Materials and methods

The first trial was planted in the El Cerrillo Experimental Field, of the Autonomous University of the State of Mexico (19° 10’ 25” N - 99° 37’ 02” W), on April 7, 2016 2016. A split split plot experimental design was used, in which main plots included six agrochemicals (Aliette, Actigard, Poncho Votivo, Serenade, Messenger and Consist Max) and one absolute control. As subplots, two application methods were included (foliar and on the soil), applying onto the soil by spraying the bottom of the furrow when planting, and foliar application was by spraying the plant 50 days after its emergence. Split splitplots consisted of 3 doses of the products: 1. Dose recommended on the product label, 2. Low dose, with 50% of the recommended dose, and 3. High dose, with the recommended dose plus 50% of this dose (Table 1). The experimental unit consisted of two 3 m long rows, 0.80 m between rows, with an area of 4.8 m² and three repetitions. The maize seed used was of the commercial hybrid BG1384W (Biogene). The control treatments consisted of only water. The infection was of a natural incidence.

The second planting cycle (April 5^th, 2017) was set up in the same Experimental Field. Due to a low plant emergence due to an attack of the corn seed worm (Hylemia sp.), the trial was entirely replanted on May 10^th, repeating the same experimental design. In order to register the severity of the diseases, scales were used for common rust (Puccinia sorghi) and the tar spot complex (Phyllachora maydis and others). In rust and tar spot, severity was estimated using a visual scale of 1-5, where: 1=Resistant (with pustules in 10% of the foliar area), 2=Moderately resistant (pustules in 20-30% of the foliar area), 3=Moderately susceptible (pustules in 40-50 % of the foliar area), 4=Susceptible (pustules in 60-70% of the foliar area) and 5=Very susceptible (pustules in 100% of the foliar area). Agronomic data were collected from a yield trial (^{CIMMYT, 1985}; ^{IBPGR, 1991}), including days at 50% of male and female flowering, plant and ear height, plant and ear aspect, number of healthy and rotten ears, weight of fresh grain at harvest adjusted to 15% humidity to obtain grain yield per hectare. The variables registered were placed under an analysis of variance (ANOVA) and values obtained were placed under a Tukey test.

Results and discussion

Planting cycle TO-2016. Acibenzolar-S-methyl and Tebuconazole + Trifloxystrobin and Bacillus subtilis delayed female flowering, whereas plots treated with Fosetil-Al and Cloatianidin + Bacillus firmus showed an acceleration in female flowering (Table 2). Male flowering was uniform throughout the experimental units. The difference in days between male and female flowering was of two days, which, according to ^{López (1991}), a minimal difference between pollen emission and the appearance of silks guarantee a good pollination and grain filling. On the other hand, for ASI (Anthesis-silking interval), Fosetil-Al showed the lowest value, which, according to ^{Uribelarrea et al. (2002}), is a desirable characteristic, since a high ASI value causes the reduction of yield, due to erratic silk pollination.

Plots treated with Fosetil-Al showed a statistically significant increase in plant and ear height, whereas the Harpin Protein reduced them. For the plant balance, all agrochemicals had values of 0.50, a desirable value in the crop to obtain an adequate balance to avoid lodging.

For the ear aspect, there were no statistical differences. Between treatments, the control (untreated plots) showed the lowest number of rotten ears, while plots treated with Fosetil-Al had the highest number of rotten ears. Regardless of this, Fosetil-Al and Acibenzolar-S-metil showed a statistically higher yield with 5.7 and 5.9 t ha^-1, respectively. The lowest yield obtained was with the Harpin Protein.

Based on the scale of severity used to evaluate the damage caused by the common rust and TSC, the plants displayed a lower severity of these two foliar diseases. For TSC, the product Clothianidin + Bacillus firmus y Fosetyl-Al showed the highest severity with 1.6 (Table 2) in comparison with the average of 1.4 and the control with 1.2. Likewise for rust severity, plants from plots treated with Clothianidin + Bacillus firmus, statistically had a higher severity value (1.3), compared with the average of 1.2 and the control of 1.0. Table 2 shows that, despite this observation, the plots treated with Clothianidin + Bacillus firmus had a higher yield in comparison to the control, which has a lower disease severity value. This may be attributed to the characteristics of Bacillus, given that these organisms contribute to the productivity of crops, since they are located in the rhizosphere and colonize the roots of plants, promoting the growth of rhizobacteria that contribute to a greater absorption of nutrients (^{Schallmey et al., (2004}).

Planting cycle TO-2017. Fosetil-Al was found to accelerate flowering, while Acibenzolar-S-methyl and Trifloxystrobin + Tebuconazole delayed it (Table 3). For the variable female/male flowering, it was found that agrochemicals, means of application and dose had the same effect, indicating a minimum difference between pollen emission and the appearance of silks, which guarantee an adequate grain (^{López, 1991}). Acibenzolar-S-methyl registered the greatest plant height with 173.5 cm, followed by Tebuconazole + Trifloxystrobin with 172.3 cm., and Bacillus subtilis presented the lowest plant height with 152.4 cm. However, there was no significant difference in ear height. In plant balance, Tebuconazole + Trifloxystrobin and Acibenzolar-S-metil had the desirable plant balance value of 0.5.

Plant aspect was negatively affected with the fungicide Fosetil-Al and plants from the untreated plots had the best aspect. Ear aspect was equal in all plots, despite some of them having desireable or undesirable aspects. This coincides with ^{Guimarães et al. (2008}), who reported that the use of inducers may lead to physiological effects, as in the case of Acibenzolar-S-methyl applied on cotton plants that presented a reduction in plant height, dry and fresh weight of stems, due to the activation of the resistance imposing a demand of energy in the plants (^{Dietrich et al., 2005}), and a reduction of metabolytes for growth and other important physiological processes (^{Heil et al., 2002}).

The highest grain yield was obtained with Tebuconazole + Trifloxystrobin (5.7 t ha^-1), and the lowest, with Fosetil-Al (3.6 t ha^-1). The results for yield with the Tebuconazole + Trifloxystrobin treatment coincide with reports by ^{Couretot et al. (2013}), who mention that the mixture of triazoles and estrobilurines increases yield.

For this cycle, two evaluations were done for the severity of P. sorghi, in which, as in the first cycle, averages indicate that plants of the plot range from moderately resistant to resistant (Table 3). In the first evaluation, Serenade® (Bacillus subtilis) reduced the severity of rust, which coincides with reports by the company AgraQuest, who indicate that B. subtilis induces the natural resistance of plants. Also, ^{Maget and Peypoux (1994}) mention that compounds called Iturines, produced by this organism, display activity against plant pathogenic fungi. Various reports indicate that this bacteria can stop germination of the pathogen’s spores by interrupting the growth of the germinative tube and inhibiting the union of the pathogen with the plant leaf (^{EPA, 2006}).

In the second rust evaluation, there were no significant differences in the severities of the plants treated; however, Serenade® (Bacillus subtilis), which gave the lowest severity value in the first evaluation, is found in the same statistical group with the other products. In order to maintain the previously observed effect it may be necessary to make a second application, as mentioned by ^{Rohilla et al. (2001}), who point out that the degree of protection provided by the application of a fungicide, whether on the ground or foliar, decreases with time. There are also various reports of resistance elicitors which do not provide a significant control of the disease when compared to the control (^{Mogollón and Castaño, 2011}) since, in the field, the expression of induced resistance is influenced by the environment, genotype and nutrition (^{Walters et al., 2005}).

Fosetil-Al showed a higher efficiency by reducing the severity of the TSC, presenting the lowest value in comparison to the control, which had the highest value for severity (Table 3). The active ingredient of this product is reported as a resistance inducer in several crops, including strawberry, since it reduces the severity of crown rot (P. cactorum), root rot (P. fragariae) (^{Eikemo et al., 2003}), and in potato tubers, when applying it to control P. infestans, where it showed a greater accumulation and increase of enzymes β-1,3-glucanases, proteases, phytoalexins and phenolic compounds (^{Andreu et al., 2006}), related with plant defenses. Although Fosetil-Al displayed the lowest TSC severity value, it did not increase grain yield. ^{Guimarães et al. (2008}), mention that inducing resistance in plants may have physiological effects, since the activation of resistance imposes high demands of energy on the plant (^{Dietrich et al., 2005}) and a reduction of metabolytes for growth and other important plant processes (^{Heil and Baldwin., 2002}).

Conclusions

Both diseases in which resistance inducers were evaluated were present in both planting cycles. In the TO-16 cycle, none of the agrochemicals reduced the severity of rust and TSC, although Tebuconazole + Trifloxystrobin increased grain yield. For cycle TO-2017, Serenade proved to be the best agrochemical in reducing the severity of P. sorghi, while Fosetil-Al showed the best effect on reducing the severity of the TSC, but Tebuconazole + Trifloxystrobin induced a higher grain yield. The disease severities were not affected by the application method. There were significant differences between doses, since the severity of rust decreased when using high doses of agrochemicals.