Formulation et effet d’un biopesticide à base d’Azadirachta indica, de Sida acuta, de Cymbopogon citratus et d’extrait calcique sur la résistance du cacaoyer (Theobroma cacao L.) contre Phytophthora megakarya, agent responsable de la pourriture brune du cacaoyer

Abstract

The cultivation of cocoa (Theobroma cacao L.) has an economic importance for producing countries. Its culture is confronted with numerous constraints, such as the strong parasite pressure in the production areas. The oomycete Phytophthora megakarya is the most damaging pathogen of cocoa in Central and West Africa, causing losses of up to 80% of the production. The soil being the primary infection site of this oomycete, it inflicts severe losses in nurseries as well as in the field. The use of synthetic chemical pesticides allows to limit the losses in the nursery and in the field, but it has harmful effects on the environment, the quality of cocoa and human health. To reduce the use of these synthetic chemical pesticides, alternatives such as biopesticides are increasingly sought to increase the production of organic cocoa which has a better quality. The aim of this work was to evaluate the effect of Neem-based biopesticide, Sida weed, lemongrass and oyster shells considering their individual properties, on the growth of cocoa plants (SCA12*SNK16) and the resistance of cocoa (SNK10 and SNK413) against Phytophthora megakarya, in hybrid cocoa plants. Thus, the stability of the formulation tests was determined and the most stable and antifungal one was used to evaluate the tolerance of cocoa plants as well as the mode of action of the latter on Phytophthora megakarya. The dilution that did not cause any foliar damage and that kept its fungicidal effect was then used to evaluate the effect on the growth and the stimulation of the defenses of the hybrid SCA12*SNK16. Thus, agro-morphological growth parameters were recorded every 04 weeks for 12 weeks, followed by biochemical analysis of plant growth markers. Then, the defense markers [total phenol content, flavonoids, proteins, and activity of β-1,3-glucanase, peroxidases (POX) and Polyphenoloxidases (PPO) enzymes] were quantified in the plants. In addition, the effect of the formulation on the stimulation of cocoa defense pathways mediated by salicylic acid, jasmonic acid and inhibition of pod cell death in Phytophtora megakarya susceptible (SNK10) and tolerant (SNK413) clone was studied by transcriptomic analyse using RT-qPCR. Moreover, a metabolomic analysis of these pods before infection (SNK10 and SNK413) and after infection (SNK10) was performed. From these activities, a formulation named OBF’ which is an emulsion having 10.8% Neem oil (Nh), 8.1% aqueous extract of Neem cake (Nea), 8, 1% hydroalcoholic extract of Sida grass (Seth), 4.05% calcium extract of oyster shells (HCa), 1.35% calcium oxide (CaO), 21.6% Tween80 (Tw) and 10% essential oil of Citronella (CEO). At room temperature, the formulation is stable and has micelles of diameter ≤ 14.9 nm, pH 5.2 and conductivity 95.25 mV. The OBF’ formulation has a dose-dependent activity on the studied plant pests. Indeed, it totally inhibits the growth of Fusarium oxysporum, Pythium myriotylum, Phytophthora infestans and

Phytophthora megakarya at 1%; reduces their growth from 37.39% to 55.88% when used at 0.1%. This OBF’ formulation acts on Phytophthora megakarya by inhibiting its protein synthesis by 60% and by inhibiting the activity of ATP-H+ pumps. The production of SCA12*SNK16 cocoa seedlings with the 1% (v/v) formulation increases the agronomic parameters. Indeed, it stimulates root development after bean pre-treatment by 62.6% and increases leaf area in seedlings by at least 8.8% in 16 weeks. This increase in morphological parameters is characterize by the increase in protein content and indole acetic acid (IAA) in the roots on day 12 after bean pre-treatment compared to the chemical treatment and the negative control. In addition, it significantly increased protein and total chlorophyll content by 0.9-15% in 16 weeks. Pre treatment of the leaves of SCA12*SNK16 cocoa plants before inoculation with phytophthora megakarya spores reduce the disease severity index by making the plants susceptible to the disease into highly resistant plants with severity indexes of 3.35 and 0.86 respectively. This reduction in disease severity goes with an increase in phenolic compound, flavonoid compounds, and peroxidase, polyphenol-oxidase, and β-1,3-glucanase enzyme activity in the tissue. Furthermore, the pre-treatment of detached cocoa pods susceptible (SNK10) and tolerant (SNK413) to phytophthora megakarya makes them resistant and highly resistant respectively with severity indexes of 1 and 0.5 for SNK10 and SNK413 clones. This reduction in disease severity is accompanied by a significant increase in the expression of PR protein genes in SNK413 treated with the OBF’ formulation. But this increase is not significant in SNK10. In addition, there is a significant increase in the expression of the BI1 defense genes, and the WRKY transcription factor. However, the AOS gene expression is significantly increase only in the SNK10 variety after application of the formulation. In addition, LC-MS/MS metabolomic analysis of SNK10 and SNK413 reveal the presence of 277 compounds, including zeta-carotene, which is an precursor of the reddish pigmentation of the SNK10 clone; ifosfamide, which could be a marker of healthy pods; pterosin A which could be a marker of SNK10 clone; Hexsose + Hexsose-deoxyHexose + C20H32 which could be a marker of SNK413 clone; and Lyalosidic acid, Ormosanin and Ginsenoside Rh3 which could be markers of tolerance of SNK10 and SNK413 pods towards phytophthora megakarya. Thus, the application of the OBF’ formulation stimulates the defense mechanisms in cocoa trees. These results show that the OBF’ formulation is a good candidate in the production of cocoa based on biological substances and allow to pre immunize this plant against possible attacks in the field and to improve the quality of the seeds.