Mónica Blanco-Meneses

We present the draft genome sequences of Pseudomonas alliivorans LTM 13.1.2 (6.1 Mb) and Pseudomonas capsici LTM 78.3.2 (5.8 Mb) isolated from tomato plants in Costa Rica. These draft genomes provide important insights into the virulence factors and antibiotic resistance genes of these plant pathogens.

Tomato is one of the most important vegetable crops in Costa Rica, where favorable environmental conditions enabled year-round production but also promote bacterial diseases. In recent years, pith necrosis has been frequently observed; nevertheless, the causal agents remain unidentified in the country. This study evaluated bacteria associated with symptomatic plants collected in the Central Valley of Costa Rica. From 32 plants, 61 bacterial isolates were obtained, described morphologically, and characterized through basic biochemical tests. Partial sequencing of the 16S rRNA gene revealed diverse bacterial taxa, predominantly belonging to the genus Pseudomonas. Thirteen isolates were selected for pathogenicity assays, which confirmed variable virulence levels. Multilocus sequence analysis based on concatenated sequences of the 16S rRNA, gyrB, rpoD, and rpoB genes identified Pseudomonas alliivorans LTM 13.1.2, P. flavescens LTM 14.2.2, and P. capsici LTM 78.3.2 as causal agents of pith necrosis. Additionally, P. straminea LTM 78.2.1 and Cedecea sp. LTM 72.2.1 caused tissue discoloration. Whole-genome sequencing of the two most virulent isolates (LTM 13.1.2 and LTM 78.3.2) supported their taxonomic classification and revealed virulence-associated genes and biosynthetic clusters. This study represents the first report of these Pseudomonas species as tomato pathogens in Costa Rica and expands their known distribution and host ranges.

Tomato is one of the most widely cultivated and consumed vegetables in Costa Rica, where favorable environmental conditions enabled year-round production. However, these same conditions also promote the development of plant diseases. Diverse bacterial infections causing pith necrosis in tomato crops have become of frequent observation in recent years. Nevertheless, the causal agents in Costa Rica remain unidentified, and management strategies are poorly documented. This study evaluated the pathogenicity of bacterial isolates obtained from tomato plants collected in Costa Rica’s Central Valley. Isolates of Pseudomonas alliivorans, P. flavescens, and P. capsici were identified as causal agents of pith necrosis in tomatoes. Additionally, P. straminea and Cedecea sp. were associated with tissue discoloration. These findings provide valuable information to the tomato production sector in Costa Rica by identifying previously unreported pathogenic bacteria and expanding their known distribution and host ranges. These findings also demonstrate the ability of these isolates to cause pith necrosis.

Introduction. Costa Rica ranks among the world’s leading producers and exporters of pineapple; however, production and post-harvest handling are hampered due to phytosanitary problems caused by fungi of the genus Fusarium. Objective. To determine the pathogenicity of different Fusarium species associated with the pineapple plant and fruit, and their frequency in the main production areas of Costa Rica. Materials and methods. The experiments were conducted at the University of Costa Rica, San Jose, from 2015 to 2019. Pathogenicity tests were carried out using inoculation with six Fusarium species in bracts, peduncles, and fruits of pineapple, and were verified using Koch’s postulates. Through the collection of 120 isolates, the frequency of Fusarium species in the production areas was established. Results. All Fusarium species caused damage to bracts. The largest lesions were caused by F. oxysporum. In fruit, internal lesions were observed in the pulp with F. ananatum, and a correlation between lesion size and the concentration of inoculated conidia was found. In the peduncle and pulp, lesion diameter varied among Fusarium species. In terms of species frequency, all Fusarium species were present in the Huetar Norte region, and F. ananatum was more abundant in all production regions, except the Brunca region. Conclusions. All the species analyzed were pathogenic in the pineapple crop, in bracts, peduncles, and fruit. F. oxysporum and F. ananatum caused the greatest lesions. The presence in all pineapple-growing regions, the pathogenic potential, and the production of mycotoxins determine the importance of taking precise measures to control and prevent the spread of species from this genus.

Introduction. The identification and detection of microorganisms using molecular techniques has become a very helpful tool for the disease diagnosis and organisms present in crops. Pathogenic, non-pathogenic organisms, biological controllers and other microorganisms used as competitors, antagonists or mutualists can be isolated from agriculture, ornamental and forest crops. Objective. To identify the taxonomic level, by molecular techniques, pathogenic and non-pathogenic bacteria and yeast isolated in agriculture, ornamental and forest crops in Costa Rica, and preserve the material in a DNA bank. Materials and methods. Between 2009 and 2018, the Molecular Techniques Laboratory at the Plant Protection Research Center, Universidad de Costa Rica, received a total of 181 isolates of bacteria and yeast for detection by end-time and real-time PCR and identification through sequencing of specific regions. Results. Of the total samples, 94.2 % were analyzed by sequencing and 5.8 % by PCR. Using PCR, bacteria species were identified in rice, such as Burkholderia spp., Acidovorax avenae and Pseudomonas fuscovaginae. Through sequencing of the partial 16S region, 172 samples of bacterial species were identified, and five samples of yeast species with the ITS region of the 18S ribosomal RNA. Microorganisms isolated from eighteen species of agricultural, ornamental and forest plants were identified. The genera most identified were Pseudomonas, Bacillus and Enterobacter, and in the case of yeast, Candida, Pichia, and Wickerhamomyces. Conclusion. This research allowed the taxonomic identification of bacteria and yeast from crops in Costa Rica. In addition, a consultation input was developed, along with the possibility of future use of the microorganisms that are preserved at the DNA bank.

Celery (Apium graveolens var. dulce) is affected by several plant diseases, such as Fusarium oxysporum f. sp. apii (Foa). Four Foa races have been found in the US. The goals of this study were to determine which races are present in Costa Rica and to quantify the tolerance of the imported commercial cultivars of celery produced in the country. Isolates from 125 symptomatic celery plants from three different geographical locations were analyzed, 65 of which were selected for phylogenetic analysis. All isolates presented a short sequence of five nucleotides that differentiates Foa race 3 in the IGS rDNA region. Three different haplotypes closely related to race 3 were found, which were highly virulent, produced great losses, and affected all cultivars (resistant to races 2 and 4) of imported commercial celery. Additionally, five different cultivars of celery were evaluated against seven pathogen isolates identified as race 3 in greenhouse conditions. Two of the cultivars showed significantly less chlorosis, wilting, mortality, and higher fresh weight. Most of the Foa isolates significantly increased chlorosis, wilting, and mortality compared to non-inoculated control. Celery producers in Costa Rica lack access to seeds resistant to the Foa race 3 present in the country.

Introduction. Common beans (Phaseolus vulgaris L.) and peanuts (Arachis hypogaea L.) are legumes, widely consumed in developing countries. However, those grains could be contaminated with Aspergillus, a genus of fungus that includes mycotoxin-producing species. Objective. To identify the Aspergillus species that colonize the grains of common beans and peanuts in Costa Rica and to identify atoxigenic strains of A. flavus. Materials and methods. Eighty-three samples of common bean grains and nineteen samples of peanuts were collected during the years 2019 and 2020 in regions where those grains are produced in Costa Rica. All samples were used to isolate and identify Aspergillus species by partial sequences of ITS (internal transcribed spacer) and cmd (calmodulin) genes. Atoxigenic strains of A. flavus were also identified by chemical and molecular methods. Results. 46 % and 32 % of the collected common beans and peanuts samples, respectively, were contaminated with Aspergillus spp. In common beans, 85 isolates of Aspergillus were obtained, most of them belonging to A. flavus and A. niger species. Most of the A. flavus isolates were obtained from the Cabécar (fifteen isolates) and Nambí (seven isolates) common bean varieties, while A. niger was mostly isolated from the Cabécar variety (eleven isolates). In the case of peanuts, thirteen isolates were obtained, most of them identified as A. niger. No atoxigenic strains were found in peanuts, however, in common beans, five strains of A. flavus with this characteristic were found. Conclusions. The present study demonstrated the diversity of Aspergillus species that colonize the grains of common beans and peanuts in Costa Rica. Most of the species isolated are producers of mycotoxins that cause adverse effects on human health.

Introduction. The Laboratory of Molecular Techniques applied to Phytoprotection (LTM), Crop Protection Research Center, Universidad de Costa Rica; receives agricultural, ornamental, and forest crops for the identification of microorganisms using molecular techniques. Objective. To identify pathogenic and non-pathogenic fungi, oomycetes, and protozoa in agricultural, ornamental, and forest crops in Costa Rica by molecular methods. Materials and methods. Between 2009 and 2018, plant parts (root, stem, leaves, fruits) and other materials such as water, substrate, and soil were received. A total of 805 isolates were obtained for identification of microorganisms by DNA extraction techniques, end-time and real-time PCR and sequencing using molecular markers. Results. The sequencing allowed the identification of a total of 154 species of fungi, 6 oomycetes, and 1 protozoa. Among the fungi identified, genera such as Fusarium predominated with fourteen different species, followed by Colletotrichum and Aspergillus with eleven species identified for each one. In the oomycetes, species of the genus Phytophthora prevailed, and within the protozoa the genus Plasmodiophora. In addition, the scientific references supporting the presence of the microorganisms identified in each particular crop have been included in the information. Conclusion. It was possible to identify by molecular techniques pathogenic and non-pathogenic fungi, oomycetes and protozoa species isolated from different agricultural, ornamental and forest crops in Costa Rica.

Introducción. La pimienta es una especie de la familia de la Piperaceae, originaria de la Costa de Malabar, situada en Kerala al sur de la India. En Costa Rica, las principales plantaciones de pimienta se localizan desde Upala hasta Talamanca, e incluye toda la franja costera y la parte baja de Sarapiquí. Según estudios de factibilidad, el país cuenta con el potencial para producir pimienta con altos parámetros de calidad. Sin embargo, las enfermedades vasculares son una de las principales limitantes del cultivo.Objetivo. El objetivo de esta investigación fue el aislamiento e identificación molecular y morfológica de las especies de Fusarium asociadas al cultivo de la pimienta (Piper nigrum L.) en2 de las principales zonas productoras del país. Materiales y métodos. Se recolectaron treinta muestras de plantas enfermas de distintas fincas en las zonas de Sarapiquí y Guatuso, se describieron detalladamente los síntomas observados en el sistema radical y/o la base del tallo para cada una de las plantas recolectadas. Los veintiún aislamientos de Fusarium spp. obtenidos se identificaron molecularmente con la secuenciaparcial del gen Factor de Elongación 1α (EF-1α)y para la identificación morfológica se utilizaron los medios de cultivo PDA y Carnation Leaf-piece Agar (CLA). Resultados y conclusiones. Se confirmó la presencia de Fusarium oxysporum en las muestras provenientes de Sarapiquí, así como la presencia de Fusarium solani en ambas zonas muestreadas. Se reporta por primera vez, la presencia de las especies Fusarium concentricum y Fusarium proliferatum asociadas a la marchitez vascular.

Introducción. La producción de mostaza(Brassica rapa sp. pekinensis) se ve afectadapor la enfermedad causada por Plasmodiophorabrassicae. Objetivo. Determinar el efecto deAbono Orgánico, Microorganismos de Montaña y Fertibiol, para el control biológico de P. brassicae. Materiales y métodos. Los tratamientos diseñados se evaluaron de manera individual yen combinación, las variables evaluadas fueronpeso fresco y seco de la parte aérea y la radical,relación raíz-tallo, índice de severidad radical ypersistencia de esporas en el suelo (rtPCR). Serealizaron 2 ciclos de cultivo y 2 evaluaciones.Se utilizaron las pruebas de Kruscal-Wallis,Shapiro-Wilks y Levene, ANDEVA y la pruebaDGC (Di Rienzo, Guzmán y Casanoves) para la separación de medias. Resultados. Los tratamientos que contenían abono orgánico (solo o en mezcla) obtuvieron los mayores valores paralas variables de peso fresco y seco, tanto parala parte aérea (más de 300% respecto al testigo)como la radical. La relación raíz-tallo (R/T) fue menor y, por ende, más favorable en el tratamiento con abono orgánico, con un valor de 0,22. Para el índice de severidad, el tratamiento con MMpresentó el menor valor en las 2 evaluaciones, mientras que los tratamientos con abono orgánico mostraron el mayor valor, pero esto no afectó negativamente el rendimiento. Para la variablepersistencia, fue posible determinar que en eltestigo hubo una disminución en la cantidad deesporas germinativas con el avance del tiempoy que los tratamientos aplicados ocasionaron respuestas variables en cuanto a este comportamiento, mostrando ecuaciones con diferente pendiente y muy diversos coeficientes de variación. El tratamiento con MM presentó para la segunda evaluación la menor cantidad promedio de esporas y el menor índice de severidad radical. Conclusión. El abono orgánico mejoró el rendimiento de las plantas y la utilización deMM redujo la severidad y mostró potencial paradisminuir la presencia de esporas en el suelo.