T. urticae showed a strong interaction of treatments in both cultivars p-value ≤ 0.0001, thus showing that AS in all its dosages directly affect the amount of oviposition of this phytophagous (Table 1). Some studies with the exogenous application of the phytohormones AS and AJ have shown promising results regarding the reduction of the number of T. urticae eggs in several crops, such as cotton (Omer et al., 2001), beans (Farouk and Osman, 2011) and tomato (Pulga et al., 2020). Still in Table 1, it is observed in the average number of eggs (߃) that the dose that most reduces the amount of eggs was the treatments T5 for Fepagro26 and T7 for Garapiá, however the doses T6 and T7 in both cultivars showed apparent toxicity in the plants. So it is justified as a dose to be used for other bioassays AS 0.5 (T5) for both cultivars because it presents good results in the control of oviposition of T. urticae in addition to not causing apparent injury to bean plants. Treatments T4 and T2 had the opposite effect, increasing the amount of eggs, which indicates that this dosage for the common bean favors the phytophagous. The polyphagous characteristic of the mite can be related to the results of this study, since it induces a response of defenses regulated by both jasmonate and salicylate that impair its performance and do not suppress the defenses induced by plants in the same way as the specialist mites (Schimmel et al. 2017). The interaction between the SA concentration factors, physicochemical characteristics of the cultivars associated with the participation of salicylic acid in the expression of systemic resistance, must be taken into account, because in this treatment T4 may not have triggered its ability to induce the synthesis of PR - Proteins (proteins related to pathogenesis) (Fernandes et al. 2009). The reduction in the performance of T. urticae, at higher concentrations of AS, can also be related to the increase in proteins that interfere with the digestibility of herbivores, such as oxidative enzymes, lipids and proteins (Thaler et al. 1996; Zhang et al. 2017; Homayoonzadeh et al. 2020).
The other treatments T5 for Garapiá, T6 for both cultivars and T7 for Fepagro26 had a negative influence on the reduction of the number of mite eggs. According to DelRío et al. (2006), if used in high concentration in plants, it results in an antagonistic effect in its protection, which may explain the negative effect of the high doses of 1mM (T6) and 2.5mM of AS (T7) used for both cultivars at the present time. study in relation to common bean defenses.
Studies with the exogenous application of AS at different doses in bean (100mg/l) and strawberry (25, 50 and 75mg L-1) plants show satisfactory results, being observed the effectiveness in reducing the oviposition of T. urticae and also report an increase in productivity in these plants (100mg/L and 1.0; 2.0; 3.0 and 4.0mM), indicating an association resulting from AS and cultivar (Farouk and Osman, 2011; Trevisan et al. 2017). In the study carried out by Wei et al. (2014) in lima bean (Phaseolus lunatus L.) with the application of 1mM of AS and jasmonic acid (AJ), a greater oviposition of T. urticae was observed at 96h with a preference for AS, which was not evidenced in the present study., since T6 was the third to reduce the amount of eggs among four doses (0.1 – T4; 0.5 – T5; 1 – T6 and 2.5Mm – T7) of AS used in quantity of eggs for the mite- brindle in both bean cultivars, indicating that AS triggers different responses for each plant species.
Analyzing the correlation Fig. 2a and b, we can infer that the increase in AS resulting from exogenous application in bean plants, in ideal doses, can reduce the oviposition of T. urticae, since the AS function is a signal, inducing the plant to express resistance against attacks. of this phytophage as evidenced by Fernandes et al. 2009 and Santamaria et al. 2020
Similar results of the influence of AS on the reduction of T. urticae eggs were observed by Favaro et al. (2019) when evaluating two strawberry cultivars Aromas and Sweet Charlie, which show a positive correlation between AS doses (25, 50 and 75mg L-1) and the reduction in the number of eggs, however the Sweet Charlie cultivar showed higher decrease in the number of eggs in relation to doses. In tomato Pulga et al. (2020) also observed a correlation between AS doses (25, 50 and 75mg L-1) and the reduction in T.urticae oviposition. In the present study, the application of AS may have activated the defense differently in the bean cultivars, since, as observed, only the cultivar Garapiá showed a positive response between the doses of AS and the reduction of oviposition. Even with the difference in doses, where mM represents the molar mass of this phytohormone and mg L-1 are milligrams diluted per liter of water, it is observed that SA directly influences this relationship and causes changes in the biology of T. urticae.
Regarding the chemotactic response of T. urticae (Fig. 3) the phytophage was attracted by plant volatiles with a dose of AS 0.5mM in 48h to cultivate Garapiá. This may have occurred due to the production of these phytohormones being induced in response to the herbivory of this mite, and these HIPVs in plants can attract or repel the herbivore (El-Sayed et al. 2018). In a study carried out by Favaro et al. (2009) with strawberry (Fragaria sp.) was evaluated the preference of T. urticae to leaves with application of different doses of AS (0; 25; 50; 75 and 100mg L-1) in comparison to clean leaves, having as result the preference by the phytophagous to clean leaves, demonstrating that AS was efficient in repelling the two-spotted spider mite. Therefore, AS can influence oviposition in addition to influencing the chemotactic response of this phytophage.
When verifying the chemotactic response of T. urticae to combinations between Citrus spp. with combinations of the presence or absence of T. urticae at the time of contrasts Cabedo López et al. (2019) resulted in T. urticae having its preference associated with the plant and the HIPVs produced by them and not by the presence of their conspecifics. Therefore, T. urticae has its response triggered as a result of the combination of HIPVs and cultivar, thus it is inferred that different cultivars offered provoke different responses in this phytophage.
The predator was responsive to bean volatiles from cv. Fepgro26 infested with T.urticae after 48h Fig. 4, compared to the other treatments. Only after this period did the herbivory of T. urticae possibly activate the plant's defense mechanisms, guiding the displacement of N. californicus. Plants under stress due to the attack of herbivores are able to activate indirect defenses, thus synthesizing and releasing complex mixtures of volatiles that will attract natural enemies (Zhang et al. 2009). Similar results to the present study were found by Nachappa et al. (2006), where the predator P. persimilis was attracted by lima bean leaves infested with T. urticae after 48 hours. Menzel et al. (2014) also observed this same response time in the phytoseiid P. persimilis to search for T. urticae.
According to McMurthy et al. (2013) N. californicus has a generalist feeding habit with specificity linked to the genus Tetranychus, which may have caused a different response from the one shown in the work by Zhang et al. (2009) where the predator Phytoseiulus persimilis Athias-Henriot, 1957, also a generalist phytoseiid used in the biological control of T. urticae responded only to lima bean (Phaseolus lunatus) leaves infested with T. urticae in contrast to lima bean and Bemisia tabaci (Genn., 1889) (Hemiptera, Aleyrodidae).
In this study, the different responses triggered in bean cv. Fepagro26 and Garapiá by the herbivory by T. urticae may have been due to AS in addition to factors such as the physiological characteristics of each cultivar because, even though both exhibited intermediate resistance to the diseases, different responses were obtained. There are no studies of these cultivars regarding their resistance to mites, but there are field observations of damage caused by them (personal information from Juarez Fernandes de Souza). According to Pinto-Zevallos et al. (2013) herbivores are continually adapting to avoid being detected by their predators, thus evading plant defenses.
In the study carried out by Shimoda et al. (2005) it was observed that the predator N. californicus could exploit a variety of volatiles related to T. urticae, as it was responsive to lima bean leaves infested with T. urticae and its visible products (webs, eggs and feces), where they remained three days under the plants until they were contrasted with fresh air. The attractiveness of this predator is related not only to its ability to exploit a variety of volatiles originating from its prey, but also from the prey and host plant complex.
The difference in response in cv. Fepagro26 (Fig. 5) responsive and Garapiá (Fig. 6) where the predator responded similarly, may be due to the low induced response emitted by the plant. Where these volatiles were not intense enough for N. californicus to perceive T. urticae.
However, the different responses must be extrapolated to different plants, as in the work by Rocha et al. (2022) where different contrasts were offered between density of T. urticae (10, 100 or 200 individuals) in corn plants with 24 hours of previous infestation. N. californicus showed no preference to the treatments tested, and the authors relate this fact to the possible inability of the predator to identify the volatile compounds to go in search of this phytophage or the infestation time of 24 hours was insufficient to activate the plant's defenses.
In the present work, N. californicus presented contradictory responses (Figs. 5 and 6) to infested plants, probably this is due to the defense of plants that had their mechanism stopped after herbivory in cv. Fepagro26 or that the predator only responds to kairomones that were emitted from the presence of T.uticae at the time of contrast.
The results of this study indicate that the cultivar factor is of great relevance to the predator's response (Figs. 5 and 6), since the phytophage improves its ability to nullify or delay the plant's defense response (Dicke, 2015; Santamaria et al. 2020), as well as between polyphagous phytophagous species and specialists that trigger activations of different defense responses in plants. This effect was reported by Oliveira et al., 2015 where Tetranychus evansi Baker and Pritchard, 1960 in tomato, which is a key pest in this crop, does not activate plant defenses, whereas for the polyphagous T. urticae it activates plant defenses, making the tomato (Solanum lycospersum) has opposite effects on its defense due to the mite species (Oliveira et al. 2015).
In this work, the exogenous application of phytohormones did not trigger the defense mechanisms of the plants, it was expected that AS and T. urticae would amplify this volatile, thus triggering the attraction by N. californicus, however it was attracted by plants infested only with T. urticae, but not by T. urticae and AS in cv. Fepagro26 (Fig. 7). This fact can be explained by the activation of plant defenses that is triggered by herbivory by T. urticae, which has specific signals that are modulated by jasmonic acid (AJ), salicylic acid (AS) and ethylene (ET), where the difference in proteins in plants such as bean induce different responses (Santamaria et al., 2020).
Results like Shimoda (2010) where N. californicus did not differ between lima bean infested leaves with T. urticae contrasted with methyl salicylate, as well as in the present study where the predator did not differentiate between T. uticae and AS + T. urticae demonstrate that the same AS associated with the prey T. urticae triggers in the predator a contradictory response to that found when only its prey is present, this is due to the change in the chemical profile caused by the addition of AS. In the work by Shimoda, 2010, when testing the contrasts between different mixtures of volatiles, which had already been identified from lima bean plants infested with T. urticae, with common bean infested by this phytophagous, the predator N. californicus responded to the leaves infested, then N. californicus has its response associated with the presence of T. urticae. Silveira (2017) when comparing the response of Phytoseiulus macropilis (Banks, 1904) to the treatments methyl jasmonate (MeJA), methyl salicylate (MeSA) and the combination of the two (MeJA and MeSA) with application of these phytohormones with 48 hours and 60 females of T. urticae on tomato (Solanum lycopersicum), found that the predator P. macropilis was more attracted and recaptured (after seven days under the plant, the predators were counted again), in addition to higher oviposition in plants with MeSA, unlike what was recorded in the current study. study where N. californicus had its response altered due to the addition of AS in common bean. Botti et al. (2019) when verifying the response of N. californicus to maize and Bt maize (genetically modified) plants, realized that this predator was not able to distinguish the chemical differences between maize and Bt maize with the presence of T. urticae, as well as demonstrating that alterations in this prey and plant system with the inclusion of phytohormone as in our study or genetic modifications as in this work cause a change in the behavior of N. californicus.
The present work demonstrates that further research is needed regarding the interactions between T. urticae, N. californicus and AS for use in biological control programs, since phytophagous and predator responded to AS in both cultivars. In relation to T. urticae, it presented a contradictory relationship in the use of AS, since the dose of 0.5 mm (T5) reduces oviposition and even so it was attracted by plants atomized with AS. AS also deserves more attention since the bean plants seem to have released different profiles, as it did not respond to the phytophagous when T. urticae + AS was associated.