Pepper (Capsicumspp.), a horticultural crop of wide distribution and easy handling that represents a key source of income for small and medium producers, sees its production threatened by phytopathogens such as Rhizoctonia solaniand Sclerotinia sclerotiorum, responsiblefor root rot and white mold, respectively. Trichodermaspp., fungi widely recognized for their antagonistic capacity, are effectively used as biological control agents due to mechanisms such as competition, antibiosis, and mycoparasitism. The objective ofthe study was to evaluate the production of metabolites by native isolates of Trichodermaspp. and determine their antifungal activity against R. solaniand S. sclerotiorum. All the organic extracts evaluated showed some degree of antifungal activity. In particular, the organic extract of the isolate Trichodermasp. FCQ14 completely inhibited the growth of both pathogens, while the extract of T. asperellumFCQ42 achieved 100% inhibition against S. sclerotiorum. In the extract of Trichodermasp. FCQ14, a major compound was identified whose spectroscopic properties match those of trichodermin, a trichothecene known to inhibit protein synthesis in eukaryotes. This metabolite is suggested to be responsible for the observed antifungal activity.

In agriculture, the presence of soil-borne phytopathogenic fungi represents a limiting factor that causes significant crop losses. In this context, Trichoderma brevicompactum represents a viable alternative to chemical fungicides, acting as an effective biological control agent for managing fungal diseases. The objective of this work was to characterize the antifungal activity of a native isolate of T. brevicompactum FCQ18, obtained from soil associated with tomato cultivation, against Macrophomina phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum. Direct confrontation assays showed inhibition of the growth of all evaluated phytopathogens. Additionally, antibiosis assays using cellophane membranes and dialysis indicated that the inhibitory effect is mainly due to low molecular weight molecules, as similar inhibition percentages were observed in both assays. Furthermore, a bio-guided isolation of the metabolite involved in antibiosis was performed. The organic extract of T. brevicompactum FCQ18 was fractionated using chromatographic techniques and its antifungal activity was evaluated, leading to the identification of a fraction with 100% inhibitory activity against the tested phytopathogens. Subsequent purification of the fraction with the highest antifungal activity allowed the identification of the principal compound responsible for the observed antibiosis, whose spectroscopic data corresponds to the mycotoxin trichodermin. The antifungal activity of trichodermin against the agriculturally important pathogens M. phaseolina and S. sclerotiorum is described for the first time.

Introduction. The charcoal rot fungus, Macrophomina phaseolina, is a ubiquitous necrotrophic phytopathogen that infecting soybean and other plant species. Despite its significant impact on crops, limited progress has been made in understanding the factors that influence phytotoxic molecule secretion by this phytopathogen. Objective. To evaluate the effect of soybean leaf infusion in the culture medium on the differential secretion of phytotoxic molecules of M. phaseolina. Materials and methods. The study was conducted between 2016 and 2023 at the Departamento de Química Biológica, Universidad Nacional de Asunción, Paraguay. Two fungal isolates were cultured in vitro using potato dextrose broth (PDB) and Czapek-Dox broth media, with or without soybean leaf infusion. Phytotoxic activity of secreted molecules was a using soybean leaf discs. The crude organic extract from the cultures was separated using chromatographic techniques, and purified metabolites were characterized by UHPLC-PDA/MS, HRMS (APGC), HRMSESI, 1HNMR and 13CNMR. Results. Molecules secreted by M. phaseolina FCQ11 cultured in infusion-enriched PDB induced the highest percentage of necrosis. Under these conditions, three differentially secreted metabolites were isolated and identified: (R)-mellein, (3R,4R)-hydroxymellein, and (-)-botryodiplodin. Conclusions. Soybean leaf infusion presence in M. phaseolina growth media stimulates phytotoxic metabolite production and alters the profile of secreted metabolites.

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.

Macrophomina phaseolina (Tassi) Goid. is a fungal pathogen that causes root and stem rot in several economically important crops. However, most of disease control strategies have shown limited effectiveness. Despite its impact on agriculture, molecular mechanisms involved in the interaction with host plant remains poorly understood. Nevertheless, it has been proven that fungal pathogens secrete a variety of proteins and metabolites to successfully infect their host plants. In this study, a proteomic analysis of proteins secreted by M. phaseolina in culture media supplemented with soybean leaf infusion was performed. A total of 250 proteins were identified with a predominance of hydrolytic enzymes. Plant cell wall degrading enzymes together peptidases were found, probably involved in the infection process. Predicted effector proteins were also found that could induce plant cell death or suppress plant immune response. Some of the putative effectors presented similarities to known fungal virulence factors. Expression analysis of ten selected protein-coding genes showed that these genes are induced during host tissue infection and suggested their participation in the infection process. The identification of secreted proteins of M. phaseolina could be used to improve the understanding of the biology and pathogenesis of this fungus. Although leaf infusion was able to induce changes at the proteome level, it is necessary to study the changes induced under conditions that mimic the natural infection process of the soil-borne pathogen M. phaseolina to identify virulence factors.

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.

The globally distributed necrotrophic fungus Macrophomina phaseolina is the causal agent of economically important crop diseases such as soybean charcoal rot. This fungus secretes a wide variety of proteins and metabolites that allow it to invade the plant and initiate the infection process. The role of fungi secreted proteins with hydrolytic activity in the infection process has been extensively studied; proteins without enzymatic activity could also play an important role in this process. The analysis of total proteins would allow to broaden the knowledge about this pathogen and establish more efficient strategies for its control. The objective of the present work was to evaluate three methods for the extraction of proteins secreted by M. phaseolina. The fungus was grown in potato dextrose broth (PDB) and Czapek-Dox (CZP) with and without soybean leaf supplementation. Proteins were extracted from the lyophilized filtrate of PDB medium using three extraction methods and analyzed by SDS-PAGE. The protein precipitation with trichloroacetic acid in acetone was selected because it showed a better resolution of the protein profile. The filtrate of M. phaseolina grown in PDB supplemented with soybean (MpPDBs) presented the highest yield of protein extraction of secreted proteins among all conditions evaluated. The protein profiles of PDB medium with and without supplementation showed seven differential bands, one of the specific, detected in MpPDBs. These results constitute a basis for studies on the implication of proteins secreted by the fungus in the infection process.

El hongo necrotrófico, mundialmente distribuido, Macrophomina phaseolina es el agente causal de enfermedades de cultivos de importancia económica como la podredumbre carbonosa de la soja. Este hongo secreta una amplia variedad de proteínas y metabolitos que le permiten invadir la planta e iniciar el proceso de infección. La contribución de las proteínas con actividad hidrolítica secretadas por este hongo en el proceso de infección ha sido ampliamente estudiada, las proteínas sin actividad enzimática también podrían poseer un papel importante en este proceso. El análisis de proteínas totales permitiría ampliar el conocimiento sobre este patógeno, y establecer estrategias más eficientes para su control. El objetivo del presente trabajo fue evaluar tres métodos de extracción de proteínas secretadas por M. phaseolina. El hongo fue crecido en medio líquido PDB y Czapek-Dox (CZP) con y sin suplemento de hojas de soja. Las proteínas fueron extraídas del filtrado liofilizado del medio PDB utilizando tres métodos de extracción, y analizadas mediante SDS-PAGE. Se seleccionó el método de precipitación de proteínas con ácido tricloroacético en acetona ya que mostró una mejor resolución del perfil proteico. El filtrado de M. phaseolina crecido en PDB suplementado con hojas de soja (MpPDBs) presentó un mayor rendimiento de extracción de proteínas secretadas entre todas las condiciones evaluadas. Los perfiles proteicos del medio PDB con y sin suplemento, presentaron siete bandas diferenciales, una de ellas específica, detectada en MpPDBs. Estos resultados constituyen una base para estudios sobre el papel de proteínas secretadas por el hongo en el proceso de infección.

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.

Los hongos del género Trichoderma son ampliamente utilizados como agentes de control biológico para controlar diversos fitopatógenos, sin embargo, muchas moléculas implicadas en el biocontrol aún se desconocen. Existen numerosos trabajos en los cuales se evalúan las moléculas secretadas por el hongo, entre las que se encuentran las proteínas. Para evaluar estas moléculas se deben extraer del sistema experimental en el cual crece el hongo, y la metodología de extracción, la cual es altamente dependiente de diversos factores, requiere el ajuste de las condiciones experimentales. Así, se planteó la evaluación de dos metodologías para el análisis del perfil de proteínas secretadas por aislados de Trichoderma spp. al medio de cultivo, partiendo de filtrado sin liofilizar y liofilizado. Los perfiles proteicos obtenidos a partir de material liofilizado, aplicando la precipitación de proteínas con ácido tricloacético en acetona seguida de una limpieza con metanol y cloroformo, generó un perfil adecuado para el análisis de las proteínas secretadas al medio de cultivo.

La pandemia producida por el coronavirus SARS-CoV-2 está causando estragos sanitarios y económicos en todo el mundo, obligando a la reorientación de recursos para disminuir el contagio y superar los problemas económicos.

El compromiso de la Universidad Nacional de Asunción (UNA), para con la investigación científica y tecnológica, se halla activo y en constante construcción. Con profunda firmeza y convicción se ha obrado en virtud del ideal que, el verdadero desarrollo del Paraguay, se logrará gracias al progreso científico-académico sistematizado mediante la investigación y la innovación.Lo sentenciado, se ha materializado en acciones concretas a través de novedosas iniciativas, publicaciones y galardones asignados. Solo por citar el último mes completo de trabajo, el 26 de noviembre se llevó a cabo el Webinar “Premio Nacional de Ciencia 2020”, evento en el cual reconocidos investigadores de la UNA presentaron sus artículos seleccionados tanto local como internacionalmente. Igualmente, se ha destacado la firma de la carta de compromiso entre la UNA y la Dirección Nacional de Propiedad Intelectual (DINAPI) con el objetivo de fijar las condiciones hacia el fortalecimiento de las actividades de desarrollo y formación de talentos humanos en materia de propiedad intelectual.Como se ha podido apreciar, la labor de la UNA en cuanto a la investigación es diversa, vanguardista y multidisciplinaria. En palabras de la Rectora, la Prof. Dra. Zully Vera de Molinas: “Fortalecer la investigación a través de alianzas estratégicas, en referencia a las gestiones con instituciones públicas y privadas, que apoyan el quehacer científico”.El presente número de la Revista Estudios e Investigaciones –UNA, editada por la Dirección General de Investigación Científica y Tecnológica (DGICT), se enmarca en el fin propuesto. Así, en la presente edición, se podrá observar novedosos trabajos de investigación que abordaron al Paraguay desde todas sus áreas del saber. Los mismos, son el reflejo de la rigurosidad científica y del esfuerzo diario por innovar científicamente.

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).

Lapachol acetate [systematic name: 3-(3-methylbut-2-enyl)-1,4-dioxonaphthalen-2-yl acetate], C17H16O4, was prepared using a modified high-yield procedure and its crystal structure is reported for the first time 80 years after its first synthesis. The full spectroscopic characterization of the molecule is reported. The molecular conformation shows little difference with other lapachol derivatives and lapachol itself. The packing is directed by intermolecular π–π and C—H...O interactions, as described by Hirshfeld surface analysis. The former interactions make the largest contributions to the total packing energy in a ratio of 2:1 with respect to the latter.

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.

A thorough study of the fermentation broth of three strains of Botrytis cinerea which were grown on a modified Czapek-Dox medium supplemented with 5 ppm copper sulphate, yielded five undescribed metabolites. These metabolites possessed a sesquiterpenoid (+)-4-epi-eremophil-9-ene carbon skeleton which was enantiomeric to that of the phytoalexin, capsidiol. The isolation of these metabolites when the fungus was stressed, suggests that they may be potential effectors used by B. cinerea to circumvent plant chemical defences against phytopathogenic fungi. The biosynthesis of these compounds has been studied using 2H and 13C labelled acetate.

SummaryTrichoderma arundinaceum (Ta37) and Botrytis cinerea (B05.10) produce the sesquiterpenoids harzianum A (HA) and botrydial (BOT), respectively. TaΔTri5, an HA non‐producer mutant, produces high levels of the polyketide compounds aspinolides (Asp) B and C. We analyzed the role of HA and Asp in the B. cinerea‐T. arundinaceum interaction, including changes in BOT production as well as transcriptomic changes of BcBOT genes involved in BOT biosynthesis, and also of genes associated with virulence and ergosterol biosynthesis. We found that exogenously added HA up‐regulated the expression of the BcBOT and all the virulence genes analyzed when B. cinerea was grown alone. However, a decrease in the amount of BOT and a down‐regulation of BcBOT gene expression was observed in the interaction zone of B05.10‐Ta37 dual cultures, compared to TaΔTri5. Thus, the confrontation with T. arundinaceum results in an up‐regulation of most of the B. cinerea genes involved in virulence yet the presence of T. arundinaceum secondary metabolites, HA and AspC, act separately and together to down‐regulate the B. cinerea genes analyzed. The present work emphasizes the existence of a chemical cross‐regulation between B. cinerea and T. arundinaceum and contributes to understanding how a biocontrol fungus and its prey interact with each other.