The COVID-19 pandemic has led to the search for new molecules with antiviral activity against SARS-CoV-2. The entry of the virus into the cell is one of the main targets for inhibiting SARS-CoV-2 infection. Natural products are an important source of new therapeutic alternatives against diseases. Pseudotyped viruses allow the study of SARS-CoV-2 viral entry inhibitors, and due to their simplicity, they allow the screening of a large number of antiviral candidates in Biosafety Level 2 facilities. We used pseudotyped HIV-1 with the D614G SARS-CoV-2 spike glycoprotein to test its ability to infect ACE2-expressing HEK 293T cells in the presence of diverse natural products, including 21 plant extracts, 7 essential oils, and 13 compounds from plants and fungi. The 50% cytotoxic concentration (CC₅₀) was evaluated using the resazurin method. From these analyses, we determined the inhibitory activity of the extract of Stachytarpheta cayennensis, which had a half-maximal inhibitory concentration (IC₅₀) of 91.65 µg/mL, a CC₅₀ of 693.5 µg/mL, and a selectivity index (SI) of 7.57, indicating its potential use as an inhibitor of SARS-CoV-2 entry. Moreover, our work indicates the usefulness of the pseudotyped-virus system in the screening of SARS-CoV-2 entry inhibitors.

Trichothecene mycotoxins are recognized as highly bioactive compounds that can be used in the design of new useful bioactive molecules. In Trichoderma brevicompactum, the first specific step in trichothecene biosynthesis is carried out by a terpene cyclase, trichodiene synthase, that catalyzes the conversion of farnesyl diphosphate to trichodiene and is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin, a trichothecene-type toxin, which is a valuable tool in preparing new molecules with a trichothecene skeleton. In this work, we developed the hemisynthesis of trichodermin and trichodermol derivatives in order to evaluate their antimicrobial and cytotoxic activities and to study the chemo-modulation of their bioactivity. Some derivatives with a short chain at the C-4 position displayed selective antimicrobial activity against Candida albicans and they showed MIC values similar to those displayed by trichodermin. It is important to highlight the cytotoxic selectivity observed for compounds 9, 13, and 15, which presented average IC₅₀ values of 2 μg/mL and were cytotoxic against tumorigenic cell line MCF-7 (breast carcinoma) and not against Fa2N4 (non-tumoral immortalized human hepatocytes).

Introducción. Macrophomina phaseolina es un hongo necrotrófico de difícil control. Hongos biocontroladores, como las especies del género Trichoderma, son una alternativa para cultivos afectados por este fitopatógeno. Objetivo. El objetivo del presente trabajo fue determinar la capacidad de antibiosis de Trichoderma arundinaceum, T. brevicompactum y T. harzianum, contra dos aislamientos de M. phaseolina. Materiales y métodos. El trabajo se efectuó entre octubre de 2015 y marzo del 2016. Se utilizaron tres cepas de referencia de Trichoderma: T. arundinaceum (IBT40837), T. brevicompactum (IBT40841) y T. harzianum T34 (CECT2413) y de M. phaseolina dos aislamientos (FCQ6 y FCQ9). Se realizaron ensayos de confrontación directa, antibiosis y análisis del perfil de proteínas y metabolitos secretados por Trichoderma. Resultados. Las especies de Trichoderma empleadas inhibieron de forma significativa el crecimiento de los dos aislamientos de M. phaseolina en los ensayos de confrontación directa, membrana de celofán y/o membrana de diálisis. En el ensayo de confrontación directa la mayor inhibición del crecimiento de los hongos se observó a las 96 h. El hongo aislado de sésamo (Sesamun indicum L., variedad Escoba blanca), permitió la evaluación de la actividad antifúngica de las moléculas de alto y bajo peso molecular incluso hasta las 120 h, donde T. arundinaceum mantuvo 100% de inhibición del crecimiento; en este mismo tiempo de exposición, los hongos T. brevicompactum y T. harzianum demostraron la importancia de las moléculas de alto peso molecular para el mantenimiento de la actividad antifúngica. Los resultados mostraron la diversidad de metabolitos secundarios y proteínas secretadas por las tres especies de Trichoderma. Conclusión. Este trabajo constituye la primera descripción de la actividad antifúngica de T. arundinaceum y T. brevicompactum contra M. phaseolina, y además, se destaca el potencial de hongos aislados de suelo nativo como alternativa biológica para el control de hongos fitopatógenos de importancia agrícola.

Stevia (Stevia rebaudiana [Bertoni] Bertoni) is a perennial plant originating in Paraguay. Stevia is primarily cultivated for the production of non-caloric sweeteners. In December 2018, wilted stevia cv. ‘PC4’ were recovered from two separate fields of 0.3 ha (24.66 S 56.46 W) and 0.5 ha (24.69 S 56.44 W), both with 3 years history of stevia production in San Estanislao County, San Pedro, Paraguay. The wilted plants were randomly distributed in beds covered with plastic mulch and a 30% disease incidence was recorded. Dark brown septate hyphae and microsclerotia were observed on stem bases and black necrotic roots of the wilted plants. Root and crown regions were washed, cut into 0.5 to 1.0 cm pieces, and then surface-disinfested with 0.6% NaOCl before placing them in Petri dishes containing acidified potato-dextrose-agar. Plates were incubated for one week at 25 ± 5°C under fluorescent light with a 12 h photoperiod yielding five isolates SP1PY, SP2PY, SP3PY, SP4PY and SP5PY with gray-black colonies without conidia but showing numerous microsclerotia. Twenty microsclerotia from pure cultures of five isolates were measured, with mean width 38.8 ± 4.7 µm and length 68.8 ± 15.5 µm. Fungal DNA was extracted from mycelia of five isolates for PCR amplification of the internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF1-α) using ITS4/ITS5 and EF1-728F/EF-2 primers (Machado et al. 2019). The resultant amplicons were sequenced at Eton Bioscience (Research Triangle Park, NC) and deposited in the NCBI GenBank database (ITS: MT645815, OM956150, OM956151, OM956152, OM956153; and TEF1-α: MT659121, OM959505, OM959506, OM959507, OM959508). Sequences were aligned with several isolates of Macrophomina spp. previously reported (Huda-Shakirah et al. 2019; Machado et al. 2019; Santos et al. 2020; Poudel et al. 2021) using ClustalW. Alignments (ITS and TEF-1α) were concatenated to generate a maximum likelihood tree using MEGA7. The novel isolates grouped into the M. euphorbiicola clade with 95% of bootstrap support. Stevia plants cv. ‘Katupyry’ were grown in 10 cm-diameter nursery bags containing autoclaved sandy soil and kept under greenhouse conditions (28 ± 5°C; 16 h photoperiod). Fifteen plants per isolate (n=75) were inoculated by adding 20 g of rice infested with M. euphorbiicola to each plant. Infested grains were distributed around the crown of the plant at a depth of 0.5 cm; non-infested rice was added to four control plants. Lower-stem lesions and microsclerotia of M. euphorbiicola developed on all inoculated plants. No lesions or microsclerotia were observed on control plants. The M. euphoribiicola fungus was re-isolated from inoculated stevia plants but not from the non-infested rice treated plants. Koch’s postulates were repeated twice with similar results. Previously, M. phaseolina was reported causing charcoal rot on stevia in Egypt (Hilal and Baiuomy 2000), and in North Carolina, USA (Koehler and Shew 2017). However, Paraguayan isolates grouped with isolates of M. euphorbiicola based on the combined sequences of the ITS and TEF-1α regions. Machado et al. (2019) reported M. euphorbiicola causing charcoal rot on castor bean (Ricinus communis) and bellyache bush (Jatropha gossypifolia) in Brazil, which borders northeast Paraguay, a major stevia production area. This pathogen has a significant impact on stevia production during hot, dry weather by reducing the number of harvestable plants and increasing replanting costs in perennial production systems.

There is a growing interest in the development of sustainable alternatives to the use of chemical pesticides for pest management in agricultural systems. This research aimed to isolate and characterize native strains of Trichoderma spp. from different soils of Paraguay using morphological and molecular criteria. We processed plant and soil samples from eight commercial farms distributed in different departments of Paraguay and isolated 14 monosporic isolates of Trichoderma spp., obtaining two isolates from the Department of Alto Paraná (FCQ36 and FCQ37), four isolates from Cordillera (FCQ42, FCQ43, FCQ44, and FCQ46), one isolate from Central (FCQ32), and seven isolates from Itapúa (FCQ13, FCQ16, FCQ18, FCQ19, FCQ21, FCQ23, and FCQ47). In addition, phylogenetic analyses using the ITS and tef1α loci were carried out. A better resolution of the tef1a gene than the ITS region was observed. Moreover, a third phylogenetic tree from the concatenated ITS and tef1α sequences matrix was generated, obtaining the same topology with higher bootstrap support values. Through this approach, we reported for the first time the presence of Trichoderma koningiopsis (FCQ19, FCQ36, and FCQ37), Trichoderma neokoningii (FCQ13), and Trichoderma asperellum (FCQ42, FCQ43, FCQ44, and FCQ46), Trichoderma brevicompactum (FCQ18 and FCQ21), and Trichoderma longibrachiatum (FCQ 47) in Paraguay. The Trichoderma species identified in this study can be used to develop effective biocontrol products for agricultural and industrial purposes in Paraguay.