Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.     

Net CO2 assimilation of cacao seedlings during periods of plant water deficit

The effect of leaf water potential () on net CO₂ assimilation rate (A), stomatal conductance (g), transpiration (E) and water-use efficiency (WUE) was measured for three cultivars of cacao (Theobroma cacao L.) seedlings during three recurrent drought cycles. Net assimilation varied greatly at high water potentials, but as dropped below approximately -0.8 and -1.0 MPa, A was reduced to less than 1.5 mol CO₂ m^-2 s^-1. The relation between g and A was highly significant and conformed to an asymptotic exponential model, with A approaching maximal values at stomatal conductances of 55–65 mmol H₂O m^-2 s^-1. Net assimilation varied linearly (r=0.95) with transpiration, and the slope of the A-E relation (WUE) was approximately 3.0 mol CO₂ mmol^-1 H₂O throughout the range of stomatal conductances observed. C _i was insensitive to water stress, even though both g and A were strongly affected. Under the experimental conditions used here, mesophyll photosynthesis did not appear to control g through changes in C _i. As stress intensified within each drying cycle, WUE of nonirrigated seedlings did not decline relative to that of controls even though CO₂ and water vapor exchange rates underwent large displacements. The effect of seed source was highly significant for WUE, and the basis for observed differences among genotypes is discussed.Abbreviations ABA Abscisic Acid     

Effects of temperature on growth and water relations of cacao (Theobroma cacao var.Comum) seedlings

Growth of 55-day-oldTheobroma cacao var.Comum seedlings varied with temperature regimes, various plant parts, growth parameters, and time of harvesting. Over a 60-day period the optimal day-temperature regimes were near 33.3°C for dry weight increase and relative growth rates of seedlings and leaves; 30.5°C for increase in leaf area, height growth, and leaf abscission; 22.2°C for dry weight increase of stems or roots, stem diameter growth, and root-shoot ratio. The rates of increase in dry weights of stems or roots as well as root-shoot ratios declined progressively at temperatures above 22.2°C Partitioning of dry matter was affected by temperature regime, with proportionally more photosynthate retained by shoots and less translocated to roots at high temperatures. The progressive decrease in the root-shoot ratio at temperatures above 22.2°C may decrease drought tolerance of seedlings because roots will be less capable of absorbing endugh water to replace transpirational losses. This was shown by more negative shoot water potentials at high temperatures. Research supported by the College of Agricultural and Life Science. University of Wisconsin, Madison, Wi, USA and the Centro de Pesquisas do Cacau (CEPLAC/CEPEC), 45600 Itabuna, Ba, Brazil.     

Somatic embryogenesis and plant regeneration from floral explants of cacao (Theobroma cacao L.) using thidiazuron

Summary A procedure for the regeneration of cacao (Theobroma cacao) plants from staminode explants via somatic embryogenesis was developed. Rapidly growing calli were induced by culturing staminode explants on a DKW salts-based primary callus growth (PCG) medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and thidiazuron (TDZ) at various concentrations. Calli were subcultured onto a WPM salts-based secondary callus growth medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and 1.4 nM kinetin. Somatic embryos were formed from embryogenic calli following transfer to a hormone-free DKW salts-based embryo development medium containing sucrose. The concentration of TDZ used in PCG medium significantly affected the rate of callus growth, the frequency of embryogenesis, and the number of somatic embryos produced from each responsive explant. A TDZ concentration of 22.7 nM was found to be the optimal concentration for effective induction of somatic embryos from various cacao genotypes. Using this procedure, we recovered somatic embryos from all 19 tested cacao genotypes, representing three major genetic group types. However, among these genotypes, a wide range of variation was observed in both the frequency of embryogenesis, which ranged from 1 to 100%, and the average number of somatic embryos produced from each responsive explant, which ranged from 2 to 46. Two types of somatic embryos were identified on the basis of their visual appearance and growth behavior. A large number of cacao plants have been regenerated from somatic embryos and established in soil in a greenhouse. Plants showed morphological and growth characteristics similar to those of seed-derived plants. The described procedure may allow for the practical use of somatic embryogenesis for clonal propagation of elite cacao clones and other applications that require the production of a large number of plants from limited source materials.     

Effects of VA mycorrhiza on the growth of cacao seedlings under nursery conditions in Venezuela

The effects of vesicular-arbuscular mycorrhizae inoculation on the growth of cacao seedlings (Theobroma cacao var. Ocumare 60) grown for 5 months in a nursery were studied. The effects of introduced VAM fungi (Glomus occultum, Acaulospora appendicula, Glomus manihotis, Acaulospora morrowae and Scutellospora pellucida) in soils treated with copper oxychloride or methyl bromide, were compared with the indigenous VAM and with their respective non-inoculated controls.Cacao seedlings responded well to indigenous VAM fungi, which included Scutellospora calospora as the dominant species, inducing increases significant in height, dry weight and foliar uptake of P, Cu and Zn in relation to the sterile control. G. occultum and A. appendicula increased the height of cacao seedlings but to an extent not yet statistically significant through the duration of the experiment. S. pellucida and A. appendicula doubled the phosphorus uptake of cacao seedlings. The methyl bromide sterilization induced the lowest Cu and Zn uptake in the cacao seedlings but this effect was ameliorated with VAM inoculation. Manifest deficiency symptoms were not observed in the treatments.Copper oxychloride treatment depressed growth to the same level as the sterile control although its residual effects did not kill VAM. It could change the competitive relations among the VAM species and in this case seemed to affect adversely the more efficient native fungi.The influence of the presence and long permanence of large cotyledons in cacao on the results obtained is discussed.     

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.     

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.     

A genetic linkage map of Theobroma cacao L.

A linkage map of the cocoa genome comprising 193 loci has been constructed. These loci consist of 5 isozymes, 101 cDNA/RFLPs, 4 loci from genes of known function, 55 genomic DNA/RFLPs and 28 RAPDs. A population of 100 individuals derived from a cross between two heterozygous genotypes was used. Segregation analyses were performed with the JoinMap program. Ten linkage groups, which putatively correspond to the ten gametic chromosomes of cocoa, were identified. The map covers a total length of 759 cM with a 3.9 cM average distance between 2 markers. A small fraction (9%) of the markers deviated significantly from the expected Mendelian ratios.     

dsRNA-induced gene silencing in Moniliophthora perniciosa, the causal agent of witches’ broom disease of cacao

The genome sequence of the hemibiotrophic fungus Moniliophthora perniciosa revealed genes possibly participating in the RNAi machinery. Therefore, studies were performed in order to investigate the efficiency of gene silencing by dsRNA. We showed that the reporter gfp gene stably introduced into the fungus genome can be silenced by transfection of in vitro synthesized gfpdsRNA. In addition, successful dsRNA-induced silencing of endogenous genes coding for hydrophobins and a peroxiredoxin were also achieved. All genes showed a silencing efficiency ranging from 18% to 98% when compared to controls even 28 d after dsRNA treatment, suggesting systemic silencing. Reduction of GFP fluorescence, peroxidase activity levels and survival responses to H₂O₂ were consistent with the reduction of GFP and peroxidase mRNA levels, respectively. dsRNA transformation of M. perniciosa is shown here to efficiently promote genetic knockdown and can thus be used to assess gene function in this pathogen.     

Effect of metabolites ofAspergillus niger andTrichoderma viride on development and structure of radicle of cocoa (Theobroma cacao) seedlings

Both the volume and concentration of filtrate ofAspergillus niger andTrichoderma viride influenced the development of the radicles of cocoa seedlings. Radicles placed in Petri dishes containing filter paper moistened with 20 and 30 ml of undiluted filtrate and filtrate dilutions of 1∶1 and 1∶2 failed to develop lateral roots and eventually died. The culture filtrate ofA. niger was more repressive. Radicles in the same volume of filtrate (20 and 30 ml) of higher dilutions (1∶5 and 1∶10) developed lateral roots and survived. Radicles placed in less volume (≤10 ml) survived and produced lateral roots irrespective of concentration of filtrates. Development of the radicles and roots in the control was consistently better (P=0.05) than in filtrate solutions of eitherA. niger orT. viride. The hypocotyls of seedlings under the influence of metabolites ofA niger showed greater cambial activity and formed xylem vessels and tracheids with larger lumina.     

Combining-ability for cacao (Theobroma cacao L.) yield components under southern Bahia conditions

The objective of this study was to assess five cacao cultivars (selfs) and 20 hybrids with regard to their general-and specific-combining ability for yield components using method 1, model I, of the diallel analysis system. The selfings and the hybrids were obtained through controlled crossings, tested in the field in a random block design with four replications and plots containing 16 plants. The experiment was set up in the Centro de Pesquisas do Cacau, in Itabuna, Bahia, Brasil, in 1975. The characteristics studied were: the number of healthy and collected fruits per plant (NHFP and NCFP), the weight of humid seeds per plant and per fruit (WHSP and WHSF), and the percentage of diseased fruits per plant (PDFP), for 5 years (1986–1990). The F-test values, highly significant for general combining ability (GCA) and specific combining ability (SCA), demonstrated the existence of variability for both effects. However, the effects of SCA were greater than those of GCA, when compared in terms of the average squared effects. This condition held for the characteristics NHFP, NCFP and WHSP, which shows the relative importance of the non-additive genetic effects over the additive effects. The reciprocal effects did not show significance. Breeding methods which explore the additive portion of genetic variance should be employed for obtaining higher-yielding cacao and high seed weight. For this, the segregant populations should involve cultivars CEPEC 1, SIAL 169 and ICS 1. Combinations involving the cultivar ICS 1 presented the most favorable results for the characteristics WHSP and WHSF, where the hybrid SIAL 169 x ICS 1 and its reciprocal were outstanding.     

Selection of Trichoderma stromaticum isolates for efficient biological control of witches’ broom disease in cacao

Witches’ broom is the most devastating disease of cacao in Brazil, and losses to it entail serious socio-economical and environmental problems. Biological control of the causal agent Moniliophthora perniciosa (Mp) using the antagonistic fungus Trichoderma stromaticum (Ts) is promising, although the identification of superior isolates is necessary. Here, we report a study on the selection of more effective Ts isolates based on field experiments. Sixty-three Ts isolates from a local collection were applied on brooms and placed under typical conditions of shaded-cacao plantations in southeastern Bahia State (Brazil), during two periods of three months each. The percentages of Ts sporulation and incidence and severity of Mp were the parameters used for biocontrol assessments. The results from both experiments were very distinct, indicating a high phenotypic variation in this collection and suggesting a significant effect of the environment in the Ts–Mp interaction. Ts-sporulation rates were negatively correlated with the presence of Mp in the brooms and a number of isolates reduced Mp incidence more efficiently than the reference isolate. Contrasting isolates in their efficiency of reducing Mp incidence were selected and further tested in four subsequent field trials for validation purposes. The results partially confirmed their biocontrol phenotypes but also suggested isolate-specific responses to environmental variations. Inhibition of Mp-basidiospore germination by total protein secreted in culture supernatants of Ts isolates correlated well with field results and revealed a potentially useful procedure for pre-screening of large collections towards selection of better biological control isolates. The characteristics and efficiency of the method as a reliable protocol for identification of superior BCAs in the witches’ broom—cacao pathosystem is discussed.     

Chloroplast and mitochondrial DNA diversity in Theobroma cacao

The variability of cocoa (Theobroma cacao) cytoplasmic genomes has been investigated. A total of 177 cocoa clones was surveyed for restriction fragment length polymorphism (RFLP) in chloroplast DNA and in mitochondrial DNA using two restriction endonucleases and various heterologous cytoplasmic probes. A high level of polymorphism was found for the mitochondrial genome. This study points up a structuring of the species that fits with the distinction between the Criollo and Forastero populations. In contrast to all previous analyses, a higher level of polymorphism is found among the Criollo clones while the Forastero clones form quite a homogeneous group.     

The relationship between lignin and morphological characteristics of the tracheary elements from cacao(Theobroma cacao L.) Hulls

Tracheary elements (TEs) were physically separated from the hulls of cacao pods(Theobroma cacao L). Their morphological features were extensively investigated with scanning electron microscopy and chemical characterization. Spiral TEs were covered with a thin layer of primary wall that had a web-like structure on its outer surface. These TEs had a spiral circularity diameter of 8.2 ± 0.6 μm and an estimated secondary wall thickness of about 2.1 ± 0.2 μm. Polarized microscopy analysis revealed that the cellulose microfibrils were aligned parallel to that thickening. Lignin content was 36.1%, with a 0.13:1.00 molar ratio of syringyl to guaiacyl units and a 1.09:1.00 molar ratio of erythronic acid and threonic acid. Total yields of the alkaline nitrobenzene oxidation and ozonation products were 324.5 and 148.8 μmol g^-1 of extract-free TEs, respectively. Based on these morphological and lignin characteristics, we conclude that fully ripened cacao hulls exhibit the same features of secondary wall thickening as those seen at an earlier stage.     

Recovery of species richness and conservation of native Atlantic forest trees in the cacao plantations of southern Bahia in Brazil

The Atlantic forests of southern Bahia in Brazil present great species richness and a high degree of endemism. A large part of these native forests were transformed into cacao plantations in an agroforestry system known locally as cabrucas, where native trees were culled and cacao was planted under the shade of remaining trees. The present study analyzed the influence of time of implantation (age) and time of abandonment of management practices on tree species diversity of cabruca plantations to evaluate the capacity for conservation and recovery of species richness of native Atlantic Forest trees in cabrucas. Phytosociological surveys were conducted in five cabrucas with different conditions of age and state of abandonment. All trees, including hemiepiphytes and excluding the cacao plants, with a minimum stem diameter of 10 cm at breast height, were surveyed within a 3-ha sampling area in each plantation. A total of 2514 individual trees belonging to 293 species and 52 families were recorded in the five cabrucas. The Shannon diversity index varied from 3.31 to 4.22 among the cabrucas and was positively correlated with the time of abandonment (r = 0.97). The new cabrucas showed the highest values of estimated total richness (Chao) and the highest proportion of late successional species than the old ones. All areas preserved a very high proportion of native forest species while the three old cabrucas showed a higher proportion of exotic species than the two new ones. Thus the exotic species seem to replace more of the native species in the long run because of management practices and local preferences. The cabrucas presented also a high capacity for the regeneration of tree species richness after abandonment. Simple alterations in management practices could improve the recruitment of late successional species in these areas. Economic incentives may be necessary for the farmers to adopt management practices to retain native species which bring no economic returns.     

Mapping of Quantitative Trait Loci for Butter Content and Hardness in Cocoa Beans (Theobroma cacao L.)

Cocoa butter is an important raw material for the chocolate, pharmaceutical, and cosmetic industries. The butter content and quality in cocoa beans are genetically controlled characteristics, and affect its commercial value and industrial applicability. In the present work, an F₂ population derived from the cross between the ICS-1 and Scavina-6 cocoa clones was used for molecular mapping. A linkage map was constructed based on amplified fragment length polymorphism, random amplified polymorphic DNA, and simple sequence repeat markers, resulting in a total of 273 markers, distributed in 14 linkage groups (LGs). Phenotyping of butter content was performed after ether extraction and butter hardness was determined by sweeping differential calorimetry. One quantitative trait locus (QTL) associated to butter content was mapped at linkage group 9 (LG9) and two QTLs for butter hardness were identified at linkage groups 9 and 7 (LG9 and LG7). The two QTLs mapped at the LG9 explained 51.0% and 28.8% of the phenotypic variation for butter content and hardness, respectively. These QTLs were concentrated in the same map region, suggesting a close genetic linkage or pleiotropic effect. The QTLs identified may be useful in further marker-assisted selection breeding programs aimed at cocoa butter quality improvement.     

Molecular characterisation of fungal endophytic morphospecies associated with the indigenous forest tree, Theobroma gileri, in Ecuador

Fungal endophytes were isolated from healthy stems and pods of Theobroma gileri, an alternative host of the frosty pod rot pathogen of cacao. Non-sporulating isolates were grouped into 46 different morphological species according to their colony morphology. Many of these morphospecies were assumed to be basidiomycetes and, therefore, were of particular interest. Basidiomycetous endophytes have received far less attention than ascomycetes and also have potential as biological control agents of the basidiomycetous pathogens of T. cacao: Moniliophthora roreri (frosty pod rot pathogen) and M. perniciosa (witches' broom disease). The morphospecies were further characterised by molecular analyses. Amplification of the nuLSU was undertaken for phylogenetic placement of these non-sporulating cultures and revealed a total of 31 different taxa of which 15 were basidiomycetes belonging to the class Agaricomycetes, and 16 ascomycetes primarily belonging to the Sordariomycetes.     

Bird diversity in cacao farms and forest fragments of western Panama

Theobroma cacao plantings, when managed under the shade of rainforest trees, provide habitat for many resident and migratory bird species. We compared the bird diversity and community structure in organic cacao farms and nearby forest fragments throughout mainland Bocas del Toro, Panama. We used this dataset to ask the following questions: (1) How do bird communities using cacao habitat compare to communities of nearby forest fragments? (2) To what extent do Northern migratory birds use shaded cacao farms, and do communities of resident birds shift their abundances in cacao farms seasonally? (3) Do small scale changes in shade management of cacao farms affect bird diversity? Using fixed radius point counts and additional observations, we recorded 234 landbird species, with 102 species that were observed in both cacao and forest fragments, 86 species that were only observed in cacao farms, and 46 species that were restricted to forest fragments. Cacao farms were rich in canopy and edge species such as tanagers, flycatchers and migratory warblers, but understory insectivores were nearly absent from cacao farms. We observed 27 migratory species, with 18 species in cacao farms only, two species in forest only, and seven species that occurred in both habitats. In cacao farms, the diversity of birds was significantly greater where there was less intensive management of the canopy shade trees. Shade tree species richness was most important for explaining variance in bird diversity. Our study shows that shaded cacao farms in western Panama provide habitat for a wide variety of resident and migratory bird species. Considering current land use trends in the region, we suggest that action must be taken to prevent conversion away from shaded cacao farms to land uses with lower biodiversity conservation value.     

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.     

Characterisation and genetic mapping of resistance and defence gene analogs in cocoa (Theobroma cacao L.)

Disease resistance and defence gene analog (RGA/DGA) sequences were isolated in cocoa using a PCR approach with degenerate primers designed from conserved domains of plant resistance and defence genes: the NBS (nucleotide binding site) motif present in a number of resistance genes such as the tobacco N, sub-domains of plant serine/threonine kinases such as the Pto tomato gene, and conserved domains of two defence gene families: pathogenesis-related proteins (PR) of classes 2 and 5. Nucleotide identity between thirty six sequences isolated from cocoa and known resistance or defence genes varied from 58 to 80%. Amino acid sequences translated from corresponding coding sequences produced sequences without stop codons, except for one NBS –like sequence. Most of the RGAs could be mapped on the cocoa genome and three clusters of genes could be observed : NBS-like sequences clustered in two regions located on chromosomes 7 and 10, Pto-like sequences mapped in five genome regions of which one, located on chromosome 4, corresponded to a cluster of five different sequences. PR2-like sequences mapped in two regions located on chromosome 5 and 9 respectively. An enrichment of the genetic map with microsatellite markers allowed us to identify several co-localisations of RGAs, DGAs and QTL for resistance to Phytophthora detected in several progenies, particularly on chromosome 4 where a cluster of Pto-like sequences and 4 QTL for resistance to Phytophthora were observed. Many other serious diseases affect cocoa and the candidate genes, isolated in this study, could be of broader interest in cocoa disease management.