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Introduction
Tomato (Solanum lycopersicon L.; Solanales: Solanaceae) is one of the horticultural crops of great importance worldwide, due to its high consumption in both fresh and processed, with a planted area of approximately 5 million hectare and a production of more than 177 million t in 2016. In the same year, Mexico contributed with more than 4 million t (2.3 %) harvested in an area of more than 90,000 ha (FAO, 2016). This crop, like many others, is susceptible to the attack of a great diversity of phytopathogenic fungi. These microorganisms can cause economic losses up to 100 % in tomatoes (Martínez-Ruiz et al., 2016). Some of these fungi are causing root and crown diseases, which obstruct the vascular bundles of the plant, preventing the normal flow of water and nutrients, which is reflected in symptoms such as defoliation, wilting, foliar chlorosis, reduced growth, low fruit production, among other negative effects (Shafique et al., 2016). Among the most important pathogenic fungi of tomato are Fusarium oxysporum f. sp. lycopersici, Alternaria solani and Phytophthora infestans (El_Komy et al., 2015). Chemical fungicides are the most commonly used for the control of these phytopathogens with satisfactory results; however, the excessive use of these substances may induce the development of resistance by pathogens, elimination of non-target microorganisms, pollution of agricultural soil and water bodies, and other negative effects (Al-Rahmah et al., 2013; Shafique et al., 2016). An alternative for the control of these pathogens can be the use of antagonistic bacteria of the genus Bacillus, recognized as excellent biological control agents, quality enhancers of agricultural soils and promoters of plant growth, among other attributes (Cabra-Cendales et al., 2017). However, several Bacillus strains have been evaluated mainly in vitro, individually and in few studies have been considered the effects of the application of these on the bromatological composition of the fruits and its potential to decrease the indirect damage in fruits caused in situ by phytopathogenic microorganisms (Esitken et al., 2010; Ju et al., 2014; El_Komy et al., 2015). Derived from the above, the objective of the study was to determine the effect of three Bacillus strains alone and in interaction with three phytopathogenic fungi on the growth of tomato plants and fruit quality.
Material and Methods
Plant and microbial material
The microbial strains were provided by the ceparium from the Centro de Investigación en Alimentación y Desarrollo (CIAD), Chihuahua Headquarters, Cuauhtémoc Campus, and the Universidad Michoacana de San Nicolás Hidalgo, Michoacán, Mexico. Three bacterial strains of the genus Bacillus [B. amyloliquefaciens (Ba); B. methylotrophicus (Bm) and B. subtilis subsp. inaquosorum (Bs)] were used. The pathogenicity of the phytopathogens and the antagonistic effects of Bacillus spp. were confirmed in previous studies (Rios-Velasco et al., 2016; Ruiz-Cisneros et al., 2017) and three phytopathogens isolated of tomato crops in Mexico [Alternaria solani (As), Fusarium oxysporum (Fo) y Phytophthora infestans (Pi)].
Tomato seeds (Solanum lycopersicon L. cv. Merlice), were provided by the Company DeRuiter™ (Monsanto Holland), while the pollinating agents Bombus terrestris L. (Hymenoptera: Apidae), were purchased with Koppert Mexico, S.A. de C.V. (El Marqués, Querétaro, Mexico).
Establishment and management of the tomato cultivation
The experiment was performed in a greenhouse (tunnel type of 20 × 8 × 5 m, without heating) in Cuauhtémoc, Chihuahua, Mexico located at 28° 26’ 51” N; 106° 49’ 43” W and 2,020 masl. Tomato seeds were germinated in trays of 200 cavities until the state of 2-3 true leaves. Subsequently, they were transplanted in black plastic bags with a capacity of 5 L with a mixture of sterilized substrate (121 °C, 15 psi, 1 h) composed of loamy soil, vermiculite (Termolita, S.A. de C.V., Santa Catarina, Nuevo León, Mexico) and peat moss (Lambert Peat Moss Inc.-Turbines Lambert Inc., Quebec, Canada) in a ratio of 1: 1: 1. Fertilization was performed every 30 days post-transplantation (dpt), [250 mL of Urea (6 g/L) (Ferti-urea, Productora de Fertilizantes del Noroeste, S.A. de C.V., Cd. Obregón, Sonora, Mexico) and Triple 19 (2 g/L) (Poly-Feed, greenhouse grade, soluble solid fertilizer NPK 19-19-19, Haifa Mexico, S.A. de C.V., Mexico) and 10 mL of Murashige and Skoog medium (4.4 g/L) (Sigma-Aldrich, Mexico)], during the 2016 production cycle. The pollination was carried out by a bumblebee colony B. terrestris.
Inoculation of the substrate with microorganisms
The Bacillus strains were grown in LB broth (Sigma-Aldrich, St. Louis, MO, USA) at 28 °C, in constant shaking (180 rpm) for 2 d. For their increase, the fungi were inoculated into a nutrient broth (BD Bioxon, Becton Dickinson de Mexico, S.A. de C.V., Cuautitlán Izcalli, Estado de Mexico, Mexico), except P. infestans that was grown in a vegetable broth V8 (V8 juice, Campbell´s™) added with calcium carbonate (3 g/L), at 28 °C, in constant shaking (180 rpm) for 5 d.
Pathogens and antagonists were applied individually to the substrate of 150 pots, at 8 dpt, according to the concentrations shown in Table 1. Thirty milliliters (4.5-6.0×1010 CFU) of the suspension of the antagonistic bacterium and 20 mL (2.6-7.8×107 conidia) of the suspension of the phytopathogen were applied. Ten plants were used for each microorganism (antagonist or phytopathogen), where every plant was an experimental unit (T2-T7, Table 1) y 90 plants for the interaction tests between antagonists and pathogens (T8-T16, Table 1). Additionally, 10 plants without inoculum were used as controls (T1, Table 1).
Table 1 Treatments and concentration (CFU or conidia/mL) of inoculations on tomato plants with Bacillus strains and phytopathogenic fungi.
| Treatment | Antagonists (CFU/mL) |
Phytopathogens (conidia/mL) |
||
|---|---|---|---|---|
| Code | Antagonistic bacteria | Phytopathogenic fungi | ||
| T1 | Control | - | - | - |
| T2 | Bacillus amyloliquefaciens (Ba) | - | 1.8×109 | - |
| T3 | B. methylotrophicus (Bm) | - | 2.0×109 | - |
| T4 | B. subtilis subsp. Inaquosorum (Bs) | - | 1.5×109 | - |
| T5 | - | Fusarium oxysporum (Fo) | - | 1.3×106 |
| T6 | - | Alternaria solani (As) | - | 3.9×106 |
| T7 | - | Phytophthora infestans (Pi) | - | 2.6×106 |
| T8 | Ba | Fo | 1.8×109 | 1.3×106 |
| T9 | Ba | As | 1.8×109 | 3.9×106 |
| T10 | Ba | Pi | 1.8×109 | 2.6×106 |
| T11 | Bm | Fo | 2.0×109 | 1.3×106 |
| T12 | Bm | As | 2.0×109 | 3.9×106 |
| T13 | Bm | Pi | 2.0×109 | 2.6×106 |
| T14 | Bs | Fo | 1.5×109 | 1.3×106 |
| T15 | Bs | As | 1.5×109 | 3.9×106 |
| T16 | Bs | Pi | 1.5×109 | 2.6×106 |
CFU= Colony forming units.
The interaction treatments (antagonists vs pathogens), consisted in the application of the corresponding phytopathogen at 18 dpt (i.e. 10 d after the application of the Bacillus strains). The population of antagonists was maintained by three applications of the inoculum at intervals of 20 d, after the first inoculation. It should be noted that the phytopathogens were inoculated once. The plants were maintained under greenhouse conditions and were evaluated during the production cycle (April to October 2016).
Agronomic measurements
The plants were evaluated for height and weight of the aerial part, length and weight of roots, stem diameter, to the end of the production cycle. Chlorophyll (SPAD units) was estimated four times (every 20 dpt) in 10 random leaves per plant, of each treatment, using a Spad 502DL Plus chlorophyll meter (Konica Minolta Brand, Spectrum Technologies, Inc., Aurora Illinois, USA). The number of fruits produced per plant was also recorded and based on this, the yield was calculated, considering all the fruits harvested over time during the evaluation period.
Tomatoes were harvested in the red point, according to the specifications of the United States Department of Agriculture (USDA, 2015) and were stored for 3 d at 4 °C until analysis. The fruits were evaluated for weight, polar and equatorial diameter, color, number and weight of seeds, total soluble solids (TSS, °Brix), titratable acidity (% citric acid), firmness and thickness of the mesocarp. The polar and equatorial diameters were measured using a diameter meter measurer (Cranston Machinery Co., Oak Grove, Oregon, USA). The color [L* (lightness from black to white), a* (goes from red to green) y b* (goes from blue to yellow)]. was measured at four equidistant points on the equatorial axis of each fruit, using a Minolta CR300 colorimeter. The number of seeds was determined in 10 random fruits of each treatment. The total soluble solids (TSS, °Brix) were determined, using a PAL-1 digital table refractometer (Atago Co. Ltd., Tokyo Japan). For the titratable acidity, 10 g of flesh (from 20 fruits taken randomly from each treatment) were homogenized with 250 mL of sterile distilled water and 50 mL of the obtained solution were titrated with 0.1 N NaOH, using phenolphthalein 0.5 % as color indicator. The titratable acidity was expressed as % citric acid (NMXF-102-S-1978). For the firmness of the fruits, 50 tomatoes taken randomly from each treatment were evaluated and was determined by a puncture test using a texture analyzer Ta XTPlus (Texture Analyzer, Surrey, RU), starting at 30 cm/min, at a distance of 15 cm at two opposite points along the equatorial plane and the maximum force (N) was recorded. The thickness of the mesocarp was determined in four sections of 10 random fruits from each treatment, using a digital Vernier (Knova 0-150 mm).
Bromatological analysis
A composite sample of 250 g was obtained from 20 fruits taken randomly from each treatment. The samples were placed into Ziploc™ bags and stored at -80 °C until analysis. Subsequently, the samples were homogenized using a LB10 blender (Waring Laboratory, Torrington, CT, USA) and puree sub-samples were obtained (from 1 to 10 g, depending on the determination). These were subjected to measurements of humidity, ash, proteins (N × 6.25), fats (lipids) and total fiber, according to the official methods (925.10, 923.03, 991.20 and 920.35) of the Association of the Official Analytical Chemistry (AOAC, 2002).
Statistical analysis
The experiment consisted of 16 treatments (Table 1). The assays were performed with 10 tomato plants per treatment, considering each plant as an experimental unit, using a completely random design. Data were analyzed using the software Statistical Analysis System version 9.0 (SAS, 2002) to balance the analysis of variance (ANOVA), and the means were separated by the Tukey test (p=0.05). The fruit measurements were determined in triplicate and the data obtained were subjected to an ANOVA and a Tukey separation test (p=0.05).
Results and Discussion
Plant growth promotion and yield of the plant
The three Bacillus strains showed positive effects in at least one of the agronomic variables evaluated (Figures 1 and 2). The fresh and dry weight of the plants ranged from 104 to 246.6 g and from 29.7 to 47.9 g, respectively. The plants inoculated with B. subtilis obtained the highest values, causing the most notable effect in those response variables, compared with the control plants (Figure 1). The same strain promoted the greatest root length (45 cm) and height (1.45 m, Figure 1). Also, the plants treated with B. amyloliquefaciens, showed a significant stimulus in height (1.42 m, Figure 1). The three phytopathogens reduced the height of the plant by up to 25 % in relation to those of the control (Figure 1). In addition, plants treated with A. solani and P. infestans resulted in the lowest values for root length, with 29 and 30 cm, respectively, comparatively to the controls (35 cm), but lower than those treated with the Bacillus strains (Figure 1).
Figure 1 Growth variables (height, fresh and dry weight) estimated in Lycopersicon esculentum L. cv. Merlice, inoculated with Bacillus strains alone and in interaction with phytopathogenic fungi: a) plants; b) roots. The data are presented as means ± standard error.
Figure 2 Effects on agronomic variables (stem diameter, chlorophyll (SPAD units), and fruit number) of tomato plants inoculated with Bacillus strains alone and in interaction with phytopathogenic fungi. The data are presented as means ± standard error.
Likewise, the plants treated with the Bacillus strains showed the highest values in stem diameter, chlorophyll in leaves and yield, compared with those inoculated with the phytopathogens alone and in interaction (Bacillus spp. vs phytopathogens; Figure 2). Bacillus amyloliquefaciens promoted the highest fruit yield (63,000 kg Ha-1), followed by B. subtilis (52,000 kg Ha-1; Figure 2). In the rest of the treatments, the yields were variable (Figure 2).
The positive effects, resulting from the inoculation of Bacillus strains may be due to its multiple attributes as excellent root colonizers, root growth enhancers, crop yield promoters, inducers of abiotic stress resistance, among others (Beneduzi et al., 2012). On the other hand, roots produce exudates that stimulate the growth of biological control agents in a synergistic manner (Widnyana & Javandira, 2016; Yuan et al., 2015).
In our study, some agronomic variables such as plant growth, chlorophyll in leaves, yield (number and fruit diameter), root mass and stem diameter were increased by Bacillus strains. Bacillus amyloliquefaciens promoted the highest yield and fruit size, compared with those obtained from control plants. Mena-Violante & Olalde-Portugal (2007) reported an increase in yield per plant and fruit weight in 25 % and 18 %, respectively, when applying the strain B. subtilis BEB-13bs. Gül et al. (2008) and Myresiotis et al. (2014) demonstrated that the application of B. amyloliquefaciens in tomato plants increased yield by 8-9 %. Similar effects by Bacillus on yield have been reported in other crops (Esitken et al., 2010). This could be attributed to the ability of some Bacillus strains to produce hormones such as auxins, cytokinins and gibberellins, which promote root growth and fruit development. They can also solubilize inorganic phosphates and mineralize organic phosphates, among other nutrients, favoring the growth of plants (Esitken et al., 2010). In addition to its ability to produce biosurfactins (lipopeptides), responsible for increasing the mobility of Bacillus for rapid root colonization (Lugtenberg & Kamilova, 2009; Ahemad & Kibret, 2014).
Phytopathogens reduced in great manner the agronomic variables evaluated, as well as the fruit quality parameters. The plants inoculated with A. solani and P. infestans showed a negative effect on the agronomic variables, mainly on plant height, root length, stem diameter, yield and the number and size of fruits. The yield in plants treated with phytopathogens was 31 % lower than in the control, ranging from 33,000 to 39,000 kg Ha-1. Plants treated with F. oxysporum showed the lowest yield (Figure 2). This could be attributed to the deficient nutritional assimilation of diseased plants, caused by the mycotoxins (fusaric acid) and enzymes (cutinases and esterases) produced by phytopathogens (Beneduzi et al., 2012).
On the other hand, the yield in plants treated with the Bacillus strains and subsequently with the phytopathogens, was lower than that observed with the Bacillus strains alone and the controls. However, the symptoms of the disease caused by the phytopathogens were reduced and plant growth was even promoted in some treatments (Figure 1). This could be due to some strains of Bacillus being highly competitive and capable of colonizing the rhizosphere forming a protective layer in the roots producing fungicidal compounds, which acts as a natural barrier against phytopathogens (Widnyana & Javandira, 2016).
Fruit quality
The data obtained for the polar and equatorial diameters of fruits, are shown in Figure 3. The size of fruits of plants inoculated with Bacillus strains, was higher (21 %) than the rest of the treatments. The biggest fruits were produced by plants inoculated with B. subtilis (>55 mm in diameter). In contrast, the plants inoculated with phytopathogens, produced smaller fruits. The fruits of plants treated with B. subtilis, had the greatest mesocarp thickness, while those of plants inoculated with phytopathogens were thinner. The thinnest mesocarp was observed in fruits of plants treated with A. solani (Figure 3).
Figure 3 Effects on agronomic variables (polar and equatorial diameter, number of seeds/fruit, seeds weight and mesocarp thickness) of tomato fruits obtained from plants inoculated with Bacillus strains alone and in interaction with phytopathogenic fungi. The data are presented as means ± standard error.
The number of seeds per fruits fluctuated from 38 to 151 (Figure 3). The greatest number was obtained from the treatment with B. methylotrophicus. The weight of seeds per fruit fluctuated from 0.18 to 1.04 g. The highest weight was obtained in fruits of plants treated with Bacillus strains, particularly with B. methylotrophicus, while the lowest were observed in plants inoculated with F. oxysporum and the interaction B. amyloliquefaciens vs A. solani. In this regard, Tewksbury et al. (2008) mentioned that pathogens could reduce the number and size of seeds in fruits, or even, obtain empty seeds, presumably because of a deficiency in the assimilation of the nutrients caused by the disease.
In general, TSS and titratable acidity did not show a clear trend, although TSS were higher in the fruits of plants treated with the pathogens A. solani and P. infestans. This may be due to a higher carbohydrate hydrolysis rate, which may have implications in the fresh tomato market (Tigist et al., 2013). The titratable acidity was higher in fruits of plants treated with the three Bacillus strains, than in the controls (Table 2). Where the highest values were observed in fruits of plants treated with B. amyloliquefaciens (0.48 % citric acid), while the lowest values were observed in fruits from plants treated with F. oxysporum (0.24 % citric acid, Table 2).
Table 2 Total soluble solids (TSS, ºBrix), titratable acidity (% citric acid), firmness and fruit color by CIELAB system parameters L*, a* and b* of tomato fruits harvested from plants inoculated with Bacillus strains alone and in interaction with phytopathogenic fungi.
| Treatment | TSS (°Brix) | % citric acid | Firmness (N) | L* | a* | b* |
|---|---|---|---|---|---|---|
| Control | 4.40 ± 0.11ab | 0.34 ± 0.02ab | 37.4 ± 1.4bcdef | 49.2 ± 1.2abc | 14.4 ± 0.9ab | 19.2 ± 0.9ab |
| Antagonists alone | ||||||
| B. subtilis subsp. inaquosorum | 4.43 ± 0.53ab | 0.43 ± 0.03ab | 59.2 ± 1.0a | 48.5 ± 0.3abc | 13.8 ± 1.2abc | 19.5 ± 0.7ab |
| B. methylotrophicus | 3.78 ± 0.18b | 0.42 ± 0.09ab | 26.1 ± 0.6ef | 49.3 ± 1.1abc | 16.0 ± 0.6a | 21.6 ± 0.8ab |
| B. amyloliquefaciens | 3.95 ± 0.25ab | 0.48 ± 0.01a | 43.2 ± 1.2abcde | 53.4± 0.5a | 14.4 ± 0.6ab | 19.9 ± 1.5ab |
| Phytopathogens alone | ||||||
| F. oxysporum | 4.34 ± 0.34ab | 0.24 ± 0.03b | 23.3 ± 1.2f | 48.9 ± 0.8abc | 14.6 ± 0.7ab | 20.2 ± 1.3ab |
| A. solani | 4.77 ± 0.34a | 0.39 ± 0.04ab | 36.2 ± 1.5cdef | 50.7 ± 0.7abc | 15.0 ± 0.9ab | 22.5 ± 1.0a |
| P. infestans | 4.52 ± 0.28ab | 0.32 ± 0.03ab | 51.6 ± 1.4abc | 49.1 ± 0.2abc | 14.5 ± 0.5ab | 21.1 ± 1.5ab |
| Antagonists vs phytopathogens | ||||||
| B. subtilis vs F. oxysporum | 4.16 ± 0.22ab | 0.33 ± 0.05ab | 34.5 ± 0.8cdef | 50.1 ± 0.5abc | 13.9 ± 1.2abc | 23.6 ± 1.0a |
| B. subtilis vs A. solani | 4.68 ± 0.23ab | 0.39 ± 0.18ab | 41.7 ± 1.3abcdef | 49.6± 1.9abc | 16.3 ± 0.4a | 19.5 ± 0.3ab |
| B. subtilis vs P. infestans | 3.96 ± 0.25ab | 0.33 ± 0.05ab | 55.4 ± 1.1ab | 50.5 ± 0.8abc | 14.8 ± 0.6ab | 21.0 ± 0.4ab |
| B. methylotrophicus vs F. oxysporum | 4.34 ± 0.24ab | 0.27 ± 0.09ab | 52.1 ± 1.4abc | 51.5 ± 1.5abc | 12.1 ± 1.4bcd | 22.8 ± 1.0a |
| B. methylotrophicus vs A. solani | 4.18 ± 0.11ab | 0.36 ± 0.05ab | 32.0 ± 0.6def | 41.8 ± 1.0bc | 9.9 ± 1.5d | 18.5 ± 1.6ab |
| B. methylotrophicus vs P. infestans | 4.07 ± 0.49ab | 0.35 ± 0.03ab | 46.7 ± 1.7abcd | 40.3 ± 0.7b | 10.7 ± 1.3cd | 15.5 ± 1.9b |
| B. amyloliquefaciens vs F. oxysporum | 3.79 ± 0.22b | 0.40 ± 0.05ab | 52.7 ± 1.1abc | 48.9 ± 1.1abc | 14.7 ± 0.5ab | 21.1 ± 1.4ab |
| B. amyloliquefaciens vs A. solani | 4.69 ± 0.10ab | 0.32 ± 0.12ab | 33.6 ± 1.4cdef | 49.3 ± 1.1abc | 13.9 ± 1.0abc | 19.8 ± 1.3ab |
| B. amyloliquefaciens vs P. infestans | 3.83 ± 0.13ab | 0.38 ± 0.13ab | 58.6 ± 0.9a | 50.2 ± 2.0abc | 13.3 ± 0.9abcd | 20.9 ± 0.3ab |
Means with the same literal between columns, are statistically equal, according to the Tukey’s test (p=0.05). ± Standard error.
The firmness of the fruits ranged from 23.3 to 59.2 N without showing a clear trend. The fruits of plants treated with B. subtilis, showed the highest values, resulting in an increase in the firmness in 1.6-fold compared to control fruits. The softest fruits were obtained from plants treated with F. oxysporum (Table 2). Similar results were obtained by Mena-Violante & Olalde-Portugal (2007) in tomato plants inoculated with B. subtilis. According to these authors, the firmer fruits are more resistant to the attack of pathogenic microorganisms, which improves its shelf life. This effect can be attributed to changes in the production of phytohormones, mainly ethylene, which is responsible of maturation genes in plants. The color of the fruits was not affected by the microorganisms evaluated (Table 2), which is consistent with the existing literature. This is the attribute of quality most closely related to appearance, sugar content, acidity, pH, texture, flavor and juiciness (Araujo et al., 2014).
Bromatological composition of fruits
The moisture content in fruits of plants treated with Bacillus strains ranged from 95.5 to 96.8 %. Plants treated with B. methylotrophicus produced fruits with the lowest water content (Table 3). However, the moisture content in fruits from plants treated with Bacillus strains and in interaction with phytopathogens was higher than 98 %. This can be attributed to hydric stress induced in plants, by the disease caused by these pathogens, which could inhibit the normal transpiration of plants and their fruits (Yadeta & Thomma, 2013).
Table 3 Bromatological analysis of tomatoes harvested from plants inoculated with Bacillus strains alone and in interaction with phytopathogenic fungi.
| Treatment | Bromatological composition | ||||
|---|---|---|---|---|---|
| Moisture (%) | Ashes (%) | Proteins(%) | Fats (Lipids) (%) | Fiber (%) | |
| Control | 96.8 ± 0.13cde | 0.09 ± 0.01b | 0.35 ± 0.09cd | 0.08 ± 0.01bcd | 1.18 ± 0.01a |
| Antagonistic microorganisms | |||||
| B. subtilis subsp. inaquosorum | 95.7 ± 0.20de | 0.41 ± 0.06a | 0.93 ± 0.04a | 0.02 ± 0.04d | 1.19 ± 0.03ª |
| B. methylotrophicus | 95.5 ± 0.18e | 0.43 ± 0.01a | 0.68 ± 0.07abc | 0.18 ± 0.03a | 1.16 ± 0.03ª |
| B. amyloliquefaciens | 96.8 ± 0.71cde | 0.41 ± 0.07a | 0.92 ± 0.11a | 0.08 ± 0.02abcd | 1.14 ± 0.03ª |
| Phytopathogenic microorganisms | |||||
| F. oxysporum | 97.1 ± 0.33bcd | 0.38 ± 0.06a | 0.70 ± 0.15ab | 0.15 ± 0.04abc | 1.58 ± 0.05a |
| A. solani | 97.7 ± 0.05abc | 0.08 ± 0.01b | 0.33 ± 0.02d | 0.04 ± 0.01d | 1.76 ± 0.02ª |
| P. infestans | 97.4 ± 0.25abc | 0.10 ± 0.03b | 0.72 ± 0.06ab | 0.06 ± 0.01cd | 1.29 ± 0.04ª |
| Antagonists vs phytopathogens | |||||
| B. subtilis vs F. oxysporum | 98.3 ± 0.07ab | 0.03 ± 0.04b | 0.51 ± 0.04bcd | 0.03 ± 0.04d | 1.32 ± 0.54a |
| B. subtilis vs A. solani | 98.5 ± 0.06a | 0.02 ± 0.04b | 0.44 ± 0.04bcd | 0.08 ± 0.02bcd | 1.40 ± 0.23ª |
| B. subtilis vs P. infestans | 98.6 ± 0.10a | 0.03 ± 0.05b | 0.42 ± 0.04bcd | 0.03 ± 0.01d | 1.47 ± 0.20ª |
| B. methylotrophicus vs F. oxysporum | 98.7 ± 0.05a | 0.04 ± 0.01b | 0.40 ± 0.04bcd | 0.04 ± 0.01d | 1.32 ± 0.54a |
| B. methylotrophicus vs A. solani | 98.5 ± 0.09a | 0.04 ± 0.05b | 0.47 ± 0.04bcd | 0.10 ± 0.01abcd | 1.24 ± 0.10a |
| B. methylotrophicus vs P. infestans | 98.5 ± 0.03a | 0.03 ± 0.02b | 0.44 ± 0.04bcd | 0.08 ± 0.01abcd | 1.27 ± 0.39ª |
| B. amyloliquefaciens vs F. oxysporum | 98.5 ± 0.03a | 0.03 ± 0.02b | 0.51 ± 0.02bcd | 0.05 ± 0.70d | 1.44 ± 0.13ª |
| B. amyloliquefaciens vs A. solani | 98.6 ± 0.09a | 0.02 ± 0.01b | 0.46 ± 0.08bcd | 0.17 ± 0.04a | 1.48 ± 0.72ª |
| B. amyloliquefaciens vs P. infestans | 98.5 ± 0.1a | 0.08 ± 0.04b | 0.45 ± 0.06bcd | 0.06 ± 0.02cd | 1.54 ± 0.98ª |
Means with the same literal between columns, are statistically equal, according to the Tukey’s test (p=0.05). ± Standard error.
The ash content was higher in fruits from plants treated with Bacillus strains in 95 %, than those obtained from the interaction treatments (Bacillus vs phytopathogens) and in the control plants (Table 3).
The protein content ranged from 0.33 to 0.93 % (Table 3), where it was observed that tomatoes from plants treated with the Bacillus strains, contained more protein (0.68-0.93 %) than those obtained from the control plants. The fruits of plants treated with pathogens had a high protein content, except with the treatment of A. solani, resulting in the lowest values (Table 3). While the content of fats (lipids) ranged from 0.02 to 0.18 % without a clear trend (Table 3), where the highest value was observed in tomatoes of plants treated with B. methylotrophicus. The bromatological composition of the fruits analyzed was similar to that reported in literature (Guil-Guerrero & Rebolloso-Fuentes, 2009; Pinela et al., 2012). Pinela et al. (2012), reported high moisture levels, ranging from 90.6 to 93.7 %, ash content ranged between 0.54 and 0.74, a low lipid content ranging from 0.03 to 0.17 in tomato cultivars. Additionally, Hernández-Suárez et al. (2007) mentioned that many factors such as cultivar and cultural practices could influence in the bromatological composition of the tomato fruits, mainly in the mineral concentration (ashes), protein and fat content. Therefore, the ability of Bacillus strains to fix nitrogen, solubilize minerals such as phosphorus and produce hormones that regulate plant growth, may be involved in the increase of protein and ash content in fruits (Sivasakthi et al., 2014). On the other hand, the efficacy of the antagonistic bacteria evaluated under greenhouse conditions, without heating, could be affected by temperature, internal humidity and concentration of the antagonistic and phytopathogenic microorganisms (Inam-ul-Haq et al., 2009).
Conclusion
The Bacillus strains evaluated in this study promoted fruit yield and size in tomatoes under greenhouse conditions, emphasizing on variables such as plant height, leaf chlorophyll, yield, root mass, stem diameter, size and fruit quality. Therefore, strains B. amyloliquefaciens and B. subtilis subsp. inaquosorum can be a sustainable alternative to be used to increase the yield and size of the fruits in tomato.
