Antimicrobial Activity of Syzygium aromaticum and Zanthoxylum xanthoxyloides Essential Oils Against Phytophthora megakarya

The aim of this study was to assess the chemical composition and the antimicrobial activity of essential oils of dried fruits and buds of Zanthoxylum xanthoxyloides (Z. xanthoxyloides) and Syzygium aromaticum (S. aromaticum or clove), respectively, against Phytophthora megakarya (P. megakarya). Essential oils were extracted by hydrodistillation, and their composition was determined by gas chromatography and by gas chromatography coupled with Mass Spectrometry. The minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) of the essential oils against P. megakarya were assessed by the Agar dilution method. The in vivo efficacy study consisted of spraying the essential oil emulsions on cocoa pod husk pieces (CPHP), followed by the inoculation with P. megakarya zoospores. The hydrodistillation yielded 10.54 and 1.89% of essential oils for S. aromaticum and Z. xanthoxyloides, respectively. Both oils were mainly made up of oxygenated monoterpenes (89.58 and 88.2%, respectively). Eugenol (83.02%) and eugenyl acetate (9.15%) were the main components of clove oil while α‐citronelol (25.83%) and trans‐geraniol (16.49%) were mostly found in the Z. xanthoxyloides oil. Clove oil exhibited stronger antimicrobial activity with a MIC of 250 μl/l than Z. xanthoxyloides with MIC of 350 μl/l. The symptoms were totally suppressed on pod husk treated with clove oil at 2000 μl/l. The decrease in the growth rate of the necrosis (GRN) and the sporulation of P. megakarya (PS) on cocoa husk after the successful infection was significant after the treatment with essential oils. These results are promising and indicate that the studied essential oils might be further investigated as natural alternatives to synthetic fungicides for the control of cocoa black pod diseases.     

Application of glycerol as a foliar spray activates the defence response and enhances disease resistance of Theobroma cacao

Previous work has implicated glycerol‐3‐phosphate (G3P) as a mobile inducer of systemic immunity in plants. We tested the hypothesis that the exogenous application of glycerol as a foliar spray might enhance the disease resistance of Theobroma cacao through the modulation of endogenous G3P levels. We found that exogenous application of glycerol to cacao leaves over a period of 4 days increased the endogenous level of G3P and decreased the level of oleic acid (18:1). Reactive oxygen species (ROS) were produced (a marker of defence activation) and the expression of many pathogenesis‐related genes was induced. Notably, the effects of glycerol application on G3P and 18:1 fatty acid content, and gene expression levels, in cacao leaves were dosage dependent. A 100 mm glycerol spray application was sufficient to stimulate the defence response without causing any observable damage, and resulted in a significantly decreased lesion formation by the cacao pathogen Phytophthora capsici; however, a 500 mm glycerol treatment led to chlorosis and cell death. The effects of glycerol treatment on the level of 18:1 and ROS were constrained to the locally treated leaves without affecting distal tissues. The mechanism of the glycerol‐mediated defence response in cacao and its potential use as part of a sustainable farming system are discussed.     

Genetic diversity and relationships of cacao (Theobroma cacao L.) in southern Mexico

 Neotropical tree crops are affected by a combination of biological and human factors that complicate the study of genetic diversity and crop evolution. Genetic diversity and relationships among southern Mexican populations and horticultural collections of Theobroma cacao (chocolate, cocoa, cacao) are examined in light of the agricultural practices of the Maya. Collections of cacao were obtained from the extremes of its geographic range including archeological sites in southern Mexico where cacao was first domesticated. Genetic diversity was assayed by 57 informative random amplified polymorphic DNA (RAPD) marker loci. A unique sample of the total diversity found in this study exists in the southern Mexican populations. These populations are significantly different from all other cacao with regards to their profile of RAPD bands, including the ‘criollo’ variety, their morphological and geographical group. A population of cacao found in a sinkhole (cenote) in northern Yucatan with genetic affinities to populations in Chiapas suggests the Maya maintained plants far away from their native habitat. This finding concurs with known agroforestry practices of the Maya. Modern efforts to increase germplasm of tropical tree crops such as cacao should carefully examine archeological sites where genetic diversity, either deliberately or by chance, was collected and maintained by ancient cultures. Received: 21 May 1997 / Accepted: 9 October 1997     

Doxorubicin induced neuro- and cardiotoxicities in experimental rats: Protection against oxidative damage by Theobroma cacao Stem bark

80 rats, randomly selected, were divided into 3 treatment groups: pre-, co- and post-treatment; consisting of 6 sub-groups each (5 rats per sub-group): baseline, normal saline (2 mL), α-lipoic acid (20 mg/kg body weight), 200 mg/kg, 400 mg/kg or 800 mg/kg body weight Theobroma cacao stem bark aqueous extract (TCAE). All rats except for baseline group were intoxicated with 20 mg/kg body weight doxorubicin (DOX) intraperitoneally. The animals in pre- or post-treatment group received a single dose of DOX (20 mg/kg body weight) intraperitoneally 24 h before or after 7 days’ oral administration with TCAE respectively while those in co-treatment group were co-administered 2.86 mg/kg body weight of DOX with either normal saline, α- lipoic acid or TCAE orally for 7 days. Animals were sacrificed (pre- and post- treatment groups were sacrificed on the ninth day while the co-treatment group sacrificed on the 8th day). Brain and heart tissue samples were harvested for enzyme markers of toxicity, oxidative stress and histopathological examinations. DOX intoxication caused significant decrease in activities of LDH and ACP, and increase in γGT and ALP activities in brain tissues while causing a significant increase in LDH, ACP, γGT activities and decrease in ALP activity in the cardiac tissues. DOX intoxication caused a significant increase in concentrations of H₂O₂ generated, MDA and PC, XO, MPx and NOX activities with concomitant decrease in CAT, SOD, GPx and GST activities, and in concentrations of GSH, AsA and α-Toc in brain and cardiac tissues. Pre-, co- and post-treatment with TCAE at either 200 mg/kg, 400 mg/kg or 800 mg/kg body weight significantly reversed the oxidative damage to the organs induced by DOX-intoxication. The result affirmed that T. cacao stem bark aqueous extract protected against DOX induced oxidative damage in brain and cardiac tissues of experimental rats.     

Comparison of two cacao (Theobroma cacao L.) clones for the effect of pollination intensity on fruit set and seed content

We compared the influence of pollination intensity (PI) on fruit set and seed number per pod in two cacao (Theobroma cacao L.) clones, IFC5 (Forastero Lower-Amazon Amelonado), which is self-compatible and known to produce a high number of seeds per pod under open pollination, and SCA6 (Forastero Upper-Amazon), which is self-incompatible and known to produce a low number of seeds per pod under open pollination. With both clones, PI had a positive effect on fruit set, with the maximum rate requiring more than 150 pollen grains per pod. One-half of the maximum rate of fruit set was reached with 39 pollen grains per pod for SCA6, and 78 for IFC5. With SCA6, a significant positive effect of PI on seed number per pod was also observed, with maximum seed number requiring more than 200 pollen grains per pod. In contrast, seed numbers with IFC5 were approximately equal over the PI range 100–800 pollen grains per pod. Patterns of seed number per pod were compared after: (1) open insect pollination, (2) low-intensity hand pollination and (3) high-intensity hand pollination. The patterns obtained with IFC5 under open pollination showed a peak of 40–50 seeds per pod, whereas the distribution patterns were more even with SCA6. The pattern of seed number under open pollination was similar to that obtained with high-intensity hand pollination for IFC5, and with low-intensity hand pollination for SCA6. We concluded that the high number of seeds per pod observed with IFC5 under natural insect pollination may be explained by a high number of compatible self-pollen grains on the stigma and by a severe drop of low-pollinated flowers eliminating potential lowfilled fruits. With SCA6, however, the number of compatible pollen grains deposited on the stigma was probably low under open pollination, and the flowers required lower pollen quantities to set fruit, which resulted in a high frequency of low seed numbers per fruit. This difference in the capacity to set low-seeded fruits might be considered as an adaptive trait related to the mode of reproduction.     

Growth Enhancement,Amino Acid Synthesis and Reduction in Susceptibility Towards Phytophthora megakarya by Arbuscular Mycorrhizal Fungi Inoculation in Cocoa Plants

The effects of some selected arbuscular mycorrhizal (AM) fungi, Gigaspora margarita and Glomus mossae on the growth and the role of soluble amino acids of two contrasting cocoa cultivars (ICS84 tolerant and SNK10 sensitive) against black pod disease caused by Phytophthora megakarya were investigated. Root colonization by AM fungi is between 50 and 70% 18 weeks after planting. Tested AM fungi significantly increased all the plant growth parameters (height, number of leaves, shoot and root matter) and P uptake as compared to non‐inoculated plants in pot experiments. AM fungi inoculated cocoa reduced the disease severity. Compared to the control, the soluble amino acid levels increased with inoculation of the AM fungi strains in the necrotic stems of disease on inoculated cocoa plants. Significant relationships between amino acids and disease severity observed for two cocoa cultivars imply that the induction of specific amino acids synthesized by leaves, such as arginine, cysteine and glutamic acid, may represent potential candidate molecules for adaptation of such cultivars to P. megakarya disease. Inoculating seedlings with AMF in nurseries could enhance the development of cocoa plants protected against P. megakarya.     

Effect of Fruiting Traits on the Field Resistance of Cocoa (Theobroma cacao L.) Clones to Phytophthora megakarya

The effects of some traits of field resistance (precocity and duration of the fruiting cycle, age of diseased fruit and vertical pod distribution on the tree) to Phytophthora megakarya of four known cocoa clones were studied in an on‐station clonal plot planted in 1982 in the south‐west of Cameroon. Weekly observations of fruit set and development, black pod and rainfall were carried out during three growing seasons (1999, 2000 and 2001). The study confirmed the previous field and laboratory assessments of resistance of these clones based on the mean percentages of rotten pods obtained annually. The present study has permitted the identification of fruit aged 2–3 months as the highly susceptible stage of development in the most susceptible clone. In addition, precocity and pod cycle duration varied significantly among the clones. The earlier the pod cycle began, the more susceptible was the clone: the most resistant clone started flowering 1 month after the most susceptible clone and therefore escaped the peak of disease severity. Rainfall intensity greatly modified the incidence of the disease in 2001, with high yield losses occurring in all four clones (70–93%), but their ranking remained stable over the 3 years. The spatial distribution of pods on the trees showed that pods on the trunk were more likely to become diseased than those on the branches, but its effect as a clone resistance component is variable among the four clones; the resistant clone producing more pods on the trunk and the susceptible clone more in the canopy.     

Evaluation of the extent of genetic variability among Theobroma cacao accessions using RAPD and RFLP markers

 Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to evaluate genetic relationships within the Theobroma cacao species and to assess the organization of its genetic diversity. Genetic variability was estimated with 18 primers and 43 RFLP probes on 155 cocoa trees belonging to different morphological groups and coming from various geographic origins. The majority of the RFLP probes issued from low-copy DNA sequences. On the basis of on the genetic distance matrices, the two molecular methods gave related estimates of the genetic relationship between genotypes. Although an influence of cocoa morphological groups and geographical origins of trees was observed, a lack of gene differentiation characterized the T. cacao accessions studied. The continuous RFLP variability observed within the species may reflect the hybridization and introgressions between trees of different origins. Nevertheless, the Nacional type was detected to be genetically specific and different from well-known types such as Forastero, Criollo and Trinitario. Some of those genotypes were characterized by a low heterozygosity rate and may constitute the original Nacional pool. These results also provide information for the constitution of a cocoa tree core collection. Received: 10 June 1996/Accepted: 11 October 1996     

Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.     

Characterization of the Proteome of Theobroma cacao Beans by Nano‐UHPLC‐ESI MS/MS

Cocoa seed storage proteins play an important role in flavour development as aroma precursors are formed from their degradation during fermentation. Major proteins in the beans of Theobroma cacao are the storage proteins belonging to the vicilin and albumin classes. Although both these classes of proteins have been extensively characterized, there is still limited information on the expression and abundance of other proteins present in cocoa beans. This work is the first attempt to characterize the whole cocoa bean proteome by nano‐UHPLC‐ESI MS/MS analysis using tryptic digests of cocoa bean protein extracts. The results of this analysis show that >1000 proteins could be identified using a species‐specific Theobroma cacao database. The majority of the identified proteins were involved with metabolism and energy. Additionally, a significant number of the identified proteins were linked to protein synthesis and processing. Several proteins were also involved with plant response to stress conditions and defence. Albumin and vicilin storage proteins showed the highest intensity values among all detected proteins, although only seven entries were identified as storage proteins. A comparison of MS/MS data searches carried out against larger non‐specific databases confirmed that using a species‐specific database can increase the number of identified proteins, and at the same time reduce the number of false positives. The results of this work will be useful in developing tools that can allow the comparison of the proteomic profile of cocoa beans from different genotypes and geographic origins. Data are available via ProteomeXchange with identifier PXD005586.     

Genetic diversity and structure of managed and semi-natural populations of cocoa (Theobroma cacao) in the Huallaga and Ucayali Valleys of Peru

BACKGROUND AND AIMS: Cocoa (Theobroma cacao) is indigenous to the Amazon region of South America, and it is well known that the Peruvian Amazon harbours a large number of diverse cocoa populations. A small fraction of the diversity has been collected and maintained as an ex-situ germplasm repository in Peru. However, incorrect labelling of accessions and lack of information on genetic diversity have hindered efficient conservation and use of this germplasm. This study targeted assessment of genetic diversity and population structure in a managed and a semi-natural population. METHODS: Using a capillary electrophoresis genotyping system, 105 cocoa accessions collected from the Huallaga and Ucayali valleys of Peru were fingerprinted. Based on 15 loci SSR profiles, genetic identity was examined for each accession and duplicates identified, population structure assessed and genetic diversity analysed in these two populations. KEY RESULTS: Ten synonymous mislabelled groups were identified among the 105 accessions. The germplasm group in the Huallaga valley was clearly separated from the group in Ucayali valley by the Bayesian assignment test. The Huallaga group has lower genetic diversity, both in terms of allelic richness and of gene diversity, than the Ucayali group. Analysis of molecular variance suggested genetic substructure in the Ucayali group. Significant spatial correlation between genetic distance and geographical distances was detected in the Ucayali group by Mantel tests. CONCLUSIONS: These results substantiate the hypothesis that the Peruvian Amazon hosts a high level of cocoa genetic diversity, and the diversity has a spatial structure. The introduction of exotic seed populations into the Peruvian Amazon is changing the cocoa germplasm spectrum in this region. The spatial structure of cocoa diversity recorded here highlights the need for additional collecting and conservation measures for natural and semi-natural cocoa populations.     

Heritability of resistance against ectoparasitism in the Drosophila-Macrocheles system

Ectoparasites are abundant in natural communities, can have pronounced deleterious fitness consequences to their host and are important vectors of transmissible parasitic disease. Yet very few studies have estimated the magnitude of heritable genetic variation underlying resistance against ectoparasitism, which significantly limits our ability to predict the evolution of this ecologically important character. The present paper reports results of artificial selection for increased resistance in Drosophila nigrospiracula against ectoparasitic, haematophagous mites, Macrocheles subbadius. In this system, which occurs naturally in the Sonoran Desert of North America, ectoparasitism significantly damages the expression of host fitness traits, including longevity, fecundity and male mating success. In the present study, resistance, which was modelled as a threshold trait, responded significantly to selection applied on either sex. Realized heritability, calculated as a mean across four replicates, was estimated to be 0.152 +/- 0.014 (SE). The heritability estimate from selection on males did not differ from that on females, but both estimates differed significantly from zero. This documented presence of additive genetic variation for resistance, coupled with knowledge of the fitness consequences of ectoparasitism, indicates that the host population possesses significant evolutionary potential. Selection was applied on the pre-attachment phase, thereby targeting behavioural forms of defence. This study therefore establishes parallels between insects and other animals in their ability to protect themselves and evolve behavioural defences against ectoparasites.     

Changes in Phenolics, Polyphenol Oxidase and its Isoenzyme Patterns in Relation to Resistance in Taro against Phytophthora colocasiae

The effect of Phytophthora leaf blight disease, caused by Phytophthora colocasiae Raciborski, on the accumulation of phenolics and polyphenol oxidase (PPO) activity in ex vitro plants was studied in three resistant (DP‐25, Duradim and Jhankri) and one susceptible (N‐118) genotypes of taro [Colocasia esculenta (L). Schott]. The inoculation of taro leaves with P. colocasiae spores resulted in a quantitative change in both biochemical parameters and induction of PPO isoforms in resistant genotypes. The amount of phenolics was increased owing to blight by 68.02%, 58.87%, 52.67% and 11.50% in DP‐25, Duradim, Jhankri and N‐118, respectively. The per cent increase in PPO under stress over non‐stress condition was also highest in DP‐25 (49.14%) followed by Duradim (41.56%), Jhankri (40.55%) and N‐118 (17.08%). The resistant genotypes showed higher activity of PPO as compared with susceptible ones, which was reflected through its banding pattern in isoenzyme analysis, detecting four different isoforms. The intensity of the bands was higher in the resistant genotypes than in susceptible N‐118. The appearance of high intensity bands and/or reduction in the intensity of particular isoform(s) in the zymograms of all the three resistant taro genotypes studied, led to the apparent conclusion of linking PPO isoenzyme expression with blight resistance in taro. The blight incidence (per cent leaf infection and leaf area infection) was lower in the resistant genotypes than in susceptible, N‐118. The yield reduction owing to blight was below 20% in DP‐25, Jhankri and Duradim, while the same was more than 40% in N‐118. The phenolics and PPO activity was negatively correlated with disease incidence and yield reduction owing to blight. Based on the results of disease incidence, biochemical contents and yield, the pattern of stress tolerance was DP‐25 > Duradim > Jhankri > N‐118. The studied parameters, i.e. phenolics and PPO could be used as biochemical markers for leaf blight stress tolerance studies in taro.     

Distribution of assimilate during the flush cycle of growth in Theobroma cacao L.

The growth of the shoot and roots of seedling plants of cocoa (Theobroma cacao L.) under constant glasshouse conditions showed a rhythmic cycle, with the maximum growth stages of each alternating in a regular sequence. When the growth cycle of the shoot was upset by removing all new leaves immediately after unfolding, the roots showed a high constant growth rate during this period, suggesting that normally the rapidly expanding leaves exert an inhibitory influence on the roots. Conversely removal of portions of the root delayed the production of new leaves in the shoot. The level of soluble and starch carbohydrate in the mature leaves, roots and stem declined during the period of expansion of the flush leaves, but accumulated again at the end of the leaf expansion stage. It is likely that this reserve carbohydrate was remobilised and translocated to the flush leaves during their period of expansion. A large proportion of newly formed photoassimilate, as shown by the distribution of ¹⁴C radioactivity from different source leaves, was also translocated to the young leaves during expansion. The large sink created by these leaves may cause photoassimilate and reserve carbohydrate to be diverted from the roots, thereby inhibiting root growth during the stage of leaf expansion. It is suggested that the rhythmic leaf production at the apex may control the growth cycle of the roots.     

Antimicrobial Activities of the Essential Oils of Various Plants against Tomato Late Blight Disease Agent Phytophthora infestans

The aim of this study was to find an alternative to synthetic fungicides currently used in the control of devastating oomycete pathogen Phytophthora infestans, causal agent of late blight disease of tomato. Antifungal activities of essential oils obtained from aerial parts of aromatic plants such as oregano (Origanum syriacum var. bevanii), thyme (Thymbra spicata subsp. spicata), lavender (Lavandula stoechas subsp. stoechas), rosemary (Rosmarinus officinalis), fennel (Foeniculum vulgare), and laurel (Laurus nobilis), were investigated against P. infestans. Both contact and volatile phase effects of different concentrations of the essential oils used were determined by using two in vitro methods. Chemical compositions of the essential oils were also determined by GC-MS analysis. Major compounds found in essential oils of thyme, oregano, rosemary, lavender, fennel and laurel were carvacrol (37.9%), carvacrol (79.8), borneol (20.4%), camphor (20.2%), anethole (82.8%) and 1,8-cineole (35.5%), respectively. All essential oils were found to inhibit the growth of P. infestans in a dose-dependent manner. Volatile phase effect of oregano and thyme oils at 0.3 μg/ml air was found to completely inhibit the growth of P. infestans. Complete growth inhibition of pathogen by essential oil of fennel, rosemary, lavender and laurel was, however, observed at 0.4–2.0 μg/ml air concentrations. For the determination of the contact phase effects of the tested essential oils, oregano, thyme and fennel oils at 6.4 μg/ml were found to inhibit the growth of P. infestans completely. Essential oils of rosemary, lavender and laurel were inhibitory at relatively higher concentrations (12.8, 25.6, 51.2 μg/ml respectively). Volatile phase effects of essential oils were consistently found to be more effective on fungal growth than contact phase effect. Sporangial production was also inhibited by the essential oil tested. Light and scanning electron microscopic (SEM) observation on pathogen hyphae, exposed to both volatile and contact phase of oil, revealed considerable morphological alterations in hyphae such as cytoplasmic coagulation, vacuolations, hyphal shrivelling and protoplast leakage.     

Tracing the native ancestors of the modern Theobroma cacao L. population in Ecuador

The native Theobroma cacao L. population from Ecuador, known as Nacional, is famous for its fine cocoa flavour. From the beginning of the twentieth century, however, it has been subjected to genetic erosion due principally to successive introductions of foreign germplasm whose hybrid descendants gradually replaced the native plantations, implying a decrease in cocoa quality. We attempted to trace this native cacao within a wide pool of modern Ecuadorian cacao population. Three hundred and twenty-two cacao accessions collected from different geographical areas along the pacific coast of Ecuador and maintained in two living collections were analysed using 40 simple-sequence repeat markers. Most of Ecuadorian cacao accessions displayed a high diversity and heterozygosity level. A factorial analysis of correspondence (FAC) showed a continuous variation among them, with a few ones, grouped at an extreme side of the FAC cloud, showing higher levels of homozygosity and lower introgression level by foreign cacaos. A paternity analysis revealed that these highly homozygous individuals are the most probable ancestors of the modern Nacional hybrid pool. These particular accessions studied could represent the native Nacional cacao present in Ecuador before the foreign introductions. Their identification will help to conserve valuable genetic material and to improve cocoa quality in new cacao varieties.