Elicitor-Induced Defense Response in Soybean Plants Challenged by Bemisia tabaci

The whitefly, Bemisia tabaci Biotype B (Hemiptera: Aleyrodidae), is considered one of the world’s major agricultural pest groups, attacking a wide range of crop hosts and causing considerable crop loss. Understanding the interactions between whiteflies and host plants promotes the development of novel strategies for controlling whiteflies. This study aimed to evaluate the biochemical alterations caused by induced resistance in soybean plants, challenged by B. tabaci. The experiment was performed at the Federal University of Technology, Parana. Soybean seeds (cv. BRS 284) were sown in polyethylene pots in the greenhouse. The elicitors sprayed on the soybean leaves were: acibenzolar-S-Methyl (ASM-0.005%); Salicylic Acid (SA-2Mm); foliar phosphite (ULTRA K-0.004%); Chitosan (CH-1%); and silicon (SI-0.25%); whilst the control group was sprayed with distilled water. Plants were infested with 50 adult whiteflies in the cages that were released after applying the treatments. At 0, 24, 48 and 96 hours after the beginning of the experiment, biochemical analyses of total proteins and the activity of the phenylalanine ammonia-lyase (PAL), peroxidases (PO), phenolic compounds, chitinase, and β-1,3-glucanase were made. The results showed that the application of elicitors increased the route of the phenylpropanoids with the activation of PAL and formation of phenolic compounds. It was also verified the activation of pathogenicity-related enzymes such as peroxidases and chitinase.


Introduction
Brazil is one of the greatest producers of soybean (Glycine max (L.) Merrill) in the world.In 2017/2018, Brazil produced 119.28 million metric tons (CONAB, 2018).Nevertheless, insect pests represent a huge challange to soybean productivity and sustainability due to losses and problems related to the abuse of chemical insecticides.
The whitefly Bemisia tabaci (Genn.)Biotype B (Hemiptera: Aleyrodidae) is a polyfagous pest and it is responsible for great losses in production in soybean crops by causing direct and indirect damage (Oliveira et al., 2014;Wang et al., 2015).
Although the whitefly causes little mechanical damage to plants, along with its saliva the insect injects toxins into the phloem; besides, indirect damages are caused by excretion of honeydew, which favours the development of pathogenic fungi, reducing crop productivity (Bôas & Branco, 2009).This hemipteran is traditionally controlled through insecticides application.There are some limitations though, such as high cost, low efficiency, ecological imbalance and insect resistance (Moraes, Ferreira, & Costa, 2009).Plant defense induction is a method that can be used alongside other pest control methods in IPM.Its objective is to increase the plant ability to defend itself against biotic stresses such as pathogen and insect attack.These defenses can be activated by treatment with biotic or abiotic agents.These molecules, capable of activating plant defense responses, are called elicitors (Sticher et al., 1997).Among the most studied elicitors, there are acibenzolar-S-methyl (ASM), salicylic acid (SA), phosphites, chitosan and silicon (Cruz et al., 2011;Terra, 2010).The elicitors, when applied to plants, are identified by receptors and then trigger the processes of plant defense (Trouvelot, Héloir, & Poinssot, 2014).
The induction of resistance against herbivorous can be expressed in two different ways: direct and indirect.Direct induced resistance is mediated by the accumulation of secondary metabolites and defense-related proteins (Mithöfer & Boland, 2012).Indirect induced resistance results in the release of a mixture of volatile organic compounds (VOCs) that specifically attract natural enemies, and are also involved in the mediation of various ecological interactions.Exogenous defense elicitors are biodegradable, used in low quantities and does not lead to pest resistance.Thus, they show potential for the development of new pest control strategies (Mithöfer & Boland 2012;Pinto et al., 2013).
Research regarding resistance induction of soybean plants against pests are still scarce considering induction potential; thus, this study aimed to evaluate the biochemical changes caused by abiotic resistance elicitors in soybean plants challenged with B. tabaci.

Material and Methods
The experiment was conducted at the Federal University of Technology Parana.Soybean seeds from cultivar BRS 284 were sown in polyethylene pots (10 L) containing moist soil obtained from the topsoil (ca.0-20 cm in depth) of a soybean crop located in Dois Vizinhos, Paraná, Brasil.Each pot contained five soybean plants,.and they were kept in a greenhouse until the beginning of the experiment (25±2 ºC).The experiment was performed when plants reached phenological phase V6.The treatments used were: T1: acibenzolar-S-Methyl (ASM-0.005%);T2: salicylic acid (AS-2 Mm); T3: phosphite Potassium (ULTRA K®-0.004%);T4: chitosan (CH-1%); T5: silicon (SI-0.25%);T6: control group (distilled water)-challenged and not challenged by B. tabaci.The treatments were sprayed over the above ground part of the plant.The vases were kept individually in cages with anti-aphid screens.For treatments that plants were challenged by B. tabaci, 50 adult flies were added per cage, collected from the field.The experimental design was completely randomized in a two-by-two factorial design, the factors being the treatments and the presence or absence of B. tabaci with five replicates per treatments.
For biochemical analysis (total proteins, total and reduced sugars, phenolic compounds and the activity of enzymes related to plant defense-peroxidase (PO), phenylalanine ammonia-lyase (PAL), chitinase and β-1,3-glucanase), plant tissue (0.5 g), were collected at intervals of 0, 24, 48, 96 and 168 hours after application of the elicitors.Samples were frozen and stored in liquid nitrogen.All biochemical analyses were performed according to classical methodology: Protein content (Bradford, 1976); PAL (Kuhn, 2007).PO and phenolic compounds (Matsuno & Uritani, 1972).Otal soluble sugars (Dubois et al., 1956); Reducing sugars (Miller, 1959).The concentration of reducing sugars was calculated as a function of the standard glucose curve.
For quantification of chitinase and β-1,3-glucanase activities, the samples were macerated in 4.0 mL of 100 mM acetate buffer (pH 5.0), with subsequent centrifugation (20,000 g for 25 min at -4 °C ).The supernatant was collected and used for the evaluation of enzyme activity.The enzymatic activity of chitinase was assessed by the release of soluble "CM-chitin-RBV" fragments from carboxymethylated chitin labeled with RBB-Remazol Brilant Blue.The readings were performed with a spectrophotometer (595 nm).For quantification of β-1,3-glucanase (600 nm), bright blue carboxymethylcurdlan-remazol solution (CM-Curdlan-RBB 4 mg/ml, Loewe Biochemica GmbH ) was used as substrate, according to a methodology developed by Wirth and Wolf (1992) and with the procedure described by Guzzo and Martins (1996).
The collected data were submitted to ANOVA (p ≤ 0.05) and presented as descriptive statistics (mean±standard deviation) and regression analysis (p ≤ 0.05) by the Assistat ® program (Silva & Azevedo, 2009).

Proteins Activity
There was a significant interaction between the factors evaluated in the experiment.The activity of proteins was affected both by the treatments and by time.As observed in Figure 1, there were alterations in protein synthesis, since the measured values presented variation in relation to the application of the inductors and the attack of the whiteflies.In general, the highest activation occurred at 96 hours, both with the presence and absence of insects.
Proteins are polymers formed by amino acids and are the most abundant macromolecules in plant tissue.Proteins present structural and dynamic functions; some proteins are enzymatic catalysts, participating in several biochemical reactions; and some, are precursors for many secondary metabolites involved in local and systemic responses (Zhou et al., 2015).
At 168 hours, an increase in protein levels was observed, including in the control.This reason can be explained by the plant's ability to release volatile organic compounds (VOCs), a way of signaling non-elicited plants.In addition to direct action on induction of resistance, they may act indirectly attracting predators of herbivores (Heil, Bueno, 2007).treatments were tried: foliar fertilization with silicon acid at 1%; soil fertilization with 250 ml silicic acid solution at 1%; foliar fertilization with silicon acid at 1% + soil fertilization with 250 ml silicic acid solution at 1%; and a control.The results show the lignin percentage increased in the leaves of plants fertilized with silicon via soil and/or foliar and the percentage of tannins increased only in the leaves fertilized via soil plus foliar.The silicon acted as a resistance inducer to M. persicae in potato.
Peroxidases are the only enzymes that polymerize the alcohols in lignin, being present in lignified plant cells.They also participate in the oxidation of indole acetic acid (AIA) and phenolic compounds (3).Lignin, in addition to cellulose and other polysaccharides that occur in the cell wall of the upper plants, works as a physical barrier and acts to heal wounds caused by fungi and insects (Gaspar et al., 1982;Kao, 2003).
Both stressors (phytopathogen and insect) when they attack the plants make them respond quickly with an "oxidative explosion", which constitute the production of reactive oxygen species (ROS), mainly superoxide anion (O 2 •-) and peroxide of hydrogen (H 2 O 2 ) (Hu et al., 2009).The accumulation of these substances in plant cells can be toxic to both (plant and stressors).The H 2 O 2 is the main reactive oxygen species that activates molecules for the induction of defense genes and the polymerization of proteins that make up the cell wall (Łukasik et al., 2012).
The herbivory has been linked to changes in intracellular EROs and in peroxidase activity in plants and the largest group of insects studied corresponds to hemiptera (Torres, 2010;War et al., 2012).War et al. (2011) analyzed the peroxidase activity of three peanut genotypes (Arachis hypogaea L.) in response to feeding of Spodoptera litura (Fab.)(Lepidoptera: Noctuidae) and observed that there was an increase in the activity of this enzyme at 96 hours after infestation.
It is evident that the peroxidases play an important and dinamino role in defense against the attack of insect pests.Peroxidases have the potential to be used as markers for selecting insect resistant turfgrasses, and may help explain how plants defend themselves against biotic stresses, such a tolerant plant's defense response to insect feeding, including the signaling of plant defense reactions to injury, efficient removal of reactive oxygen species, or both.

Chitinase Activity
The chitinase levels had interference when the elicitors were applied, and with absence of the insects the activation was pronounced with 168 hours.When the plants were challenged with the inductors and the presence of the insects, there was activation from 48 hours for all the inductors except for the phosphite with 168 hours.
These results demonstrate that when the inductors are applied with the presence of the insect, the preferential defense mechanism activated is the chitinases, a fact that can be explained by the presence of chitin in the exoskeleton of the insects, acting in the process of signaling and expression of this hydrolytic enzyme in plant defense.Factor that also indicates the non-expression of glucanases.
Activation in the presence of the insect may also be linked to another form of plant recognition, as they may recognize specific components in insect saliva.Most plant responses to insects involve the response to both injury and the recognition of certain compounds abundant in insect saliva or regurgitation.These compounds belong to the group of molecules called elicitors.For insect-derived elicitors, the molecular patterns associated with herbivores are HAMPs (herbivore associated molecular patterns) and effectors, which directly and indirectly influence plant defense (Taiz & Zeiger, 2017). jas.ccsenet.

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Figure 1.P resistance Figure 5. resistance