Population structure of Moniliophthora perniciosa in the main cacao producing departments of Colombia

The witches’ broom (Moniliophthora perniciosa) is considered as one of the main threats for cacao production and, consequently, for chocolate production worldwide.. In this work, the genetic diversity and population structure of M. perniciosa were analyzed for 59 isolates collected in five departments of Colombia and using 10 microsatellite markers. Analyses revealed 35 multilocus genotypes (MLGs) and clonal populations structure according to linkage disequilibrium analysis. One of the objectives of this study was to determine whether populations were differentiated by geographic origin or T. cacao host genotype. Analysis of molecular variance, Discriminant Analysis of Principal Components (DAPC) and Bruvo genetic distance suggested that the genetic structure was driven by geographic origin and not by T. cacao genotype. The results of this study were consistent with previous findings obtained in other cocoa producing countries. Important insights were discussed regarding the dispersal patterns of the pathogen in Colombia and the genetic change of its populations due to different environmental conditions.

Mitochondrial Genomics of Six Cacao Pathogens From the Basidiomycete Family Marasmiaceae

Thread blight disease has recently been described as an emerging disease on cacao (Theobroma cacao) in Ghana. In Ghana, thread blight disease is caused by multiple species of the Marasmiaceae family: Marasmius tenuissimus, M. crinis-equi, M. palmivorus, and Marasmiellus scandens. Interestingly, two additional members of the Marasmiaceae; Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches’ broom disease), are major pathogens of cacao in the Western hemisphere. It is important to accurately characterize the genetic relationships among these economically important species in support of their disease management. We used data from Illumina NGS-based genome sequencing efforts to study the mitochondrial genomes (mitogenomes) of the four cacao thread blight associated pathogens from Ghana and compared them with published mitogenomes of Mon. roreri and Mon. perniciosa. There is a remarkable interspecies variation in mitogenome size within the six cacao-associated Marasmiaceae species, ranging from 43,121 to 109,103 bp. The differences in genome lengths are primarily due to the number and lengths of introns, differences in intergenic space, and differences in the size and numbers of unidentified ORFs (uORF). Among seven M. tenuissimus mitogenomes sequenced, there is variation in size and sequence pointing to divergent evolution patterns within the species. The intronic regions show a high degree of sequence variation compared to the conserved sequences of the 14 core genes. The intronic ORFs identified, regardless of species, encode GIY-YIG or LAGLIDADG domain-containing homing endonuclease genes. Phylogenetic relationships using the 14 core proteins largely mimic the phylogenetic relationships observed in gene order patterns, grouping M. tenuissimus with M. crinis-equi, and M. palmivorus with Mon. roreri and Mon. perniciosa, leaving Mar. scandens as an outlier. The results from this study provide evidence of independent expansion/contraction events and sequence diversification in each species and establish a foundation for further exploration of the evolutionary trajectory of the fungi in Marasmiaceae family.

Caracterización de un jardín clonal de cacao (Theobroma cacao L.) en la amazonía peruana

La variabilidad genética de clones de cacao (<em>Theobroma cacao</em> L.), que existe en el jardín clonal de la Estación Experimental Pichanaqui, de procedencia nacional e internacional, plantea la necesidad de realizar una caracterización morfológica a nivel de frutos, con descriptores morfológicos y escalas para cada tipo de tejido colectado, que usaron como auxiliares las siglas, los colores y formas que permitieron diferenciar las características de cada individuo. Las variables evaluadas fueron número de frutos por planta, peso de mazorcas maduras, numero de granos por mazorca, peso de almendras seca por mazorca, peso de 10 semillas secas, presencia de plagas como la Moniliasis (<em>Moniliophthora roreri</em>), Escoba de bruja (<em>Moniliophthora perniciosa</em>), Mancha parda (<em>Phytophthora palmivora</em>), Perforador (<em>Carmenta foraseminis</em>), Chinche amarillo (<em>Monalonium dissimulatum</em>) y calidad organoléptica en pulpa fresca. Además, se evaluaron variables económicas como el ingreso neto del clon por hectárea. Los resultados indicaron que los mejores clones por su productividad, tolerancia media a plagas y enfermedades, rentabilidad y sabor fueron: ICS 1, ICS 60, SCA 6, ICS 39, y TSH 565. En conclusión, se debería fomentar la siembra de estos clones en los productores de la zona a fin de mejorar su competitividad en el mercado local y nacional.

Moniliophthora perniciosa, the Causal Agent of Cacao Witches’ Broom Disease Is Killed in vitro by Saccharomyces cerevisiae and Wickerhamomyces anomalus Yeasts

Cacao plantations from South America have been afflicted with the severe fungal disease known as Witches’ Broom Disease (WBD), caused by the basidiomycete Moniliophthora perniciosa. Yeasts are increasingly recognized as good fungal biocides, although their application is still mostly restricted to the postharvest control of plant and fruit decay. Their possible utilization in the field, in a preharvest phase, is nevertheless promising, particularly if the strains are locally adapted and evolved and if they belong to species considered safe for man and the environment. In this work, a group of yeast strains originating from sugarcane-based fermentative processes in Brazil, the cacao-producing country where the disease is most severe, were tested for their ability to antagonize M. perniciosa in vitro. Wickerhamomyces anomalus LBCM1105 and Saccharomyces cerevisiae strains LBCM1112 from spontaneous fermentations used to produce cachaça, and PE2 widely used in Brazil in the industrial production of bioethanol, efficiently antagonized six strains of M. perniciosa, originating from several South American countries. The two fastest growing fungal strains, both originating from Brazil, were further used to assess the mechanisms underlying the yeasts’ antagonism. Yeasts were able to inhibit fungal growth and kill the fungus at three different temperatures, under starvation, at different culture stages, or using an inoculum from old yeast cultures. Moreover, SEM analysis revealed that W. anomalus and S. cerevisiae PE2 cluster and adhere to the hyphae, push their surface, and fuse to them, ultimately draining the cells. This behavior concurs with that classified as necrotrophic parasitism/mycoparasitism. In particular, W. anomalus within the adhered clusters appear to be ligated to each other through roundish groups of fimbriae-like structures filled with bundles of microtubule-sized formations, which appear to close after cells detach, leaving a scar. SEM also revealed the formation of tube-like structures apparently connecting yeast to hypha. This evidence suggests W. anomalus cells form a network of yeast cells connecting with each other and with hyphae, supporting a possible cooperative collective killing and feeding strategy. The present results provide an initial step toward the formulation of a new eco-friendly and effective alternative for controlling cacao WBD using live yeast biocides.

Theobroma Grandiflorum Breeding Optimization Based on Repeatability, Stability and Adaptability Information

The cultivation of Theobroma grandiflorum in the Brazilian Amazon is mainly conducted by family farmers who use a range of different management straegies. Thus, breeding programs of the species must address the challenge of developing cultivars that are adapted to and stable in a variety of cultivation environments. In this context, this study aimed to estimate the optimum number of harvests for genetic selection of T. grandiflorum progenies and identify the most promising ones in terms of productivity, stability, and adaptability. The trials were implemented in three environments, using a randomized complete block design, with 25 full-sib progenies, five replications, and three plants per plot. The traits mean number of fruits/plant, mean fruit production/plant, and rate of infection with witches’ broom (Moniliophthora perniciosa) were evaluated over 11 harvests. The Restricted Maximum Likelihood/Best Linear Unbiased Prediction (REML/BLUP) mixed model method was used to estimate genetic parameters and predict genetic values, which were then applied to assess stability and adaptability. The results show that there is genetic variability among the studied T. grandiflorum progenies and that accurate genetic selection aiming at recombination is effective after three harvests, for recombination, or eleven harvests for identification of recommended progenies. Six progenies were selected that met the requirements for productivity, stability, and adaptability to different cultivation environments. These results can be used to optimize and advance T. grandiflorum breeding programs.

Theobroma grandiflorum breeding optimization based on repeatability, stability and adaptability information

The cultivation of Theobroma grandiflorum in the Brazilian Amazon is mainly conducted by family farmers who use a range of different management strategies. Thus, breeding programs of the species must address the challenge of developing cultivars that are adapted to and stable in a variety of cultivation environments. In this context, this study aimed to estimate the optimum number of harvests for genetic selection of T. grandiflorum progenies and identify the most promising ones in terms of productivity, stability, and adaptability. The trials were implemented in three environments, using a randomized complete block design, with 25 full-sib progenies, five replications, and three plants per plot. The traits mean number of fruits/plant, mean fruit production/plant, and rate of infection with witches’ broom (Moniliophthora perniciosa) were evaluated over 11 harvests. The Restricted Maximum Likelihood/Best Linear Unbiased Prediction (REML/BLUP) mixed model method was used to estimate genetic parameters and predict genetic values, which were then applied to assess stability and adaptability. The results show that there is genetic variability among the studied T. grandiflorum progenies and that accurate genetic selection aiming at recombination is effective after three harvests, for recombination, or eleven harvests for identification of recommended progenies. Six progenies were selected that met the requirements for productivity, stability, and adaptability to different cultivation environments. These results can be used to optimize and advance T. grandiflorum breeding programs.

Evaluación in vitro de 14 medios de cultivo sobre el crecimiento micelial de Moniliophthora perniciosa (Stahel ) Aime & Phillips-Mora

El cultivo de cacao en el Perú se va incrementando por la ampliación de áreas productivas y mejora de los rendimientos, sin embargo, la presencia de las tres principales enfermedades (mazorca parda, escoba de bruja y moniliasis) afectan significativamente los rendimientos en cacaotales con bajo nivel de mantenimiento, teniendo como objetivo determinar los medios de cultivo que permitan mayores crecimientos radiales de Moniliophthora perniciosa, por lo que se hace necesario contar con estrategias viables de manejo y control. Las pruebas in vitro de control químico y biológico requieren que los aislados presentan crecimientos miceliales apropiados, por lo que se planteó la comparación de 14 medios de cultivo sólidos no convencionales con 6 repeticiones. Los tratamientos fueron maltacervezneg, frejolcaraota, achiote, cascmazorca, arroz, maizamarillo, frugos, mbamilaceo, papa, melazacacao, vegetales, masabourd, escobaverde y gelatina, sobre los cuales se sembró basidiocarpos de M. perniciosa proveniente de Cacaopampa-Santa Ana, pegados en el centro de la tapa de las placas petri de 90 mm y se incubaron por 12 días a 25°C de temperatura. Los medios de cultivo que permitieron mayores crecimientos radiales de M. perniciosa fueron medio malta proveniente de cerveza negra, frejol caraota, achiote y cáscara de cacao con 90.00, 87.08, 82.58 y 66.75 mm respectivamente y el medio papa (PDA) presentó un desempeño 50% menor que el medio Maltacerveneg en el crecimiento radial de cultivo monospórico de M. perniciosa.

Adaptive evolution of Moniliophthora PR-1 proteins towards its pathogenic lifestyle

Background Plant pathogenesis related-1 (PR-1) proteins belong to the CAP superfamily and have been characterized as markers of induced defense against pathogens. Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that respectively cause the witches’ broom disease and frosty pod rot in Theobroma cacao. Interestingly, a large number of plant PR-1-like genes are present in the genomes of both species and many are up-regulated during the biotrophic interaction. In this study, we investigated the evolution of PR-1 proteins from 22 genomes of Moniliophthora isolates and 16 other Agaricales species, performing genomic investigation, phylogenetic reconstruction, positive selection search and gene expression analysis. Results Phylogenetic analysis revealed conserved PR-1 genes (PR-1a, b, d, j), shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora (PR-1f, g, h, i), as well as in recent specialization cases within M. perniciosa biotypes (PR-1c, k, l) and M. roreri (PR-1n). PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host–pathogen arms race. Furthermore, although previous work showed that MpPR-1 can detoxify plant antifungal compounds in yeast, we found that in the presence of eugenol M. perniciosa differentially expresses only MpPR-1e, k, d, of which two are not linked to pathogenicity, suggesting that detoxification might not be the main function of most MpPR-1. Conclusions Based on analyses of genomic and expression data, we provided evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Additionally, we also discuss how fungal PR-1 proteins could have adapted from basal conserved functions to possible roles in fungal pathogenesis.