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.

Effect of Cocoa Bean Shell Addition on Metabolite Profile and Antioxidant Activity of Herbal Infusions.

Cocoa bean shell (CBS) is a by-product with aromatic characteristics that can enhance the aroma and bioactivity of herbal infusions. This study was aimed to determine the effect of the addition of cocoa bean shell on the metabolite profile and antioxidant activity of infusions made with Ilex guayusa and Vernonanthura patens and their mixtures. Metabolite profile was analyzed by gas chromatography-mass spectrometry combined with multivariate analysis. Total polyphenol content and flavonoids were determined by the Folin-Ciocalteu method and by the flavonoid-AlCl3 complex, respectively. Antioxidant activities were measured by the decolorization assay of the 2,2-diphenyl-1-picrylhydrazyl radical and the ferric reducing antioxidant power. The results revealed that the addition of CBS increases the content of phenolic acids in the infusions (caffeic acid, 4-hydroxybenzoic acid, and pyrocatechol). Nonetheless, the antioxidant activity of the infusions decreased with the addition of CBS (16.21 to 2.74 TEAC). Carboxylic acids and derivatives, major compounds present in the infusions prepared with V. patens, were the metabolites that showed the highest correlation with the antioxidant activity. This study suggests that the infusions made with CBS present a profile of metabolites different from the infusions of I. guayusa, V. patens, and their mixtures.

Evaluation of the sensitivity and synergistic effect of Trichoderma reesei and mancozeb to inhibit under in vitro conditions the growth of Fusarium oxysporum.

Trichoderma is a saprophytic, soil-borne fungus with a worldwide distribution that has been extensively studied due to their capacity to synthesize secondary metabolites with antimicrobial activity, parasitize other fungi and directly interact with plant roots, inducing resistance to disease and tolerance to abiotic stresses. Fusarium wilt caused by the soil-inhabiting fungus Fusarium oxysporum is considered one of the most important diseases that affect banana cultivars. Currently, more environmentally friendly alternatives to control this disease are being proposed, these strategies include the application of low doses of synthetic fungicides and the use of biocontrol agents such as Trichoderma or Xylaria. Thus, this study aimed to evaluate under in vitro conditions the synergistic effect of the biological control agent T. reesei C2A combined with low doses of mancozeb to inhibit the mycelial growth of F. oxysporum F1. To perform the synergistic essays, 0.1 mg/mL of mancozeb was suspended in PDA plates, then plugs of T. ressei C2A were placed at the center of the Petri dishes, the plates were incubated for 7 days at 28°C. Results showed that the mycoparasitic capacity of the biocontrol strain to inhibit the mycelial growth of F. oxysporum F1 was enhanced approximately 36% compared to the control plates. Although these results are promising, future studies under greenhouse and field conditions are necessary to corroborate the effectiveness of this approach.

Comparative analysis uncovers the limitations of current molecular detection methods for Fusarium oxysporum f. sp. cubense race 4 strains.

Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4) is threatening banana production worldwide. Despite quarantine efforts, the pathogen continues to spread; thus, early diagnosis plays an essential role for the proper execution of contingency plans. Here, we assess the accuracy of four PCR-based molecular methods described in the literature for the identification and detection of race 4 strains, including Subtropical (Foc STR4) and Tropical Race 4 causing Fusarium wilt of banana. We screened a total of 302 isolates using these four markers, and performed phylogenetic analyses, Vegetative Compatibility Group (VCG) testing, sequence comparison, and pathogenicity tests for selected isolates. Our results show that three out of the four markers tested are not reliable for identification of Foc STR4 and TR4, as DNA from isolates from Ecuador, pathogenic and nonpathogenic to banana, obtained from different banana cultivars, displayed cross-reaction with these methods; that is, false positives can occur during the diagnostic process for race 4. Phylogenetic analyses, VCG testing, sequence comparison, and pathogenicity tests suggest the presence of non-target F. oxysporum isolates that share genomic regions with pathogenic strains but lack true pathogenicity to banana. The findings of this work are of foremost importance for international regulatory agencies performing surveillance tests in pathogen-free areas using the current diagnostic methods. We suggest the use of a genetic locus possibly related to virulence, previously identified by T-DNA, and amplified with primers W2987F/ W2987R, for diagnosis of Foc TR4 as the most reliable alternative. We urge the adoption of a more holistic view in the study of F. oxysporum as a plant pathogen that considers the biology and diversity of the species for the development of better diagnostic tools.

Caracterización bioquímica de hojas de clones de Theobroma cacao y su relación con los tricomas / Biochemical characterization of leaves in Theobroma cacao clones and its relation with trichomes

Theobroma cacao L. es un cultivo de gran importancia por sus posibles aplicaciones en la farmacia y la agricultura. Contiene metabolitos que resultan de gran interés por sus funciones biológicas naturales en la respuesta de la planta a los diferentes tipos de estrés, tanto bióticos como abióticos. La presencia, tipo y concentración de los metabolitos (primarios y secundarios) pueden variar de acuerdo al genotipo analizado y se pueden acumular en estructuras asociadas a la arquitectura de la hoja como son los tricomas. El objetivo de este trabajo fue caracterizar 26 clones del banco de germoplasma de Padrón, INIA-Miranda, Estado Miranda, Venezuela y estudiar la relación existente entre los indicadores bioquímicos y el número de tricomas. Se determinó el contenido de compuestos fenólicos, proteínas solubles, prolina libre, carbohidratos totales, ligninas, el número de tricomas y la relación existente entre ellos. Los resultados mostraron que en el espacio bidimensional que explica el 46,68% de la variabilidad total, se definieron cinco grupos homogéneos: diez clones se asociaron con el máximo gradiente de prolina, carbohidratos totales y contenido de ligninas, nueve con alto contenido de proteínas y fenoles y siete con mayor número de tricomas en hojas. Se comprobó que el número de tricomas no se relacionó con la concentración de compuestos fenólicos. Los clones de cacao del tipo forastero mostraron mayor concentración de fenoles totales y carbohidratos, en tanto que los trinitarios presentaron mayor concentración de proteínas y prolina.

Theobroma cacao L. is a crop of major importance for their potential applications in pharmacy and agriculture. It contains metabolites that are of great interest for its natural biological functions in plant response to different types of stress, both biotic and abiotic. The presence, type and concentration of both primary and secondary metabolites may vary depending on the genotype analyzed and these compounds can accumulate in the architecture associated structures such as leaf trichomes. The aim of this study was to characterize 26 clones of the germplasm bank of Padrón, INIA-Miranda, Miranda State, Venezuela and study the relationship between biochemical indicators assessed and the number of trichomes. The content of phenolic compounds, soluble protein, free proline, total carbohydrates, lignins, the number of trichomes and the relationship between all traits was determined. The results showed that in the two dimensional space explained 46.68% of the variability, five homogeneous groups were defined: 10 clones were associated with the maximum gradient of proline, total carbohydrate and lignin content. Nine clones were associated with high content of phenols and a group of seven clones with the highest number of trichomes on leaves. It was found that the number of trichomes was not related to the concentration of phenolic compounds. The clones of Forastero cocoa type showed higher concentration of total phenols and carbohydrates, meanwhile the Trinitario type presented higher concentration of proteins and prolina.