Characterization of the microbiota dynamics associated with Moniliophthora roreri, causal agent of cocoa frosty pod rot disease, reveals new viral species.

Introduction: Theobroma cacao, the cocoa tree, is a target for pathogens, such as fungi from the genera Phytophthora, Moniliophthora, Colletotrichum, Ceratocystis, among others. Some cacao pathogens are restricted to specific regions of the world, such as the Cacao swollen shoot virus (CSSV) in West African countries, while others are expanding geographically, such as Moniliophthora roreri in the Americas. M. roreri is one of the most threatening cacao pathogens since it directly attacks the cacao pods driving a significant reduction in production, and therefore economic losses. Despite its importance, the knowledge about the microenvironment of this pathogen and the cocoa pods is still poorly characterized. Methods: Herein we performed RNA sequencing of spores in differential stages of culture in a medium supplemented with cacao pod extract and mycelium collected of the susceptible variety ICT 7121 naturally infected by the pathogen to evaluate the diversity and transcriptional activity of microorganisms associated with the in vitro sporulation of M. roreri. Results: Our data revealed a great variety of fungi and bacteria associated with M. roreri, with an exceptional diversity of individuals from the genus Trichoderma sp. Interestingly, the dynamics of microorganisms from different kingdoms varied proportionally, suggesting they are somehow affected by M. roreri culture time. We also identified three sequences similar to viral genomes from the Narnaviridae family, posteriorly confirmed by phylogenetic analysis as members of the genus Narnavirus. Screening of M. roreri public datasets indicated the virus sequences circulating in samples from Ecuador, suggesting a wide spread of these elements. Of note, we did not identify traces of the viral sequences in the M. roreri genome or DNA sequencing, restricting the possibility of these sequences representing endogenized elements. Discussion: To the best of our knowledge, this is the first report of viruses infecting the fungus of the genus Moniliophthora and only the third description of viruses that are able to parasite elements from the Marasmiaceae family.

Growth and nutritional responses of wild and domesticated cacao genotypes to soil Cd stress.

Cadmium (Cd), a toxic non-essential metal, is easily accumulated in cacao tissues. This represents a risk for cacao exportation, and consequently it affects the economic well-being of the resource-poor-small-producers in Latin America. A greenhouse experiment was conducted with 53 wild and domesticated cacao genotypes to determine their response to Cd in terms of growth and Cd and essential nutrients accumulation. Cacao seedlings were grown for 6 months in an acidic soil with or without added Cd. The total concentration of macro (Ca, K, Mg, N and P) and micronutrients (B, Cu, Fe, Mn and Zn) as well as Cd were measured in shoots along with growth (biometric) parameters after harvest. The results revealed that even if there was a wide range of Cd concentrations among genotypes, there was a reduction in the concentration of essential nutrients in genotypes grown in Cd spiked soils, however these concentrations were not significantly different from the control. In the case of growth parameters, the effects of Cd were diverse across all genotypes some of them being more tolerant to Cd stress than others. Thus, different growth responses to Cd stress are related to a genotype effect. Based on their lower Cd concentration, a total of 11 cacao genotypes (AYP-22, PAS-105, UGU-126, ICT-1026, ICT-1087, ICT-1189, ICT-1292, PH-17, CCN-51, ICS-39 and TSH-565) are proposed here as low Cd-accumulating genotypes. Therefore, these genotypes are potentially useful as rootstock to reduce uptake and transport of Cd, especially in economically important cacao cultivars.

Genome sequence and effectorome of Moniliophthora perniciosa and Moniliophthora roreri subpopulations.

BACKGROUND: The hemibiotrophic pathogens Moniliophthora perniciosa (witches' broom disease) and Moniliophthora roreri (frosty pod rot disease) are among the most important pathogens of cacao. Moniliophthora perniciosa has a broad host range and infects a variety of meristematic tissues in cacao plants, whereas M. roreri infects only pods of Theobroma and Herrania genera. Comparative pathogenomics of these fungi is essential to understand Moniliophthora infection strategies, therefore the detection and in silico functional characterization of effector candidates are important steps to gain insight on their pathogenicity. RESULTS: Candidate secreted effector proteins repertoire were predicted using the genomes of five representative isolates of M. perniciosa subpopulations (three from cacao and two from solanaceous hosts), and one representative isolate of M. roreri from Peru. Many putative effectors candidates were identified in M. perniciosa: 157 and 134 in cacao isolates from Bahia, Brazil; 109 in cacao isolate from Ecuador, 92 and 80 in wild solanaceous isolates from Minas Gerais (Lobeira) and Bahia (Caiçara), Brazil; respectively. Moniliophthora roreri showed the highest number of effector candidates, a total of 243. A set of eight core effectors were shared among all Moniliophthora isolates, while others were shared either between the wild solanaceous isolates or among cacao isolates. Mostly, candidate effectors of M. perniciosa were shared among the isolates, whereas in M. roreri nearly 50% were exclusive to the specie. In addition, a large number of cell wall-degrading enzymes characteristic of hemibiotrophic fungi were found. From these, we highlighted the proteins involved in cell wall modification, an enzymatic arsenal that allows the plant pathogens to inhabit environments with oxidative stress, which promotes degradation of plant compounds and facilitates infection. CONCLUSIONS: The present work reports six genomes and provides a database of the putative effectorome of Moniliophthora, a first step towards the understanding of the functional basis of fungal pathogenicity.

Heavy metal accumulation in leaves and beans of cacao (Theobroma cacao L.) in major cacao growing regions in Peru.

Peru is one of the leading exporters of organic cacao beans in the world. However, the accumulation of heavy metals in cacao beans represents a problem for cocoa bean export and chocolate quality. The aim of this study was to investigate the distribution and accumulation of heavy metals in cacao leaves and cocoa beans in three major cacao growing regions of Peru. The study was conducted in cacao plantations of 10 to 15years old in three regions of Peru: North (Regions of Tumbes, Piura, Cajamarca, and Amazonas); Center (Regions of Huánuco and San Martin) and South (Junin and Cuzco). Samples of leaf and cacao beans were collected from 70 cacao plantations, and the nature of cacao clone or genotype sampled was recorded. The concentrations of heavy metals such as Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn in leaves and beans were determined using atomic absorption spectrophotometer. Overall, concentrations of heavy metals were below the critical limits; however, the presence of high levels of Cd in cacao grown in Amazonas, Piura, and Tumbes regions is of primary concern. Plantations of cacao with different cacao clones show differences in Cd accumulation both in leaves and cocoa beans. Therefore, it is promising to screen low Cd accumulator cacao genotypes for safe production of cacao on lightly to moderately Cd contaminated soils. Also, synergism between Zn and Cd present both in plant and soil suggests that Zn has a direct effect on Cd accumulation in cacao.

Changes in soil physical and chemical properties in long term improved natural and traditional agroforestry management systems of cacao genotypes in Peruvian Amazon.

Growing cacao (Theobroma cacao L.) in an agroforestry system generates a productive use of the land, preserves the best conditions for physical, chemical and biological properties of tropical soils, and plays an important role in improving cacao production and fertility of degraded tropical soils. The aim of this study was to evaluate the impact of two long term agroforestry systems of cacao management on soil physical and chemical properties in an area originally inhabited by 30 years old native secondary forest (SF). The two agroforestry systems adapted were: improved natural agroforestry system (INAS) where trees without economic value were selectively removed to provide 50% shade and improved traditional agroforestry system (ITAS) where all native trees were cut and burnt in the location. For evaluation of the changes of soil physical and chemical properties with time due to the imposed cacao management systems, plots of 10 cacao genotypes (ICS95, UF613, CCN51, ICT1112, ICT1026, ICT2162, ICT2171, ICT2142, H35, U30) and one plot with a spontaneous hybrid were selected. Soil samples were taken at 0-20, 20-40 and 40-60 cm depths before the installation of the management systems (2004), and then followed at two years intervals. Bulk density, porosity, field capacity and wilting point varied significantly during the years of assessment in the different soil depths and under the systems assessed. Soil pH, CEC, exchangeable Mg and sum of the bases were higher in the INAS than the ITAS. In both systems, SOM, Ext. P, K and Fe, exch. K, Mg and Al+H decreased with years of cultivation; these changes were more evident in the 0-20 cm soil depth. Overall improvement of SOM and soil nutrient status was much higher in the ITAS than INAS. The levels of physical and chemical properties of soil under cacao genotypes showed a marked difference in both systems.

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.