Materials and Methods

Establishment of the experiments. Every 15 d, pepper (C. annuum) seeds of the Camino Real (Harris Moran) hybrid and a creole cultivar from Tetela de Ocampo, Puebla state, Mexico, were planted in 125 ml polyurethane cups with sterilized peat moss (PREMIER) and perlite (AGROLITA) at a 3:1 proportion. The seedlings were transplanted at 45, 30, and 15 d age into polyurethane pots with 1 L soil from the pepper producing region in Vega de Metztitlán, Hidalgo, Mexico (VMH), where pepper wilt is a serious problem. To define treatments, a factorial arrangement of the following factors was constructed: 1) cultivar (Camino Real and creole from Tetela de Ocampo), 2) seedling age (transplanted at 45, 30, and 15 d after seeding), 3) resistance inducer (with or without ASM), and 4) P. capsici inoculation (Strain 1, Strain 2, and a control without inoculation). A total of 36 combinations (2 x 3 x 2 x 3) were evaluated. The experiment was established under greenhouse conditions, under a completely random design with ten replicates, and it was done twice.

In the corresponding treatments, the leaves were sprayed weekly with 1 mg ASM/plant to the seedlings of all three ages, beginning one week before transplanting and until the end of the experiment (51 d after transplanting). Actigard(r) 50 (Syngenta) was used as a source of ASM. The inoculation of P. capsici was done 45 d after transplantation. The strains used were isolated from Serrano pepper plants from the VMH and they were previously tested as pathogenic in the abovementioned cultivars. Before inoculation, all the plants were irrigated with distilled water. Each seedling was inoculated with 1 mL of a suspension with 105 zoospores/mL. The suspension was injected into the base of the stem at a depth of 2 cm. After inoculation, all plants were irrigated until the soil in each pot was saturated. The controls without inoculation were treated with sterile distilled water. Daily temperature conditions in the greenhouse varied from 12 to 35 °C.

Evaluated variables. After transplanting, wilt severity, plant height, and the number of leaves per plant were evaluated every third day. At the end of the experiment, the dry weight of the aerial part was evaluated. The severity was evaluated based on the scale of ^{Moran et al. (2010)}. From these results, the area under the disease progress curve (AUDPC) was calculated by means of the trapezoidal integration method (^{Shaner and Finney, 1977}). To estimate the dry weight of the plants, the plant material was placed in perforated paper bags and dried in a forced air oven at 70 °C for 72 h. At the end of the experiment, root segments were collected and incubated at room temperature (24 °C) in PARPH medium (^{Kannwischer and Mitchell, 1978}). Also, root subsamples were placed in Petri dishes with sterile distilled water to verify the occurrence of coenocytic mycelia with sporangia typical of P. capsici.

Data analysis. The data obtained were analyzed statistically by the variance analysis technique and the Tukey multiple range test (^{Steel and Torrie, 1980}). The information was processed with the SAS (Statistical Analysis System, v. 9.3 Cary, North Carolina) statistical software.

Results

Table 1 shows a summary of the results of the variance analysis for the main variables evaluated in the present work. Figures 1 to ⁷ report the results for the highest order significant interactions.

Table 1 Summary of the variance analysis (P > F) of the variables evaluated in Serrano pepper plants (Capsicum annuum L.). 

¹Trasplante a 45, 30 y 15 días después de la siembra.

²cv Camino Real (Harris Moran), cv criollo de Tetela de Ocampo, Puebla.

³Con acibenzolar S-metil, sin Inductor de resistencia.

⁴Testigo no Inoculado, Cepa1 y Cepa 2 de Phytophthora capsici.

** Interacción utilizada para la comparación de medias y preparación de gráficas.

Figure 1 Experiment 1. Effect of the Plant age X Genotype X Resistance inducer X Phytophthora capsici inoculation interaction on the area under the disease progress curve of Serrano pepper plants (Capsicum annuum L.) cultivated in unsterilized soil from the Vega de Metztitlán, Hidalgo (VMH). The oomycete strains were isolated from Serrano pepper plants from the VMH. The inoculation was done with 10⁵ zoospores per plant 45 days after transplanting. Means from ten replicates. Means with the same letter are statistically equal (Tukey α = 0.5). Age 1, 2, and 3 = transplant at 45, 30, and 15 days after seeding. CR = Camino Real (Harris Moran) cv. TO = Tetela de Ocampo, Puebla, creole cv. TNI = control without P. capsici inoculation, unsterilized soil. 

Figure 2 Experiment 2. Effect of the Plant age X Genotype X Phytophthora capsici inoculation interaction on the area under the disease progress curve of Serrano pepper plants (Capsicum annuum L.) cultivated in unsterilized soil from the Vega de Metztitlán, Hidalgo (VMH). The oomycete strains were isolated from Serrano pepper plants from the VMH. The inoculation was done with 10⁵ zoospores per plant 45 days after transplantation. Means from 20 replicates. Means with the same letter are statistically equal (Tukey α = 0.5). Age 1, 2, and 3 = transplant at 45, 30, and 15 days after seeding. CR = Camino Real (Harris Moran) cv. TO = Tetela de Ocampo, Puebla, creole cv. TNI = control without P. capsici inoculation, unsterilized soil. 

Figure 3 Experiment 2. Effect of the Plant age X Resistance inducer X Phytophthora capsici inoculation interaction on the area under the disease progress curve of Serrano pepper plants (Capsicum annuum L.) cultivated in unsterilized soil from the Vega de Metztitlán, Hidalgo (VMH). The oomycete strains were isolated from Serrano pepper plants from the VMH. The inoculation was done with 10⁵ zoospores per plant 45 days after transplantation. Means from 20 replicates. Means with the same letter are statistically equal (Tukey α = 0.5). Age 1, 2, and 3 = transplant at 45, 30, and 15 days after seeding. Cl=with acibenzolar S-methyl. Sl = with no resistance inducer. TNI = control without P. capsici inoculation, unsterilized soil. 

Figure 4 Experiment 1. Effect of the Plant age X Genotype X Resistance inducer interaction on the dry weight of the aerial part of Serrano pepper plants (Capsicum annuum L.) cultivated in unsterilized soil from the Vega de Metztitlán, Hidalgo (VMH). Means from 30 replicates. Means with the same letter are statistically equal (Tukey α = 0.5). Age 1, 2, and 3 = transplant at 45, 30, and 15 days after seeding. CR = Camino Real (Harris Moran) cv. TO = Tetela de Ocampo, Puebla, creole cv. Cl = with acibenzolar S-methyl. Sl = without resistance inducer. 

Figure 5 Experiment 1. Effect of the Plant age X Genotype X Phytophthora capsici inoculation interaction on the dry weight of the aerial part of Serrano pepper plants (Capsicum annuum L.) cultivated in unsterilized soil from the Vega de Metztitlán, Hidalgo (VMH). The oomycete strains were isolated from Serrano pepper plants from the VMH. The inoculation was done with 10⁵ zoospores per plant 45 days after transplantation. Means from 20 replicates. Means with the same letter are statistically equal (Tukey α = 0.5). Age 1, 2, and 3 = transplant at 45, 30, and 15 days after seeding. CR = Camino Real (Harris Moran) cv. TO = Tetela de Ocampo, Puebla, creole cv. TNI = control without P. capsici inoculation, unsterilized soil. 

Figure 6 Experiment 1. Effect of the Resistance inducer X Phytophthora capsici inoculation interaction on the dry weight of the aerial part of Serrano pepper plants (Capsicum annuum L.) cultivated in unsterilized soil from the Vega de Metztitlán, Hidalgo (VMH). The oomycete strains were isolated from Serrano pepper plants from the VMH. The inoculation was done with 10⁵ zoospores per plant 45 days after transplantation. Means from 60 replicates. Means with the same letter are statistically equal (Tukey α = 0.5). TNI = control without P. capsici inoculation, unsterilized soil. Cl = with acibenzolar S-methyl. Sl = without resistance inducer. 

Figure 7 Experiment 2. Effect of the Plant age X Genotype X Resistance inducer interaction on the dry weight of the aerial part of Serrano pepper plants (Capsicum annuum L.) cultivated in unsterilized soil from the Vega de Metztitlán, Hidalgo (VMH). Means from 30 replicates. Means with the same letter are statistically equal (Tukey α = 0.5). Age 1, 2, and 3 = transplant at 45, 30, and 15 days after seeding. CR = Camino Real (Harris Moran) cv. TO = Tetela de Ocampo, Puebla, creole cv. Cl = with acibenzolar S-methyl. Sl = without resistance inducer. 

Area under the disease progress curve (AUDPC). In experiment 1, the variance analysis indicated highly significant effects of the age X cultivar X resistance inducer X inoculation interaction (P<0.0004). This result indicates that, in general, the plants of age 1 (transplanted 45 d after seeding) had a greater AUDPC than those of ages 2 and 3 (transplanted 30 and 15 d) (Figure 1). At age 1, the inoculation with P. capsici had a greater AUDPC than the controls with ASM and no inoculation, particularly when the plants were infected with Strain 1 (Figure 1). When ASM was not applied, the differences between treatments with or without inoculation were only significant in the Camino Real cultivar when it was inoculated with Strain 1. With the exception of this treatment, the response pattern among cultivars at this age was similar. In ages 2 and 3, although AUDPC was greater in the inoculated treatments, the difference with respect to the control were only significant in the plants of the Tetela de Ocampo cultivar inoculated with Strain 1, without ASM.

The application of ASM decreased the AUDPC, but not in all cases. At age 1, the Camino Real plants inoculated with Strain 1 of P. capsici and the control plants with no inoculation treated with ASM had a lower AUDPC than those not treated with the inducer. However, plants inoculated with Strain 2 showed no decrease in the AUDPC by effect of ASM, and the value of this variable resulted significantly higher than the control without inoculation but with ASM. Plants at ages 2 and 3, with and without ASM, had statistically equal AUDPC.

In experiment 2, the variance analysis indicated highly significant effects of the age X genotype X inoculation (P<0.0001) and age X resistance inducer X inoculation (P<0.0015) interactions. In the first interaction, no significant differences were detected in AUDPC among the different ages, except when the plants of age 1 were inoculated with Strain 1 (Figure 2), where there was a greater AUDPC. The rest of the differences were mainly due to the inoculation with P. capsici, regardless of the strain, which caused greater AUDPC than in the controls (Figures 2 and ³ ), with the exception of Camino Real cv. plants of age 1 without inoculation, which resulted statistically equal.

When analyzing the age X resistance inducer X inoculation interaction (Figure 3), it can be seen that there was no significant decrease in AUDPC with the application of ASM, regardless of the age. Moreover, against expectations, plants of age 1 inoculated with Strain 1 and treated with ASM had a greater AUDPC than the control without inoculation and treated with ASM.

Formation of mycelia and sporangia of P. capsici from pepper roots. At the end of the experiments, there was mycelium growth in PARPH medium from plants of all ages inoculated with both strains of P. capsici, but the greatest number of segments where there was mycelial growth occurred in plants inoculated 45 d after seeding (age 1). The frequency of cases in ages 2 and 3 was considerably lower. ASM tended to decrease the number of cases with mycelium growth in age 1 but did not inhibit infection (data not shown). The formation of sporangia in water from root segments of pepper inoculated with P. capsici only occurred in age 1 plants in both experiments. These reproductive structures were not detected in plants from age 2 and 3 in any treatment.

Dry weight of the aerial part, number of leaves, and plant height. In experiment 1, the variance analysis indicated significant effects on the dry weight of the aerial part (DWAP) in the age X genotype X resistance inducer (P=0.0137), age X genotype X inoculum (P=0.023), and resistance inducer X inoculum (P=0.0344) interactions. The first interaction indicates that ASM decreased DWAP in all ages and cultivars, but only in the Camino Real cv., ages 1 and 2, and the Tetela de Ocampo cv., age 3, the differences between plants with and without ASM treatment were significant (Figure 4).

In the case of the age X genotype X inoculation interaction, the results indicate that the DWAP of the Camino Real cv., age 2, with or without P. capsici inoculation, was higher than any other treatment at ages 1 and 3 (Figure 5). In the Tetela de Ocampo cv. age 2, the results were statistically equal to those of the Camino Real. In plants inoculated with Strain 1, which showed a decrease in DWAP, the differences against the rest of the treatments were not significant according to the Tukey test, regardless of the high number of replications involved and the fact that there were relatively drastic weight decreases. It is worth mentioning that in most cases, the DWAP of the controls in all ages and cultivars was numerically higher than in the plants inoculated with the pathogen.

The results of the resistance inducer X inoculation interaction indicate that the plants with ASM had lower DWAP values than those with no ASM and that the highest value corresponded to the control without inoculation and without ASM (Figure 6). The differences within the group of plants without ASM were not significant. The non-inoculated control with no ASM had a significantly greater DWAP than the plants with ASM, with or without P. capsici inoculation. Also, the DWAP of the plants with no ASM, inoculated with Strain 1 of P. capsici was greater than that of the inoculated plants treated with ASM.

In experiment 2, the variance analysis only showed significant effect of the age X genotype X resistance inducer interaction (P = 0.0069) on the dry weight of the aerial part (DWAP). This result indicates that the application of ASM caused a significant decrease in the DWAP of plants of all genotypes and ages, with the exception of the Camino Real cv., age 1 (Figure 7). The DWAP of the Camino Real cv., age 2, without ASM had the highest value, but it was statistically equal to the plants of the same cultivar, age 1, with and without ASM and to the Tetela de Ocampo cultivar, age 2, without ASM. The DWAP of plants age 3, with ASM was significantly lower in the plants treated with this resistance inducer, regardless of the cultivar.

The results of the variance analysis of the number of leaves and the area under the plant height progress curve of showed similar trends than those described for the DWAP. There was a significant decrease in the value of these variables due to ASM application and a tendency to greater development in plants of age 2, with negative effects of P. capsici inoculation with respect to the controls with no inoculation (data not shown).

Discussion

In the present work, ASM caused a decrease of the AUDPC of pepper wilt, although the effect was not consistent against both of the evaluated strains. The plant age factor determined important changes in the expression of the disease.

ASM tended to be more effective in decreasing the severity of pepper wilt in plants of ages 2 and 3, but it did not completely inhibit symptoms expression. This result agrees with other researchers who have reported a decrease in the disease with the application of ASM against P. capsici in bell pepper (^{Matheron and Porchas, 2002}) and in squash (^{Kone et al., 2009}). In this work, ASM was applied weekly starting one week before inoculation, but it caused a decrease in dry weight, number of leaves, and plant height in plants of all ages. ^{Ramos (2013)} also observed a decrease in plant development and growth in uchuva (Physallis peruviana) plants using ASM concentrations greater than 10 mg/L. ^{Mejía et al. (2009)} also observed similar results on tree tomato (Solanum betaceum) with doses of 25 mg/L. Also, ^{Nair et al. (2007)} reported that doses of 25 mg/L delayed growth in amaranth plants (Amaranthus spp.). The activation of systemic acquired resistance through the use of ASM can lead to an additional energetic cost, thus producing smaller and lighter plants and fruits (^{Van Wees et al., 2000}; ^{Romero et al., 2001}). However, the effect of applying inducers may depend on the time of activation of the signals, the fungal strain, the inducer used, and its persistence on the plants (^{Perazzolli et al., 2008}). It is then necessary to carry out further essays to optimize the time and application dose of ASM. On the other hand, there are reports about that the combination of induced systemic resistance (ISR) inducers with microorganisms that propitiate SAR results in a greater coordination between the implicated metabolic pathways (^{Abo-Elyousr et al., 2009}); although there are reports about that the activation of one pathway inhibits the other. This type of combinations has not been evaluated in pepper, making it recommendable to test the joint action of ISR and SAR inducers, as well as the subsequent application of fungicides.

Factors associated with the biochemical and physiological changes at different seedling ages could determine variations in the susceptibility to the disease (^{Kim et al., 1989}; ^{Gevens et al., 2006}; ^{Juvany et al., 2013}). For practical purposes, the use of 30 d old plants with an optimized dose of ASM could help to decrease the damage from P. capsici. This may be particularly true if natural infection levels are as low as the ones seen in the soil used in this work which were considerably lower than those obtained with the inoculation of a high number of zoospores per plant. It is necessary, however, to evaluate this behavior under field conditions, since environmental and management factors under those conditions are not easily reproduced in the greenhouse.

Conclusions

The application of ASM decreased the AUDPC of Serrano pepper blight but it also caused, in most cases, a decrease in the accumulation of dry matter in the aerial part of the plants of the Camino Real (Harris Moran) and the creole from Tetela de Ocampo, Puebla cultivars.

Thirty day old pepper plants had greater tolerance to wilt and greater development than did 45 d and 15 d old plants, regardless of inoculation of P. capsici, 45 d after transplanting.