Comparative efficacy of essential oils of three aromatic plants as alternatives in the control of Phytophthora spp., agent of cocoa tree (Theobroma cacao L.) black pod disease in Côte d'Ivoire

This study aimed at comparing the efficacy of essential oils from three aromatic plants species (Cymbopogon citratus, Eucalyptus citriodora and Ocimum gratissimum) in vitro and in vivo against two Phytophthora strains (Phytophthora palmivora and P. megakarya) in a view to developing new biofongicides for the control of cocoa tree black pod disease. Essential oils were applied at four different concentrations (0.5; 1; 3 and 5 mL/L) in vitro and in vivo on leaf discs of three cocoa clones (NA32, PA150 and SCA6) whichsusceptibility is known. As results, all three essential oils significantly (p˂ 0.05) reduced in vitro mycelial growth of both Phytophthora strains and also reduced the leaf susceptibility to these strains. Inhibition rates in vitro of Phytophthora strains mycelial growth ranged from 83.80 to 100% for the essential oils of Cymbopogon citratus and Ocimum gratissimum species and from 17.7 to 100% for which of Eucalyptus citriodora species. In the biotests, the leaf susceptibility index of clone NA32 (susceptible) was reduced from 3.14 to 0.40. This correspond to reduction rates ranged from 64.87 to 86.70% of leaf susceptibility to both Phytophthora strains. Essential oils (HS and HO) from Cymbopogon citratus and Ocimum gratissimum species were most effective than which from Eucalyptus citriodora species. However, all three essential oils revealed antifungal properties. Therefore, they could be recommended as biofungicides for a sustainable control of cocoa tree black pod disease in Côte d'Ivoire.

​Control Activity and Antibiotic Gene Detection of Endophytic Bacteria in Suppressing Cocoa Black Pod Disease (Phytophthora palmivora Butl.)

Background: Black pod rot disease of cocoa caused by (Phytophthora palmivora Butl.) is one of the major diseases on cocoa plantations worldwide. Many attempts have been made to prevent or reduce the infection of pathogens, but they have not provided optimum results. This study aims to detect antibiotic genes in endophytic bacteria that can suppress cocoa black pod disease. Methods: Eight endophytic bacteria were isolated from healthy cocoa pods and twigs that showed potentials in suppressing P. palmivora growth in vitro were used in suppressing of black pod rot disease in vivo tests. Antibiotic biosynthesis-related genes from eight endophytic bacterial isolates were confirmed by using PCR method, which includes phenazine-1-carboxylic (PCA), pyrrolnitrin (PRN), phenazine-1-carboxamide acid (PCN), pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DPAG/Phl). Result: The endophytic bacteria, 4RSI, 5BR B3 and 2RW B2 isolates showed the highest disease suppressing index to black pod rot disease in vivo, i.e., 70.27%, 70.08% and 56.64%. The isolates 5BR B3 and 2RW B2 DNA yielded PCR product by using PCA primers (1400 bp), PRN primers (700 bp) and DAPG primers (1600 bp), while the 5RSI isolate yield PCR product using PRN primers only. Endophytic bacterial isolates 5BRB3 and 2RW B2 provided partial disease suppression to pod rot disease by inhibit pathogen growth and antibiotic compounds production.

BREEDING OF COCOA TREES (THEOBROMA CACAO L.) RESISTANTTO PHYTOPHTHORA MEGAKARYA, AGENT OF BLACK POD DISEASE IN COTE DIVOIRE

Backgrownd : Black pod disease is the cause of significant production losses of cocoa trees. This work aims to select tolerant and resistant genotypes to Phytoththoramegakarya within the main collection of cocoa trees of the National Center for Agronomic Research. Methods : The artificial inoculation test on leaf discs, was used in this study to assess the susceptibility of 52 clones of high-producing cocoa trees resistant in the field to black podcausing by P. megakarya. Results : Three groups of susceptibility to P. megakarya were demonstrated according to the reference controls. The first group is composed of two clones (IFC 1035 and CC 39) qualified as susceptible to black pod. These genotypes have respective sensitivity scores (NS) of 3 and 3.06 which are lower than those of the sensitive control NA32 (NS = 3.31). The second group is composed of 43 clones qualified as moderately resistant with sensitivity scores higher than 2.59 (PA150, moderately resistant control) and lower than 3.31 (NA32). The third group is composed of four clones qualified on the one hand as resistant (IFC 1041 and IFC 1027) with sensitivity scores higher than 1.73 (SCA6) and lower than 2.59 (PA 150) and on the other of very resistant to P. megakarya (NS> 1.73), with sensitivity scores greater than 1.73 Conclusion : These genotypes resistant to P. megakarya thus selected could constitute parents to be included in a variety improvement program with a view to the selection of plant material resistant to black pod disease.

Control of cacao (Theobroma cacao) diseases in Santo Domingo de los Tsachilas, Ecuador

Introduction. Ecuador occupies the fourth place in cocoa exports with 293,487 tons per year, produced on 559,617 hectares. However, its yield per hectare is low mainly due to the presence of pathogens that affect the pods. Objective. To evaluates different methods of control of pathogens of the Theobroma cacao cv ‘CCN-51’ pod. Materials and methods. The work was carried out in Luz de America, Santo Domingo de los Tsachilas, Ecuador, between 2016 and 2017. The treatments were the result of the combination of cultural practices + two fungicides (Chlorothalonil and Pyraclostrobin) + one biofungicide Serenade® (Bacillus subtilis QST713), with and without fertilizers. In total, 16 treatments were evaluated with 3 replicates or blocks, installed in a commercial cocoa plantation cv ‘CCN-51’ and under a Randomized Complete Block Design (RCBD). Results. The use of fungicides (chemical and biological), decreased the incidence of moniliasis (Moniliophthora roreri), black pod rot (Phytophthora spp.), and cherelle wilt; but the application of fertilizers did not increase the effectiveness of these products. Treatment with only cultural practices did not decrease the final incidence of moniliasis, nor of brown rot, in contrast, the final incidence of cherelle wilt increased. The number of pods and the yield were not directly related and the highest yield of fermented and dry cocoa, corresponded to T9 [Cultural labors + Serenade® (0.2 kg ha-1) (every 15 days) + Fertilizer (0.3 kg ha-1) + Fertilizer (1 kg plant-1)], a treatment that also had the highest net income per hectare. Conclusion. Chemical and biological control can manage cocoa pods pathogens. The integration of both control methods allowed the cocoa producer to obtain greater income.

Structural and Functional Genomics of the Resistance of Cacao to Phytophthora palmivora

Black pod disease, caused by Phytophthora spp., is one of the main diseases that attack cocoa plantations. This study validated, by association mapping, 29 SSR molecular markers flanking to QTL (Quantitative Trait Loci) associated with Phytophthora palmivora Butler (Butler) (PP) resistance, in three local ancient varieties of the Bahia (Comum, Pará, and Maranhão), varieties that have a high potential in the production of gourmet chocolate. Four SSR loci associated with resistance to PP were detected, two on chromosome 8, explaining 7.43% and 3.72% of the Phenotypic Variation (%PV), one on chromosome 2 explaining 2.71%PV and one on chromosome 3 explaining 1.93%PV. A functional domains-based annotation was carried out, in two Theobroma cacao (CRIOLLO and MATINA) reference genomes, of 20 QTL regions associated with cocoa resistance to the pathogen. It was identified 164 (genome CRIOLLO) and 160 (genome MATINA) candidate genes, hypothetically involved in the recognition and activation of responses in the interaction with the pathogen. Genomic regions rich in genes with Coiled-coils (CC), nucleotide binding sites (NBS) and Leucine-rich repeat (LRR) domains were identified on chromosomes 1, 3, 6, 8, and 10, likewise, regions rich in Receptor-like Kinase domain (RLK) and Ginkbilobin2 (GNK2) domains were identified in chromosomes 4 and 6.

Phylogenetic analysis and genetic diversity of Phytophthora palmivora causing black pod disease of cocoa in Malaysia.

Black pod, caused by Phytophthora spp., occurs worldwide and is a major problem to cocoa farmers in Malaysia. Limited studies addressed causal agents of black pod disease of cocoa in Malaysia as well as their genetic diversity. Therefore, this study was initiated to isolate and identify Phytophthora from the main cocoa plantations infected by black pod in Malaysia using sequence analyses of the ITS rDNA, EF-1α, and COX I gene regions. A total of 36 Phytophthora isolates were obtained from infected cocoa plantations from five states of Malaysia in 2016 and 14 isolates in 2013. Six Phytophthora isolates obtained from durian crop in 2013 were also used in this study. Results of phylogenetic analyses of combined dataset of the ITS rDNA, COX I and EF-1α confirmed that all Phytophthora isolates belonged to P. palmivora. P. palmivora isolates obtained from cocoa and durian clustered into different subclades based on the three regions examined. The study also examined the genetic diversity within a population of 56 P. palmivora isolates using random amplified polymorphic DNA (RAPD) and Inter-simple sequence repeat (ISSR) markers. The results of both markers indicated relatively high diversity among P. palmivora isolates. The complete separation was based on host and year of isolation. The study suggests that one species of Phytophthora viz. P. palmivora, is responsible for black pod of cocoa in Malaysia. However, the relatively high genetic diversity and separation of isolates into different clades may suggest that P. palmivora has been introduced into Malaysia via different sources.

A simple method for the extension of shelf life of cultures of Phytophthora species causing black pod disease of cacao (Theobroma cacao L.)

Black pod disease of cacao caused by Phytophthora palmivora and Phytophthora megakarya in Ghana take heavy toll of cacao production in the field. Intensive research has been carried out worldwide on these pathogens. However, viability of the cultures during prolonged storage has remained a major challenge in the research. This paper reports findings of assessment of six storage media viz sterilised distilled water (SDW), sterilised and unsterilized soil suspension (SSS and USS), vegetable 8 juice broth (V8JB), Oat Meal Agar slant under mineral oil (at 4°C) and empty tube. Viability of the cultures was assessed on V8JA and in tetrazolium chloride test. Ability of zoospores of the cultures to infect cacao leaf discs was used to assess growth vigour and pathogenicity. Phytophthora cultures stored in SDW (26 ± 2°C; alternating day light and night) were preserved for 60 days (5 years). Both P. palmivora and P. megakarya performed better on SDW and SSS than on USS due to removal of staling substances in the soil medium by the sterilisation. Vigour of growth and pathogenicity of the stored cultures required re-inoculation of host tissue (cacao pod) in order to maintain potency to continually infect host.

Observations on the Potential of an Endophytic Fungus Associated with Cacao Leaves against Phytophthora palmivora

A study was conducted to test the pathogenicity of an endophytic fungus associated with asymptomatic cacao leaves, to determine its identity through cultural, morphological and molecular characterization, and to evaluate itsantagonistic ability vs. Phytophthora palmivora causing cacao black pod rot disease. Experiments were carried out under laboratory and shade house conditions. Homogeneity of variances and normal data distribution were determined using Bartlett’s and Shapiro–Wilk tests, respectively. Inoculation of the endophyte in healthy cacao seedlings and pods at 5 × 105 to 1 × 106 conidia per mL by spraying resulted in asymptomatic infections. The endophyte was recovered from artificially inoculated tissues 14 and 26 days after inoculation (DAI) (UF18 seedlings), and at 10 (K9 seedlings) and 14 DAI from cacao pods. The endophyte was identified as Colletotrichum siamense based on its cultural, morphological and molecular characteristics. In vitroanti-pathogen assays showed that C. siamense had the potential to limit pathogen growth by antibiosis. At 3, 5 and 7 days after incubation period (DAIP), growth of the pathogen in co-cultivation with the endophyte measured 60.0, 70.0 and 71.0 mm, respectively, which wasconsiderably lower than the growth of the pathogen alone.