Genomic and Pathogenicity Mechanisms of the Main Theobroma cacao L. Eukaryotic Pathogens: A Systematic Review.

A set of diseases caused by fungi and oomycetes are responsible for large losses in annual world cocoa production. Managing the impact caused by these diseases is very complex because a common solution has yet to be found for different pathogens. In this context, the systematic knowledge of Theobroma cacao L. pathogens' molecular characteristics may help researchers understand the possibilities and limitations of cocoa disease management strategies. This work systematically organized and summarized the main findings of omics studies of T. cacao eukaryotic pathogens, focusing on the plant-pathogen interaction and production dynamics. Using the PRISMA protocol and a semiautomated process, we selected papers from the Scopus and Web of Science databases and collected data from the selected papers. From the initial 3169 studies, 149 were selected. The first author's affiliations were mostly from two countries, Brazil (55%) and the USA (22%). The most frequent genera were Moniliophthora (105 studies), Phytophthora (59 studies) and Ceratocystis (13 studies). The systematic review database includes papers reporting the whole-genome sequence from six cocoa pathogens and evidence of some necrosis-inducing-like proteins, which are common in T. cacao pathogen genomes. This review contributes to the knowledge about T. cacao diseases, providing an integrated discussion of T. cacao pathogens' molecular characteristics, common mechanisms of pathogenicity and how this knowledge is produced worldwide.

Viability of Moniliophthora roreri on Cocoa Beans Under Microfermentation and Long-Term Survival on Carrier Materials.

The viability of Moniliophthora roreri inoculum was evaluated during the microfermentation process of diseased and healthy pulp-seed masses and on a range of carrier materials: aluminum, cloth, glass, paper, plastic, raffia, and rubber tire. Fungal survival was assessed before the microfermentation (0 h) and every 24 to 96 h by the growth of colonies in potato-dextrose-agar (PDA) and sporulation in seed shells. Colonies of M. roreri and sporulation on seed shells were observed from seeds not submitted to microfermentation. No growth was recovered from diseased cocoa beans after 48 h under the microfermentation. The viability of M. roreri spores recovered from carrier materials was evaluated at 7, 15, 30, 45, and 100 days after inoculation (DAI) by collecting spores and plating them on Sabouraud dextrose yeast extract agar amended with chloramphenicol (50 mg l1). The viability was determined by counting germinated and ungerminated spores under a light microscope (40×) after incubating in a moist chamber at 26 ± 2°C for 72 h. Spores maintained long-term viability on all tested carrier materials toward the end of the experiment (overall 26%) with significant differences (<0.05) among them. Maximum spore viability occurred at 7 and 15 DAI, with cloth and plastic carrier materials considered at high risk of acting as vehicles for the fungal spread. Mathematical models of spore viability over time were fit to the data using the Bayesian information criterion. Findings confirmed the importance of the fermentation process to hamper M. roreri growth and the potential of carrier materials for fungal dispersal.

Chemical Characterization of Trichoderma spp. Extracts with Antifungal Activity against Cocoa Pathogens.

Ecuador is one of the major cocoa producers worldwide, but its productivity has lately been affected by diseases. Endophytic biocontrol agents have been used to minimize pathogenic effects; however, compounds produced by endophytes are minimally understood. This work presents the chemical characterization of the Trichoderma species extracts that proved inhibition against cocoa pathogens. Solid-liquid extraction was performed as a partitioning method using medium with the fungal mycelia of Trichoderma reesei (C2A), Trichoderma sp. (C3A), Trichoderma harzianum (C4A), and Trichoderma spirale (C10) in ethyl acetate individually. The extract of T. spirale (C10) exhibited the growth inhibition (32.97-47.02%) of Moniliophthora perniciosa at 10 µg/mL, while a slight stimulation of Moniliophthora roreri was shown by the extracts of T. reesei (C2A) and T. harzianum (C4A) at higher concentrations. The inhibitory activity could be related to alkaloids, lactones, quinones, flavonoids, triterpenes, and sterols, as indicated by chemical screening and antifungal compounds, such as widdrol, ß-caryophyllene, tyrosol, butyl isobutyrate, sorbic acid, palmitic acid, palmitelaidic acid, linoleic acid, and oleic acid, which were identified by gas chromatography-mass spectrometry (GC-MS). The results showed that the extracts, particularly T. spirale (C10), have the potential as biocontrol agents against witches' broom disease; however, further studies are needed to confirm their effectiveness.

Cacao agroforestry systems beyond the stigmas: Biotic and abiotic stress incidence impact.

Low technological knowledge in production chains, global climate change, and misinformation are concrete threats to food security. In addition, these combined threats also trigger ecological instability in megadiverse areas of the world, especially in some cacao-producing countries in South America, where this crop plays an important socio-economic role, even being used to replace illicit crops. Accordingly, the use of agroforestry systems approaches has emerged as a good alternative to maintain productivity, add high-value commodities to producers, and provide important ecosystem services for sustainable agriculture. However, limitations associated with the competition for resources between the species composing the system, and the higher incidence of some diseases, have led many producers to abandon this strategy, opting for monoculture. In this review, we seek to gather the main information available in the literature, aiming to answer the question: what is the real scientific evidence that supports the benefits and harms of adopting agroforestry systems in cacao production? We seek to make critical scrutiny of the possible negative effects of certain associations of the agroforestry system with biotic and abiotic stress in cacao. Here, we review the possible competition for light and nutrients and discuss the main characteristics to be sought in cacao genotypes to optimize these inter-specific relationships. In addition, we review the research advances that show the behavior of the main cacao diseases (Witch's broom disease, frosty pod rot, black pod rot) in models of agroforestry systems contrasted with monoculture, as well as the optimization of agronomic practices to reduce some of these stresses. This compendium, therefore, sheds light on a major gap in establishing truly sustainable agriculture, which has been treated much more from the perspective of negative stigma than from the real technological advantages that can be combined to the benefit of a balanced ecosystem with generating income for farmers.

Development of a Method for Detecting and Estimating Moniliophthora roreri Spore Loads Based on Spore Traps and qPCR.

Frosty pod rot, caused by Moniliophthora roreri, is the most damaging disease of cacao in Latin America and, to better comprehend its epidemiology, we must understand its dissemination and proliferation. However, we do not know how M. roreri spores loads fluctuate in time and space due to the lack of a reliable technique to quantify M. roreri spores in the fields. Therefore, we developed a method that relies on spore traps and qPCR to detect and quantify M. roreri spore loads. This study demonstrated that the qPCR protocol can detect down to 0.025 ng of M. roreri DNA and quantify between 0.006 ng and 60 ng. Moreover, it demonstrated that qPCR protocol can detect and quantify DNA extracted from spore suspension and spore traps containing at least 2.9 × 104 M. roreri spores. However, the variability of the estimates for spore samples was high. Finally, we described a spore-trap device designed to carry spore traps in the field. The qPCR protocol and spore-trap device here developed will help in the understanding of the M. roreri dissemination patterns since they can be used to assess the environmental loads of M. roreri spore in cacao fields.

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.

Factors Affecting the Dynamics of Frosty Pod Rot in the Main Cocoa Areas of Santander State, Colombia.

Frosty pod rot (FPR) caused by Moniliophthora roreri is the primary disease affecting cacao production in the major producing countries of the Americas and is one of the major threats to cacao worldwide. The incidence of FPR on clones with different levels of resistance was investigated in four localities of Santander State, Colombia, between July 2013 and May 2015. Dynamics of diseased pods were modeled using boosted regression trees, a machine learning technique that allows regressions to be performed without prior statistical assumptions. The results suggested that FPR epidemics varied according to plot location, clone, weeks of observation, and total pods produced. Dynamics in the phenology of pods had an effect on the epidemics, and this dynamic could partially explain the difference in resistance among clones. Although not total, partial resistance of ICS 95 was confirmed. An important wilt effect was observed, particularly in the resistant clones; consequently, differences in harvested pods were not significant among clones. Pod stripping remains a good practice for the management of the disease and this practice could also have an effect on the pod dynamics and wilt phenomenon.

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.

ANÁLISIS ESPACIAL DE LA INCIDENCIA DE ENFERMEDADES EN DIFERENTES GENOTIPOS DE CACAO (Theobroma cacao L.) EN EL YOPAL (CASANARE), COLOMBIA / Spatial Analysis of Diseases Incidence in Different Cocoa Genotypes (Theobroma cacao L.) in Yopal (Casanare), Colombia

RESUMEN Las enfermedades "pudrición negra del fruto" y "moniliasis" son reportadas como limitantes en plantaciones de cacao en Colombia. Fueron evaluados los patrones espaciales de incidencia para estas dos enfermedades en un cultivo de cacao durante un monitoreo de trece semanas en campo. Semivariogramas e interpolación kriging fueron usados para analizar el comportamiento espacial de "pudrición negra del fruto", los estadísticos Join Count e Indice de Moran se emplearon para estudiar el comportamiento espacial de "moniliasis". La incidencia de "pudrición negra del fruto" presentó estructura de dependencia espacial de acuerdo a los semivariogramas durante el tiempo de evaluación, mientras que la autocorrelación espacial de sitios de infección de "moniliasis" se distribuyó de forma aleatoria entre las plantas de cacao según el Indice de Moran. El rango de dispersión estimado de "pudrición negra del fruto" varió entre 21,74 y 40,23 m y los mapas kriging evidenciaron el progreso espacio-temporal de la enfermedad durante las semanas evaluadas. Las condiciones experimentales dadas y la aproximación de los análisis espaciales, contribuyeron al entendimiento de la dinámica espacial de dos enfermedades limitantes en tres clones de cacao en la Orinoquía Colombiana.

ABSTRACT "Black pod" and "frosty pod" diseases are reported as limiting in cocoa plantations in Colombia. Spatial patterns of incidence for these two diseases were evaluated during a thirteen weeks long period of monitoring in the field. Semivariogram and kriging interpolation were used to analyze the spatial behavior of "black pod" and Join Count and Moran's Index statistics were used to analyze the spatial behavior of "frosty pod". The incidence of "Black pod" has a spatial dependence structure according to semivariogramas during the evaluation time, while spatial autocorrelation of "moniliasis" infection sites was randomly distribuited among cocoa plants according to the Moran's Index. The estimated dispersion range of "Black pod" was from 21.74 to 40.23 m y the kriging maps showed the spatio-temporal progression of the disease during the evaluated weeks. The experimental conditions given and spatial analysis approach, contributed to the understanding of the spatial dynamics of two limiting diseases in three clones of cocoa in the Colombian Orinoquia.

Tree spacing impacts the individual incidence of Moniliophthora roreri disease in cacao agroforests.

BACKGROUND: Using conventional pesticides in crop protection has raised serious environmental concerns and there is therefore a need for integrated pest management (IPM) methods. In this paper, we found that the spacing of trees can impact disease, which could result in a reduction in pesticide applications and may act as a potential IPM method. We studied Frosty Pod Rot (FPR) in 20 cacao agroforests in Costa Rica (Upala region). RESULTS: Using a generalized linear mixed model, we analyzed the impact of the neighborhood composition and distance from a studied cacao individual on its individual FPR incidence. We found that the number of cacao tree neighbors in a radius of 3.7 m and the number of fruit trees in a radius of 4.3 m had a significant negative influence on the incidence of FPR on individual cacao trees. Moreover, cacao tree neighbors had the most significant local influence compared to the neighborhood of other taller categories such as fruit or forest trees. CONCLUSION: The mechanisms involved are related to the barrier effect, due to the effectiveness of the cacao tree's architecture as an efficient barrier against FPR spore dispersal. This paper provides new insights into optimization of the spatial environment around each host as an original IPM method. © 2017 Society of Chemical Industry.