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.

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.