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.

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.

Antifungal effect of the cumulative application of biostimulant and fertilizers on young cocoa fruits rot at Tafissou site, Centre-East of Côte d'Ivoire

The use of biostimulants as an alternative way to chemical, often toxic, remains one of the best approaches to control cocoa black pod disease caused by Phytophthora spp. This study has been carried out to evaluate the effect of biostimulant’s applications number and the cumulative action in case of applying previously fertilizers. The experimental design consisted of a Fischer block with six treatments (T01, T02, T1, T2, T3 and T4) and repeated three times. This design was replicated on two sites, one with previous fertilizer (DAE) and the other without previous fertilizer (DSE). Observations were made monthly on each test tree. The biostimulant Banzai was applied for 3 or 4 consecutive months depending on the treatment at each site. The data collected included the total number of cherelles produced and the number of rotten cherelles. From these, data were deduced the rates of healthy cherelles on which the Kruskal-Wallis test was applied to compare treatments between them and between sites. The results showed that four applications of the biostimulant with fertilizer provided better control than three applications without fertilizer. The results also showed that the majority of treatments at the DAE site were more effective than treatments at the DSE site. In conclusion, four applications Banzai coupled with fertilizer have achieved the best rates of healthy cherelles whatever the site. Regarding the cumulative effect of the previous fertilizer with Banzai, treatments of the site with the previous fertilizer were still better than the treatment site without fertilizer.

Phytophthora theobromicola sp. nov.: A New Species Causing Black Pod Disease on Cacao in Brazil

Black pod disease, caused by Phytophthora species, is among the main limiting factors of cacao (Theobroma cacao L.) production. High incidence levels of black pod disease have been reported in Brazil, being induced by Phytophthora capsici, Phytophthora citrophthora, Phytophthora heveae, and Phytophthora palmivora. To assess the diversity of Phytophthora species affecting cacao in Brazil, 40 new isolates were obtained from cacao pods exhibiting symptoms of black pod disease collected in different smallholder farms in 2017. Further, ten cacao-infecting isolates morphologically identified as P. citrophthora and P. palmivora were molecularly characterized. The genomic regions beta-tubulin, elongation factor 1 alpha, heat shock protein 90, and internal transcribed spacer, and the mitochondrially encoded cytochrome c oxidase I and II genes were PCR-amplified and Sanger-sequenced from the cacao-infecting Phytophthora isolates. The morphological characterization and evaluation of the mycelial growth rates for the Phytophthora isolates were performed in vitro. Based on the molecular analysis and morphological comparisons, 19 isolates were identified as P. palmivora (clade 4). Interestingly, 31 isolates grouped together in the phylogenetic tree and were placed apart from previously known species in Phytophthora clade 2. Therefore, these isolates are considered as a new species herein referred to as Phytophthora theobromicola sp. nov., which produced papillate, semipapillate, and persistent sporangia on simple sporangiophores. The P. palmivora isolates were identified as A1 mating type by pairing each isolate with known A1 and A2 tester strains of P. capsici, but no oogonia/antheridia were observed when P. theobromicola was paired with the different tester strains. The P. theobromicola and P. citrophthora isolates showed higher mycelial growth rates, when compared to P. palmivora, on different media at 10, 15, and 20°C, but similar values were observed when grown on clarified CA media at 25 and 30°C. The pathogenicity tests carried out on pods of four cacao clones (CCN51, PS1319, Cepec2004, and CP49) showed significant variability among the isolates of both Phytophthora species, with P. theobromicola inducing higher rates of necrotic lesion expansion, when compared to P. palmivora. Here, two Phytophthora species were found associated with black pod disease in the state of Bahia, Brazil, and the previously undescribed P. theobromicola seems to be prevalent in field conditions. This is the first report of P. theobromicola on T. cacao. Also, these findings are crucial to improve the disease control strategies, and for the development of cacao materials genetically resistant to Phytophthora.

Maximum Entropy Niche Modelling to Estimate the Potential Distribution of Phytophthora megakarya (Brasier & M. J. Griffin) in Tropical Regions

Background: Phytophthora megakarya is an invasive pathogen endemic to Central and West Africa. This species causes the most devastating form of black pod disease. Despite the deleterious impacts of this disease on cocoa production, there is no information on the geographic distribution of P. megakarya. Aim: In this study, we investigated the potential geographic distribution of P. megakarya in cocoa-producing regions of the world using ecological niche modelling. Methods: Occurrence records of P. megakarya in Central and West Africa were compiled from published studies. We selected relevant climatic and edaphic predictor variables in the indigenous range of this species to generate 14 datasets of climate-only, soil-only, and a combination of both data types. For each dataset, we calibrated 100 candidate MaxEnt models using 20 regularisation multiplier values and five feature classes. The best model was selected from statistically significant candidates with an omission rate ≤ 5% and the lowest Akaike Information Criterion corrected for small sample sizes, and projected onto cocoa-producing regions in Southeast Asia, Central and South America. The risk of extrapolation in model transfer was measured using the mobility-oriented parity (MOP) metric. Results: We found an optimal goodness-of-fit and complexity for candidate models incorporating both climate and soil data. Predictions of the model with the best performance showed that nearly all of Central Africa, especially areas in Gabon, Equatorial Guinea, and southern Cameroon are at risk of black pod disease. In West Africa, suitable environments were observed along the Atlantic coast, from southern Nigeria to Gambia. Our analysis suggested that P. megakarya is capable of subsisting outside its native range, at least in terms of climatic and edaphic factors. Model projections identified likely suitable areas, especially in Brazil and Colombia, from southwestern Mexico down to Panama, and across the Caribbean islands in the Americas, and in Sri Lanka, Indonesia, Malaysia, and Papua New Guinea in Asia and adjacent areas Conclusion: The outcomes of this study would be useful for developing measures aimed at preventing the spread of this pathogen in the tropics.