1. Introduction
In the present time, the tomato crop has gained economical and feeding importance worldwide, because the harvested surface worldwide exceeds 4 million ha per year−1, with the mean production fluctuating around 177 million tons per year (FAO, 2019). México occupies the ninth position with a 3.35 million tons production with an export value of 2613 million dollars [1] .
Although the production of this vegetable is affected by the fusariosis vascular disease, which is a cryptogamic disease caused by Fusarium oxysporum f.sp. lycopersici (FOL), being one of the factors that limit the success in the production of this vegetable, causing up to 60% of loss and in some cases total crop loss [2] .
[3] mentions that the symptoms caused by the Fusarium oxysporum f.sp. lycopersici are initially observed in the plant’s root system, which causes severe rotting and moves through the stem’s vascular bundles to the aerial part, darkening them and obstructing water and nutrients way. The root’s vascular system, stem and petioles turn brown-reddish, causing its tamponade, to cause a slight yellowness and premature aging of the lower leaves. There are 3 FOL breeds which are genetically similar and have clonal populations. In different special ways of some vegetative compatible groups (VCG) they can be found in the same polymorphism pattern of the DNA multiplied randomly, the existence of four VCG that go from 000300 to 0033 has been informed. Breeds 1 and 2 produce VCG 0030 to 0032, and breed 3 VCG 0030 and 0033 [4] .
11 FOL proteins have been identified, which have been named proteins secreted in the xylem (SIX). Three of these proteins are opposed by the tomato I genes: Avr1 (SIX4) is known by genes I and I-1 non allelic, Avr2 (SIX3) is known for I-2 and Avr3 (SIX1) is known for I-3. Avr2, Avr3 as well as SIX6, are genuine effectors, and it has been found that they contribute to the general virulence [5] .
The Fusarium species responsible for the wilting follow a similar infection pattern; penetrate through the root and colonize the plant’s stem and the vascular system. Although, the colonization is restricted in resistant crops as well as susceptible ones to the region of initial entrance of the pathogen, due to the occlusion of the vessels of gels, release of callouses and tyloses. In susceptible crops, the continuous colonization (secondary distribution) when the gels and callouses are degraded by the pectolytic enzyme effect of the pathogen and the tyloses growth is inhibited. In resistant crops, catechins type flavonoids and their oxidation products deactivate the enzymes, and the secondary distribution is confined to the initial infection points [6] .
Fusarium control is mainly based on the use of resistant varieties, soil fumigation and fungicide applications, among other techniques. In Sinaloa, the control of root diseases in tomato cultivation has been carried out through the use of pesticides (fungicides and soil fumigants), the most used fungicides are systémic: benzimidazoles, triazoles, contact: Imidazoles, ditiocarbamatos Benzimidazoles and triazoles are characterized by favoring the formation of resistant pathogen strains [7] .
Despite the above, there is no interest about the mineral nutrition having an important role in the plant disease control. The use of chemicals increases the negative impact in the environment and food safety. The deficiency of all essential nutrients affects plant’s health and susceptibility to diseases. The plants with nutritional stress are inclined to diseases, while plants with adequate nutrition are more disease tolerant [8] .
Basic scientific research has provided specific explanations about the mechanisms in which nutrition has a strong influence over the incidence and severity of the diseases of the cultivated plants. On the other hand, from this research obtained results have given the basis to add the nutritional management in commercial schemes of integrated control of the sanitary problems in several crops [9] . Also, it is pointed that the unbalances between N and K, between Ca and B and between Ca, Mg and K favor the disease’s development and have shown how the comprehensive nutrition management must be part of the management strategies for the disease control [10] .
The diffusion rate ant the composition of the exterior cytoplasmic exudates, are aspects that are affected by the nutrient availability. For example, the sugars concentration and amino acids is high in the leaves when there is potassium (K) deficiency, and how these substances favor the fungus development, its greater concentration favors fungous diseases [11] .
It was demonstrated that the CaCl2 abiotic elicitor application, decreases the damage post-harvest in pear shaped fruit, by the induction of high levels in the defense enzyme activity [12] ; the high concentration of the Ca2+ cation shows that an important event during the interaction of the hosting pathogen that induces to immune responses innate to the plant [13] .
The objective of this research investigation was to determine the effect of the nutrients (nitrogen, phosphorus, potassium and calcium) and the severity of the different Fusarium oxysporum f.sp. lycopersici breeds in tomato plants.
2. Materials and Methods
2.1. Fusarium Insulations Used
The insulations used in this study are the same used y [14] ; who confirmed that the breeds in the insulations used through pathogenicity tests and molecular tests (Table 1). The insulations are placed in the insulation collection in the Phyto protection lab in the Agronomy Faculty in the Universidad Autónoma de Sinaloa.
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Table 1. Origin, codes and accession numbers of the Genbank of Fusarium spp. insulated from tomato plants.
The insulations were obtained from commercial tomato crops (S. lycopersicum) in the state of Sinaloa, México in the fall-winter 2016-2017 cycle.
2.2. Sowing
The sowing of the four tomato varieties undetermined was done in peat and fine vermiculite substratum in trays of 200 cavities, placing one seed per cavity. They were kept under adequate greenhouse conditions the varieties used were Bony Best (BB) susceptible to the three FOL breeds, Manapal (M) resistant to one FOL breed, Walter (W) resistant to FOL breed 1 y 2 de FOL y FLA resistant to the three FOL breeds.
The pathogenicity tests done used the four tomato varieties. The plant’s roots by genotype in the two true leaves stage in sterile peat were washed and soaked in a conidial suspension (1 × 105 UFC∙mL−1) of each insulation for 10 minutes and then planted into a pot that contained sandy loam soil. The suspension was obtained by gathering the spore of each insulation. 40 days after inoculation, once the 4 true leaves showed up, they were harvested for the respective analysis.
2.3. Nutritive Solution
The nutritive solution (SN) was applied after the FOL inoculation, the used concentration were based on the Steiner universal solution (1966) using sterile distilled water, but with modifications, the concentration was increased by 15% and lowered to 0 the four elements. The fertilizers used in this experiment were, potassium nitrate KNO3, calcium nitrate (NO3)2∙4H2O, mono potassic phosphate KH2PO4, potassium sulphate K2SO4, calcium chloride CaCl2 and potassium chloride KCl (Table 2). According to [15] , post-sowing, every plant was inoculated on day 40 (when these had two true leaves) after the sowing, by the root immersion method, this was submerged for 15 minutes with FOL with an amount of 1 × 106 conidia.
2.4. Foliar Analysis
The plants were analyzed to know each elements concentration and determine N, P, K and Ca concentrations. The samples were placed in paper bags and inserted to a forced air stove at 70˚C ± 5˚C for 24 h. Once the sample dried, it was grinded in a grinder until in went through a 1.00 mm sieve. After the grinding the sample was homogenized and separated in 5 to 10 g samples for the analysis and storage. From the homogenized sample, it is placed in a 2 g melting pot, and it’s introduced in a muffle at 450˚C for two hours. The sample is retrieved and cooled down, afterwards 2 - 3 mL of distilled water is added to moist the ashes and for the estimation of each element the methodology proposed by [16] was used.
2.5. Disease’s Severity
30 days past the inoculation the plants were retrieved from the trays and were
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Table 2. Treatments and factor levels.
labeled. The severity of the disease was evaluated, according to the scale proposed by [17] ; established levels from (0 - 4) where, 0 = 0%; 1 = light necrosis (1% - 33%); 2 = moderate necrosis and discoloration (34% - 66%); 3 = extreme necrosis and discoloration (67% - 100%); 4 = dead plant.
2.6. Statistical Analysis
A completely random design was established with a factorial arrangement with six repetitions obtaining a total of 96 treatments. For variables with result of disease’s severity the data was analyzed by the non-parametrical test of Kruskal Whallis (α = 0.0), which originated a dendrogram; for the variables of the foliar analysis of the elements a variance analysis was done and a comparison Tukey mean test (α = 0.05).
3. Results and Discussion
3.1. Disease’s Severity
The severity of the vascular fusariosis caused by FOL was statistically less in treatments 93, 95 and 81. Treatments 93 (Ca at 15%, R3, variety BB) y 95 (Ca at 15%, R3, variety W), showed a decrease in disease’s severity. [18] concludes that, when calcium is increased, the FOL disease decreases, because it has an adequate amount of calcium in the tomato plant’s tissue which reduced the enzymes that fade in the cellular walls produced by FOL. Besides, it is reported that Ca deficiency increases cytoplasmic membrane permeability, which favors the sugars and amino acid accumulation in the apoplast [19] , this limits the polymer synthesis which may have a negative effect on the development of the pathogen [20] , On the other hand, Ca is fundamental in the cellular wall structure, so its deficiency may cause a stiffness loss in the cellular wall and facilitate the pathogen infection in plants [21] [22] , found that nutrition with calcium is important to the tomato bacterial withering resistance, reporting that when the calcium content in plants tissue is increased, the resistance to some diseases is induced and have shown some possible mechanisms: calcium does a direct inhibition of the polygalacturonate and other pectolytic enzymes produced by the pathogen. According to [23] , the calcium nitrate application reduces the incidence of the disease called southern putrescence caused by Sclerotium rolfsii in the carrot crop. Because of this, it can be deduced that the calcium application in the treatments may have benefited the tomato plant’s health.
3.2. Foliar Analysis
Treatment 22 consists of 193 mg∙L−1 N, R3 FOL and M variety, statistically showing a greater N content in the foliar area with a 73.5 mg concentration N g−1 plant (Table 3), this treatment had a severity level with a mean of 1, which coincides with [24] , who established an N sufficient level which oscillates between 62.5 - 100 mg of plant N g−1. This indicates that adequate nitrogen levels are less damage susceptible by Fusarium, although [25] found that nitrogen excess may promote favorable conditions for plant diseases since it encourages thinner and weaker cellular walls. On the other hand, [26] demonstrated that ammonium nitrate has no influence on the Fusarium growth in the fin and root as long as it has low levels, keeping a level of 10 to 100 mg∙L−1 N.
P concentration was greater in treatment 94 as shown in Table 4, which is made of Ca (207 mg∙L−1), P (31 mg∙L−1), R3 FOL and M variety, with 62.75 mg of plant P g−1, this showed a severity level of the disease with a 1.8 mean which agrees with [27] , where the phosphorus content in vegetal tissue, that was observed in eight treatments, values fluctuated from 24.75 of P g−1 of plant in treatment 50% Steiner solution, to 52.8 mg of P g−1 of plant in treatment 100% Steiner solution respectively.
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Table 3. Foliar analysis of Nitrogen in plant (mg).
Treatments with different letters show significant difference Tukey α = 0.05.
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Table 4. Foliar analysis of Phosphorus in plant (mg).
Treatments with different letters show significant difference Tukey α = 0.05.
These results show that when phosphorus is added to the nutrient solution in the amount of 207 mg∙L−1 it can reduce damage severity caused by Fusarium in tomato plants, as mention by [28] , P applications reduce seed disease as well as fungous diseases in root, stimulating a vigorous development that allows plants to evade the diseases since P is essential for the virus’ multiplication, excess of this may increase plant’s susceptibility to virus’ disease.
Treatment three showed a greater leave concentration of K and was statistically greater in regards of the other treatments as shown in (Table 5), which is composed by Steiner solution without addition of N, K (273 mg∙L−1) R1 FOL and W variety, such treatment showed a concentration of 110.9 mg of K g−1 of plant y and a mean severity of 0.8, this concentration agrees [29] publication, who concluded that element levels are sufficient for an adequate crop development, establishing a sufficient level from 75 to 125 mg of Kg−1 of plant. The disease’s severity in this treatment was lower, this could be explained due to the adequate potassium content, since [30] , mention in a study done with strawberry plants that K deficiency can promote root exudate production, especially phenolic acids, which can not only inhibit the strawberry’s plant’s growth, but promote the F. oxysporum growth.
The greatest Ca content in tomato plants was found in treatment 93 (Table 6) which is made of Ca (207 mg∙L−1), R3 FOL and BB variety, where 15 mg of Ca g−1 of plant were obtained, such treatment showed a disease severity of 0.8 mean, this agrees with the report done by [31] , which establishes that from 10 to 30 mg of calcium g−1 of plant, are sufficient levels so that the plant has an optimal development. The Ca increase is reported as an important event during the pathogen-host interaction inducing immune innate plant responses [32] , Ca acts as a structural element and promoter in the activation of some enzymes that participate in the defense against some pathogens and physiological disorders, according to [33] , calcium also drastically inhibits the polygalacturonate’s action, because the high levels of calcium in the cell apoplast generate a greater proportion
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Table 5. Foliar analysis of Potassium in plant (mg).
Treatments with different letter show significant difference Tukey α = 0.05.
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Table 6. Foliar analysis of Calcium in plant (mg).
Treatments with different letters show significant difference Tukey α = 0.05.
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Figure 1. Disease severity index DSI (%). Treatments with different letters show significant difference Tukey α = 0.05.
of pectates and therefore greater resistance to the wall disintegration; the Ca may act as amplifier of certain defense signals against the maturity spot and increase the speed response of the plant facing the incidence. Different scientific reports show the close relationship between the disease with the calcium deficiencies in drought seasons. The fertilization and the calcium behavior against the other soil nutrients are also incidental factors in the maturity spot, incidence that is reduced with calcium nitrate fertilizations [34] .
3.3. Disease’s Severity Index DSI
As it is observed in Figure 1 treatments 93 and 81 are made of 207 mg∙L−1 Ca, R3 of FOL, BB variety, 0Ca, R3 and BB variety presented an 8.5% value of low severity index, this agrees with the results obtained by [35] , which found low severity levels of 10%, applying low levels of N 15 mg∙L−1, while Delgado et al. (2016), obtained similar results in garlic showing that severity indexes oscillated between 2.25% to 3.5%. [36] obtained a severity index of 5% in chickpea that were inoculated with breed 5 of Fusarium oxysporum f.sp. ciceris.
4. Conclusion
Bonnie Best and Walter varieties presented lower severity of de disease with 0.5 levels, with a 207 mg∙L−1 Ca concentration; it is convenient to apply such Ca doses to reduce the disease’s severity caused by FOL. The Bonnie Best variety is susceptible to the 3 FOL races, but with optimal nutrition, it has the capacity that when attacked by microorganisms capable of activating defensive mechanisms, these mechanisms include the increase in the activation of enzymes, such as phenylalanine ammonium lyase (PAL), which is key in the synthesis of important defensive metabolites. While R2 of FOL showed greater disease severity, it is worth mentioning that the research will continue to obtain an adequate nutrient dosage to decrease the disease’s severity.
Acknowledgements
To CONACYT for the scholarship granted to Mendoza Medrano to obtain doctorate studies.