Induction of systemic resistance in Theobroma cacao L. for the management of “witches’ broom” caused by Moniliophthora perniciosa

The pathogen Moliniophthora perniciosa can cause losses in the cocoa crop of more than 90% when integrated disease management is not carried out in the crop. In the present study, the effect of sucrose as a resistance inducer in cocoa trees (Theobroma cacao L.), was evaluated for the management of the “witches’ broom” disease caused by Moniliophthora perniciosa. The experiment was developed in the municipality of Chaparral, Tolima, Colombia (854 m.a.s.l.). The treatments corresponded to 0.9 M sucrose: injected (T1), foliar spray (T2) and without sucrose (T3). A completely randomized block design with three treatments was established. The results showed that the incidence and severity of the disease in the trees treated with sucrose were lower. The polyphenol oxidase enzyme presented its highest activity in T3, while peroxidase and superoxide dismutase in T2. It was observed that sucrose as an inducer, can alter metabolic pathways involved in the defense mechanisms of cocoa trees, reducing the incidence and severity, thus establishing an alternative for the management of “witches' broom” disease in producing countries.


Introduction
The cocoa (Theobroma cacao L.) from Colombia was declared by the International Cocoa Organization (ICCO) as high quality. The organoleptic and physicochemical characteristics of cocoa determine that the country has great potential to compete in foreign markets. In this way, the areas to be investigated in order to improve the growth of the crop in the country and improve its productivity must be identified within the production process (Montoya et al. 2015;Cardona et al. 2016). In producing countries, aspects have been identified that determine the causes of the low yields of cocoa cultivation: the advanced age of the planted plantations, the type of propagation material, the low density of trees and the difficult access to technical and scientific advances such as varieties, irrigation systems and others; These factors influence the resistance of cocoa to pests and diseases such as the "witch's broom" caused by Moliniophthora perniciosa, reporting losses when the integrated disease management plan is unknown of up to 90% in production (Hernández 2016;De La Cruz et al. 2015).
In Colombia, the management of Cacao diseases has been mainly with the use of chemical synthesis inputs and to a lesser extent the use or application of biological inputs and resistance-inducing agents has been implemented (Tirado et al. 2016). In cocoa, different investigations have been carried out to determine the effect of resistance inducers for the management of diseases caused by pathogens such as Moliniophthora perniciosa and Ceratocystis cacaofunesta. Works carried out by Antonio et al. (2018) and Vieira and Valle (2006) demonstrated that the use of inducers such as glucose and sucrose can induce systemic resistance for disease management. Furthermore, they showed that plants subjected to the inducer use their enzymes linked to sugar breakdown to signal stress and defense responses.

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Thus, it is necessary to continue with the management of scientific trials to support the use of biostimulants and defense inducers. For this reason, this research aimed to evaluate the use of sucrose as an inducer of systemic resistance in cocoa plants in the presence of the M. perniciosa fungus, the causal agent of the "witch's broom" disease. For which the effect of the inducing agent on the epidemiological parameters of the disease was determined and by biochemical analysis determining the enzymatic activity of the enzyme polyphenol oxidase, peroxidase and superoxide dismutase.

Materials and methods
Geographic location Finca El Carmen, Tapias village, Chaparral, Tolima-Colombia, located in the Southwest of the department (3° 43′25 ″ N 75° 29′05 ″ W) at 854 m.a.s.l. during 2019. Average relative humidity during the experiment of 58.6% and in the sampling time for the biochemical analysis of 50.5%; average temperature of 27.9 °C and 27.8 °C at sampling time.
General characteristics of Cacao trees Technical professionals in the cultivation of Cacao, determined that the Cacao variety was "Creole". According to Cheesman (1944) the morphogeographic group "Criollo" has the greatest presence in Central America, from Venezuela to Colombia. The trees under study according to information collected with local people and after morphological analysis such as vigor, stem thickness, tree height and other characteristics, an approximate age of 30 years was determined. These trees were from seed and the crop was already established. The plantation was in a shading of approximately 30% due to poorly directed growth of tree branches due to lack of pruning. The nutritional conditions were adequate, that is, nutritional deficiencies were not reflected at the beginning of the experiment. On the other hand, there was a planting distance of 6 m between plants and 5 m between rows in the plantation.

Preliminary evaluation and selection of trees for study
In the plantation the incidence and severity of the "witch's broom" disease was evaluated to determine an initial level of damage. The severity was evaluated according to the scale developed by Loor et al. (2016). In addition to the evaluations to determine the level of affectation of "witch's broom", the presence of other pests and diseases was evaluated to determine the overall health of the crop.
Treatments Cacao trees were used in the productive stage with symptoms of the "witch's broom" disease caused by M. perniciosa. In no case was inoculation with the pathogen performed. Two treatments were applied: injected sucrose (T1), where 20 cm 3 of sucrose solution were taken with the use of high precision syringes and it was applied directly to the vascular system of the trees, 0,5 m from the ground; foliar spraying of sucrose (T2) using atomizers with a volume of 100 cm 3 of solution. For spraying, the new and old leaves of the middle and lower third of the tree were sprayed, with an average size of 75cm 2 (Vieira and Valle 2006). As T3, trees that were not applied with sucrose were considered. A 0.9 M sucrose solution was used, according to different suggestions and analyzes of physiologists of the research group and based on the studies carried out by Antonio et al. (2018) and Vieira and Valle (2006). Commercial reagent grade sucrose crystal was provided by J.T. Baker.

Disease assessment
The incidence was evaluated before the application of sucrose; it was called "zero incidence" (I0). The severity was evaluated before the application of sucrose; it was called "zero severity" (S0). After the application of sucrose, weekly evaluations of the incidence and severity were carried out. The first was determined according to the following formula: where AE: trees with the disease, AEv: Total trees evaluated by treatment.
The severity of the disease was evaluated according to the schematic scale described by Loor et al. (2016). This scale classifies the evolutionary state (vigor) of vegetative brooms into three ranges. Range 01 is represented as "mild infection" and its characteristic is an abnormal initial thickening of the apical bud. Rank 02 corresponds to a "moderate infection" and is characterized by abnormal thickening of the apical bud and deformation of the leaves in the apical bud. Finally, rank 03 is an "elevated infection" and corresponds to a fully formed broom in an apical bud.
Enzymatic response The response of the polyphenol oxidase enzymes (PPO) and the antioxidant enzymes peroxidase (POD) and superoxide dismutase (SOD) was measured. Three samples were taken randomly per experimental unit, for a total of 9 samples per treatment. Mechanical grinding of the tissue was performed with liquid nitrogen. The samples were kept at -80° C until the development of the analysis.
Buffer (potassium phosphate, pH 7, 100 mM) was added in a 20:1 ratio to the plant material, and it was homogenized. Subsequently, it was centrifuged at 10,000 rpm at 4° C for 30 min; the supernatant (enzyme extract) was taken. The protein content of the extracts was calculated using the method of Bradford (1976) with some modifications (Pande and Murthy 1994).
The quantification of enzymatic activity of PPO followed the guidelines of Jiang et al. (2015); For each 100 µL of enzyme extract, 3 mL of buffer (potassium phosphate, 0.1 M, pH 7.4, 0.1 M catechol) were added; The catechol oxidation rate was monitored at 410 nm, at 25° C for 1 min; the enzymatic activity is expressed as ΔAbs410 min −1 mg of protein −1 . For the POD activity, the method reported by Sellamuthu et al. (2013); To 36 µL of enzyme extract, 144 µL of buffer (potassium phosphate, 100 mM, pH 7, 20 mM guaiacol) were added and the solution was incubated for 5 min at 30° C; subsequently, 72 µL of hydrogen peroxide (100 mM) were added and the absorbance of the mixture 460 nm was measured every 10 s, 2 min; the enzymatic activity was expressed as ΔAbs460 min −1 mg of protein −1 . The SOD activity was developed under the report by Thangaraj (2016); 200 µL of reaction buffer (0.05 M potassium phosphate, pH 7.8, 75 µM NBT, 20 mM L-methionine, 2 mM EDTA, 50 mM Na 2 CO 3 , 2 µM riboflavin) were added to 20 µL of enzyme extract; After homogenization, the mixture was exposed to 300 µmol m-2 s-1 of irradiance (generated by a 39-W fluorescent lamp at a distance of 5 cm) at room temperature; a control without enzyme was included where maximum color develops due to the reduction of NBT, in addition to a non-irradiated control that should not develop color; activity was measured as the photochemical reduction of NBT at 560 nm and each SOD unit was defined as the amount of enzyme that inhibits 50% photoreduction of NBT.
Experimental design and data analysis A completely randomized block design was established with three treatments and three repetitions: T1: sucrose injected into the vascular system at the dose recommended by the manufacturer, T2: sucrose sprayed on the foliage at the dose recommended by the manufacturer, and T3: control without application of sucrose. Each experimental unit corresponded to a group of 10 trees for a total of 90 trees in the experiment. The applications of treatments T1 and T2 were repeated 20 days later in the 90 trees of the experiment. The samples for the enzymatic analysis were taken 45 days after the last application of Treatments T1 (inject) and Treatment T2 (spray). The results of the number of affected structures were expressed as the mean of two values ± standard deviation. All the variables were subjected to analysis of variance (ANOVA) and the means of the treatments were compared with the Tukey test (p ≤ 0.05) using the statistical software InfoStat.

Effect of sucrose on epidemiological parameters
The severity of the "witches´ broom" disease in T1 (inject) and T2 (Spray) was significantly lower then what T3 (No applic) (p ≤ 0.05) (Fig. 1). The same was with the incidence of the disease (Table 1). All the fruits present in the tree were evaluated, they presented different sizes and weights, from 15 to 25 cm in length and 12 cm to 17 cm in diameter. The flower corresponds to the unit within a flower cushion, considering that the diseased floral structure can infect the others. The green structures correspond to fully developed structures, that is, according to the scale developed by Loor et al. (2016) corresponds to range 02 corresponds to a "moderate infection" and is characterized by abnormal thickening of the apical bud and deformation of the leaves in the apical bud. Enzyme activity T1 (Inject) and T2 (Spray) treatments affect the enzymatic activity in cocoa trees. Figure 2 shows a significant difference in the PPO enzyme between the sucrose treatments T1 (Inject) and T2 (Spray) and the control T3 (No applic). The mode of application of sucrose in trees does not affect the expression of enzymatic activity. On the other hand, the POD enzyme seems to be influenced not only by the application of the carbohydrate, but also by the way it is carried out, since it is the foliar spraying that seems to have a greater effect on the activity of the enzyme. The SOD enzyme also responds to the application of sucrose, mainly by the mode of injection in the stem; however, the foliar application of the inducer also seems to affect SOD activity.

Discussion
These findings presented previously in Table 1 and Figs. 1 and 2 are possibly associated with biochemical alterations that sucrose induces in trees when affected by M. perniciosa. It was found that the control treatment had a statistically significant difference for the variables incidence and severity of the disease. Under the conditions evaluated, the form of application of sucrose in cocoa plants for the management of the "witch's broom" does not affect the management of the disease, that is, in this experiment the treatment T1 (inject) and T2 (spray) do not differ in effectiveness. Although there is no statistical difference between treatments T1 (inject) and T2 (spray) in relation to the decrease in the severity of the disease, as shown in Fig. 1C, the response of the antioxidant enzyme POD is greater through a foliar application (Thakur et al. 2016). This enzyme is responsible for the regulation of H 2 O 2 levels, so that, in the trees in which the foliar application of sucrose was carried out, there was possibly a greater regulation of this reactive oxygen species (Almagro et al. 2009;Wu et al. 2014). However, as there is no statistical difference between T1 and T2 in incidence, it can be inferred that the regulation of oxidative stress generated by H 2 O 2 is not essential in the response of cocoa trees to the pathogen M. perniciosa, decreasing the impact of the role of POD during the management of the disease. Sucrose as an inducer can alter metabolic pathways involved in the defense mechanisms of plants against the attack of pathogens and even abiotic stress, so the reduction of incidence and severity in sucrose-treated plants may be related to such biochemical events where plants are metabolically and/or structurally strengthened (Antonio et al. 2018). The decrease in the activity of the PPO enzyme implies a higher prevalence of phenolic compounds in the treatments subjected to the inducer (Jukanti 2017). These compounds can participate in processes of inhibition of the growth of the pathogen (antimicrobial compounds) and even in the generation of structures that could strengthen the cell (Rodríguez et al. 2015). The results suggest that the presence of the pathogen can elicit  the activity of the PPO enzyme, so that consequently there would be greater oxidation of phenolic compounds, being that, if these are related to the defense response of the plant, it is consistent that in T3 there is greater incidence and severity. A possible explanation for this phenomenon lies in the fact that it has been reported that the pathogen M. perniciosa can induce the accumulation of salicylic acid in the diseased tissue of the cocoa plant during the necrotrophic stage of infestation, leading to cell death and necrotization of brooms, followed by the formation of basidiocarps in the dead vegetative tissue (Chaves and Gianfagna 2006); added to this accumulation of salicylic acid, are the multiple reports of this compound as an inducer of the activity of the PPO enzyme (Gholamnezhad et al. 2016;Indunil and Vengadaramana 2017;War et al. 2011), explaining the higher levels of this enzyme in T3. The SOD enzyme, an important first-line regulator of reactive oxygen species (Alscher et al. 2002;Ighodaro and Akinloye 2018), whose activity was higher in the presence of the inducer, would mitigate the accumulation of compounds that generate oxidative stress, that leads to cell death to mitigate disease development. If the process generated to reduce the action of the pathogen is cell death, this would not depend mainly on the accumulation of ROS; this does not imply that the signaling function of these compounds is less important during the defense response of cocoa (Sánchez et al. 2012;Das and Roychoudhury 2014;Mittler 2017).
The treatments evaluated suggest that sucrose injected or sprinkled on cocoa leaves influences the reduction of the incidence and severity of the "witch's broom" disease caused by the pathogen. However, research work should be expanded and trials proposed to identify the role of sucrose as an inducer of resistance in younger plantations and in seedlings. On the other hand, the activity of the enzymes POD, SOD and PPO have been affected by the application of the carbohydrate and consequently a resistance to the disease is generated. It is suggested to modify tests to evaluate and contrast the effects found in this study in nursery stages and in different altitude zones.