andrade, 2017) where nano-technological or agrobiotechnological aspects are combined (Gouda et al., 2018), since they are part of an ecological and acceptable economic context, not for reducing the demand of agrochemicals but also for securing the quantity, quality and sustainability of the supply of innocuous foods (Guedez et al., 2008; Alvarez-Lopez et al., 2014).
This way, if the agro-technology and biotechnology are combined in a state-of-the-art agro-productive system, greater yields could be achieved, in comparison to the ones already achieved in traditional production systems. Based on the aforementioned, the purpose of this work was to assess the application of Bacillus subtilis and Rhizoglomus intraradices on growth, production and quality of the hybrid tomato fruits “Caporal” and “SV8579TE” under conditions of a semi-hydroponic cultivation system.
Material and Methods
Research area
The current study was carried out under semi-hydroponics conditions on inorganic substrate in a chapel-like greenhouse, located at 19°33’05.37’’N-96°56’40.64’’W in the city of Xalapa, Veracruz, Mexico, at 1,428 masl (meters above sea level). The average daytime temperature inside the greenhouse ranged between 25 and 35 °C and the average nocturnal temperature ranged between 18 and 20 °C from August 2017 to January 2018.
Cultivation handling
The tomato seeds of the hybrids ‘Caporal’ and ‘SV8579TE’ were placed in seed starting trays with the commercial substrate COSMOPEAT® in order to keep the conditions of germination of the seeds under control.
Twenty days after germination (DAG), the tomato seedlings were fertilized with Triple 19 (3 g•L-1) and, in order to prevent and control fungous diseases (specially Damping-off), Previcur-N (3 mL•L-1) and Cupravit (5 g•L1) were applied, just as producers from the central part of the state of Veracruz do it.
The transplant was realized 28 DAG, placing homogenous seedlings in beds with 5 cm of tezontle gravel (particle from 4 to 8 cm in size) at the bottom to facilitate drainage and 25 cm of red tezontle sand at the top (particles from 1 to 3 mm) as substrate, at a distance of 30 cm between plants and 60 cm between both rows, with 240 experimental units.
The hydric requirements of cultivation were maintained at filed capacity by means of drip irrigation applied using premium tape and automated with timer. The first two weeks, the irrigation was administered 8 times a day for 30 min, the following weeks it was brought down to 20 min and at the end for 10 min until finalizing the experiment.
According to the nutritional requirements of the crops or these production conditions, fertilization was realized with a nutritious solution (Ca[NO3]2, NaNO3, KNO3, KH2PO4, K2SO4 and micro-elements, with pH adjusted between 6-7 and CE in 2 S•m2•mol−1) chosen according to the nutritional demand of the crops (Graves, 1983; Steiner, 1984) and yellow insect traps were placed for the control of pests.
Experimental design
A completely randomized experimental design with four treatments (T) was used: T1: (Control, T), T2: (Bacillus subtilis, Bs), T3: (Rhizoglomus intraradices, Ri), T4: (Bacillus subtilis+Rhizoglomus intraradices, coinoculation Bs+Ri) and 30 repetitions in each one, with a total of 240 plants.
Inoculation with microorganisms
The inoculation with Bacillus subtilis (Bs) was realized to the transplant, applying 3 mL•plant-1 directly onto the root. The strain was provided by the Laboratorio de Fitopatología del Centro de Investigaciones Biológicas del Noroeste (CIBNOR), and was cultured in medium TSA (Trypticase soy agar) and kept shaking for three consecutive days at 28 °C. The mycorrhizal inoculum of Rhizoglomus intraradices (3 g•plant-1) applied at the time of transplanting was isolated from the rhizosphere of Jacaratia mexicana, a typical and distinctive tree species of dry deciduous forest present in a relic (surviving remnant) of the central portion of the state of Veracruz, Mexico, showing good root-colonizing capacity (≥90 %; Ri= 20-25 spores•g-1).
Evaluated variables
The evaluated variables were: height of the plant (cm), diameter of the stem (mm), number of bunches (inflorescences), number of fruits, green index (chlorophyll), qquality of fruits [area (cm), perimeter (cm), polar diameter (cm), equatorial diameter (cm), width (cm) and hardness (kg)]; as well as total production of biomass with agricultural value (kg), colony-forming units (CFU) and percentage of radicular colonization. For the clearing and staining of the roots and quantification of the mycorrhiza colonization, the technique proposed by Philips & Hayman (1970) was used. The determination of colony-forming units (CFU•g-1) was based on the methodology proposed by Glick et al. (1999).
Statistical analysis
The results were processed by means of analysis of variance (ANOVA) of simple classification and for the comparison of measures, the multiple comparison test of LSD measures by Fisher (p≤0.05) was used. The statistical analyses were carried out with the computer program Statistica v. 10.0 for Windows (StatSoft, Inc., 2011).
Results and Discussion
The ANOVA showed significant differences among the treatments for the height variable of the plants of the ‘Caporal’ hybrid (LSD by Fisher, p≤0.05), being the plants inoculated with Bs which showed superior values (22.5 %) surpassing that of the control treatment (Table 1), which can be attributed to the plant growth promoting rhizobacteria (PGPR) representing about 2-5 % of total rhizobacteria involved in improving the growth and sanity of numerous plants (Jha & Saraf, 2015; Prathap & Ranjitha Kumari, 2015) through direct and indirect mechanisms, or a combination of both (Vacheron et al., 2013; González et al., 2018).
Table 1 Average values recorded for the variables plant height, stem diameter, number of clusters, number of fruits and green index in the tomato hybrids ‘Caporal’ and ‘SV8579TE’.
| Variables | DAT¥ | ‘Caporal’ hybrid treatments | ‘SV8579TE’ hybrid treatments | ||||||
|---|---|---|---|---|---|---|---|---|---|
| T | Bs | Ri | Bs+Ri | T | Bs | Ri | Bs+Ri | ||
| Seedling height (cm) | 20 | 19.22b | 23.56a | 19.28b | 14.77c | 16.40a | 17.70a | 16.45a | 14.05b |
| 40 | 36.50b | 41.38a | 38.80b | 33.66c | 34.00a | 35.39a | 34.08a | 31.46b | |
| 72 | 80.80b | 90.40a | 87.53a | 78.66b | 91.43c | 87.33c | 105.40b | 111.53a | |
| Stem diameter (mm) | 20 | 2.92b | 3.33a | 3.07b | 2.70c | 2.32b | 2.50b | 2.80a | 2.33b |
| 40 | 4.40ab | 4.53a | 4.46ab | 4.19b | 4.33b | 4.45ba | 4.62a | 3.60c | |
| 72 | 6.33b | 6.84a | 6.37b | 6.27b | 5.79b | 6.66a | 6.57a | 6.57a | |
|
Number of bunches |
43 | 2.80ab | 3.06a | 2.86ab | 2.40b | 1.06c | 1.46b | 1.86a | 1.80a |
| 70 | 3.40a | 3.66a | 3.46a | 3.40a | 2.93cb | 2.86c | 3.26ab | 3.60a | |
| 84 | 4.33a | 5.00a | 4.80a | 3.40b | 3.60b | 4.06ba | 3.73b | 4.60a | |
| 98 | 3.33a | 3.40a | 3.40a | 2.46b | 4.06b | 3.80b | 4.20ba | 4.73a | |
|
Number of fruits |
70 | 7.60b | 12.66a | 12.53a | 7.13b | 3.33c | 6.00a | 5.86a | 4.80b |
| 84 | 13.00b | 14.93a | 13.66ab | 12.73b | 3.60c | 6.73ba | 5.60b | 7.66a | |
| 98 | 13.40a | 14.53a | 13.60a | 13.33a | 5.93c | 10.20b | 7.60c | 14.53a | |
|
Green index (CCJ¥) |
72 | 103.65c | 106.60b | 203.31a | 177.90c | 77.71d | 173.83b | 221.10a | 112.36c |
DAT¥ = Days after the transplant.
CCI¥ = Relative chlorophyll content index (0 a 999) in proportion to the transmittance of the incident light. Columns with the same letter are not significantly different according to the least significant difference test (p≤0.05).
The direct mechanisms occur when these microorganisms synthetize metabolites that favor the availability of nutritious elements required by the vegetal species in order to grow, realizing their vital functions (Gomez-Luna et al., 2012) and increasing the yield (Zahid et al., 2015), and the indirect mechanisms are characterized because rhizobacteria produce antimicrobial substances (or lithic enzymes) that impede the presence or proliferation of microorganisms that affect the normal physiological process of plants, without dismissing their capacity to compete for nutrients or species in a determined ecological niche; or else activating biocontrol mechanisms, inducing systemic resistance and/or producing siderophores, antibiotics and hydrogen cyanides that act upon phytopathogens (Esquivel-Cote et al., 2013).
The co-inoculated treatment obtained significant increases on the ‘SV8579TE’ hybrid (Bs+Ri, 21.9 %), which surpassed the control plants. This in accordance with what was reported by Kumar et al. (2015b) who after evaluating selective plant growth promoting rhizobacteria from the rhizosphere (Azotobacter chroococcum, Pseudomonas aeruginosa, Azospirillum brasilense and Streptomyces sp.) in combination with six strains from AMF (Acaulospora morrowiae, Gigaspora margarita, Glomus constrictum, Gl. mosseae, Gl. aggregatum and Scutellospora calospora) observed an increase of 82 % in height of maize plants (Zea mays L.) as a result of the synergy that could have existed to adopt greater mineral nutrition to hosts (Diaz et al., 2013).
Similarly, Falcäo et al. (2014) mention that after the inoculation of B. subtilis ALB629 in germinating cacao seeds (Theobroma cacao L.) a good growth and stem development was achieved, which coincides with the response of tomatoes for the evaluated indicators in this research (Table 1). In both tomato hybrids, it was observed that the best treatment was the one inoculated with the rhizobacteria (Bs), with increases of 4 % on the ‘Caporal’ hybrid’ and 15 % in ‘SV8579TE’ in respect to the control plants. This effect may have been related due to the activity of these microorganisms, among the ones that stand out, the synthesis of growth promoting compounds like indole-3-acetic acid (3-IAA), auxins, gibberellins and cytokinins that allow the uptake of hydrogen and phosphorous available in the soil; in addition to the presence of these phytohormones in the rhizosphere that seems to stimulate the physiological progression and development of these plants (Brimecombe et al., 2007; Vega-Celedón et al., 2016).
In the variable of number of bunches, it was observed that the best treatment for the ‘Caporal’ hybrid was Bs (Table 1) in respect to the control plants (LSD by Fisher, p≤0.05), response that could be attributed to what pointed out by Hernández-Suárez et al. (2010) about the effects of microencapsulated B. subtilis on the growth and production of fruits (yield) of L. esculentum cv. ‘Floradade’. On the other hand, in the co-inoculated treatment of the ‘SV8579TE’ hybrid, a superior response was obtained with increases of 16.5 % in respect to the control plants, due to their pre-disposition to favor symbiosis with rhizobacteria and the mycorrhizal fungi.
Regarding the number of fruits, Sánchez et al. (2012) reported that when inoculating diverse PGPRs (Enterobacter sp., E. agglomerans, Pseudomonas fluorescens, P. putida and Bacillus sp.) in tomato plants the results showed evidence of significant differences between biological and chemical fertilization, being able to confirm that the application of rhizobacteria with plant growth promoting qualities favored the production of fruits only by 50 % of the recommended dose of fertilization for the cultivation of S. lycopersicum under greenhouse, which coincides with the increase of 25 % reported by Mena-Violante & Olalde-Portugal (2007) on the number of fruits from this Solanaceae with B. subtilis and with what was found in the treatment with Bs (>14.8 %) in respect to the control plants for the ‘Caporal’ hybrid in the present study.
For the ‘SV8579TE’ hybrid, the best treatment was the coinoculated Bs+Ri with increase of 145 % in respect to the control plants, which agrees with what was reported by Lira-Saldivar et al. (2014) who upon evaluating the double coinoculation Azospirillum brasilense+Glomus intraradices (now R. intraradices) in tomato (S. lycopersicum) registered increases on the height of plants (6 %), the dry biomass (10.5 %) and the yield in comparison to the traditional fertilization under shade-house.
Likewise, the effects derived from AMF (Gl. intraradices+Gl. etunicatum) and rhizobacteria (P. putida, Azotobacter chroococcum and Azoprillum lipoferum) on phytochemical factors that have an influence on the quality of the fruit (Ordookhani et al., 2010).
In the case of the green index variable, a superior response was obtained when inoculating R. intraradices in both hybrids (Table 1) with increases of 96 % in the ‘Caporal’ hybrid and 184.5 % in the ‘SV8579TE’ hybrid in respect to the control plants, which once again can be attributed to a better exploitation of lowmobility nutrients in the soil, promoted by the radicular colonization and mycorrhizal interaction (Ri+host plant [S. lycopersicum]) (Tallapragada et al., 2016).
This agrees with what was reported on the tomato hybrid ‘El Cid’ (Lycopersicum esculentum; Saladette type) by Alvarado et al., 2014) who indicated that Rhizophagus intraradices1 significantly increased the height of the plant, the content of chlorophyll and the radicular mycorrhizal colonization, making an emphasis on the greater chlorophyll index (12 % p≤0.01) in comparison to the non-inoculated plants.
For the polar diameter variable of the fruit, the treatments that showed a favorable response on the ‘SV8579TE’ hybrid were Bs and Ri with increase of 43.8 % and 29.8 % in respect to the control plants (LSD by Fisher, p≤0.05) (Table 2). The ANOVA (LSD by Fisher, p≤0.05) expressed that the best treatment on the perimeter of the fruit was with Bs when exhibiting an increase of 16.8 % in respect to the plants of the control treatment.
Table 2 Effect of microorganisms on fruits of ‘Caporal’ and ‘SV8579TE’ hybrid tomatoes at 107 DAT.
| ‘Caporal’ hybrid | ‘SV8579TE’ hybrid | |||||||
|---|---|---|---|---|---|---|---|---|
| VARIABLES | T | Bs | Ri | Bs+Ri | T | Bs | Ri | Bs+Ri |
| Fruit area (cm) | 24.65b | 25.39b | 31.64a | 24.11b | 28.87b | 41.52a | 37.48a | 21.78c |
| Fruit perimeter (cm) | 20.28bc | 21.28ab | 22.47a | 19.54c | 21.29b | 24.88a | 24.25b | 19.04c |
| Fruit ecuatorial diameter (cm) | 0.73cb | 0.78ab | 0.80a | 0.70c | 0.79a | 0.84a | 0.80a | 0.75a |
| Fruit polar diameter (cm) | 7.33a | 7.34a | 7.53a | 6.94a | 6.82ab | 8.13ab | 7.64a | 6.54b |
| Fruit width (cm) | 5.03b | 5.26ab | 5.64a | 5.01b | 5.41b | 6.49a | 6.22a | 4.77c |
| Fruit hardness (kg) | 1.56a | 1.40a | 1.40a | 1.24a | 1.58ab | 1.29b | 1.59ab | 1.80a |
| Total production (kg) | 102.00d | 105.00c | 108.00b | 111.00a | 102.00d | 108.00b | 105.00c | 111.00a |
Columns with the same letter are not significantly different according to the least significant difference test (p≤0.05).
On the equatorial diameter of the fruit, no significant differences were found in the treatments (Table 2), whereas in the polar diameter the treatment that expressed the most desirable results was R. intraradices, with increases of 12 % in respect to the control plants. A superior response regarding the width of the fruit was obtained in the inoculated treatments with R. intraradices (14.9 %) and B. subtilis (19.9 %), while regarding its hardness the interactive factor of Bs+Ri surpassed the control plants by (13.9 %) (Table 2).
In a general sense, what was obtained was that for both hybrids with the inoculation of R. intraradices a superior response was achieved in the ‘Caporal and SV8579TE’ hybrids which agrees with what was reported by Diaz et al. (2013) who after inoculating pepper plants (‘Valeria’ hybrid) with R. intraradices boosted up the size and weight of the fruit. Thus, the quality of the fruit improved by the mycorrhizal treatment could be closely related with the greater mineral nutrition of the plants associated with these symbionts, just like Hart et al. (2015) reported after inoculating L. esculentum cv. ‘Moneymaker’ with Rhizophagus irregularis, Funneliformis mosseae or both.
For the total production of biomass with agricultural value, in both cases the best treatment was the co-inoculated with increases of 8.8 % for the ‘Caporal’ hybrid and 104 % for the ‘SV8579TE’ hybrid in respect to the control plants, aspects that once more could be related with the capacity of B. subtilis+R. intraradices) for synergistically interacting and achieving a greater availability and assimilation of nutrients (Arthurson et al., 2011; Kavatagi & Lakshman, 2014), which translate to even a lot greater yields, in comparison to what was registered in simple inoculations.
For the colony-forming units (CFU) variable, the treatment Bs+Ri reached the highest values in both hybrids (‘Caporal’ with 0.00048 CFU•g-1 and ‘SV8579TE’ with 0.00022 CFU•g-1) because when coinoculating a plant growth promoting rhizobacteria and a mycorrhizal fungus, a greater number of colonies was recorded, in comparison to when the bacterium “acts by itself”, which agrees with what was reported by Manoharachary et al. (2016) where they point out that the mycorrhizal colonization acts as a chemicalattractant of rhizospheric bacteria and, after a synergic relation occurs between both microorganisms, the nutritional and hormonal balance of its hosts is favored, intensifying its growth and development (Nadeem et al., 2014; Egamberdieva et al., 2015; 2016).
In regards to the percentage of mycorrhizal colonization the best treatment in the ‘Caporal’ treatment was Bs+Ri (87 %), in comparison with what was observed in the ‘SV8579TE’ hybrid where no significant differences presented between the treatments (LSD by Fisher, p≤0.05). This coincides with the work realized by Cano (2011) in cucumber (Cucumis sativus L.) and Saia et al. (2015) in wheat (Triticum durum Desf.) where the percentages of mycorrhizal colonization in treatments with simple inoculation (AMF; PGPR) or coinoculated (AMF+PGPR) increased the synergic relations that optimized the absorption of water and nutrients and had an influence on the growth and development of the host plants.
Finally, the results obtained in this work coincide with Bona et al. (2015; 2016) since the inoculation of both microorganisms (Bs+Ri) not only did it contribute to the vegetative development and formation of quality fruits in both tomato hybrids (‘Caporal’ and ‘SV8579TE’), but it also had an influence on the quantity of biomass with agricultural value harvested in comparison to the rest of the evaluated treatments.
