Fungal Diseases in Yerba Mate: Status and Management Strategies.

Yerba mate (Ilex paraguariensis St. Hil.), with its health benefits and socioeconomic significance, plays a crucial role in Argentina and other South American countries like Brazil and Paraguay. Its cultivation in the Province of Misiones (Argentina) supports various sectors, contributes to regional development, and provides employment opportunities. However, the transition from extractive practices to monoculture, accompanied with increased demand, has led to phytosanitary challenges. Imbalanced native microbiota, disease development, and pathogen dispersion have become prevalent issues. Understanding the known pathogens associated with yerba mate plants is crucial for developing effective agricultural strategies. The primary objective of this study is to synthesise current knowledge on prevalent fungal diseases in yerba mate cultivation, as well as to provide agricultural management recommendations for effective disease control. Fungal diseases can cause significant damage to different parts of the plant, resulting in economic losses. The proximity of neighbouring plantations to yerba mate crops may contribute to the cross-contamination of pathogens, emphasizing the need for comprehensive epidemiology and accurate diagnosis. Multiple fungal genera have been reported to cause pathologies in yerba mate. Among the fungi causing foliar diseases are Ceratobasidium niltonsouzanum, Cylindrocladium spathulatum, Pseudocercospora mate, Asterina sphaerelloides, Colletotrichum gloeosporioides aff var. yerbae, and Phyllosticta sp. Caulinary diseases are caused by Alternaria sp., Phoma sp., Colletotrichum sp., and Ceratocystis fimbriata. Regarding root rot, the genera Rhizoctonia sp., Pythium sp., Fusarium sp., and Rosellinia sp. have been reported. Proper crop management practices and monitoring are essential for effective disease control. To reduce reliance on chemical compounds, the use of biocontrol agents like Trichoderma sp. has shown promise in regulating phytopathogenic fungi populations. Continued research is vital to preserve the yerba mate industry and ensure its long-term viability while minimizing environmental impact.

De novo genome assembly of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4, two plant growth-promoting bacteria isolated from Ilex paraguariensis St. Hil. (yerba mate).

Plant growth-promoting bacteria (PGPB) are a heterogeneous group of bacteria that can exert beneficial effects on plant growth directly or indirectly by different mechanisms. PGPB-based inoculant formulation has been used to replace chemical fertilizers and pesticides. In our previous studies, two endophytic endospore-forming bacteria identified as Bacillus altitudinis were isolated from roots of Ilex paraguariensis St. Hil. seedlings and selected for their plant growth-promoting (PGP) properties shown in vitro and in vivo. The purposes of this work were to assemble the genomes of B. altitudinis 19RS3 and T5S-T4, using different assemblers available for Windows and Linux and to select the best assembly for each strain. Both genomes were also automatically annotated to detect PGP genes and compare sequences with other genomes reported. Library construction and draft genome sequencing were performed by Macrogen services. Raw reads were filtered using the Trimmomatic tool. Genomes were assembled using SPAdes, ABySS, Velvet, and SOAPdenovo2 assemblers for Linux, and Geneious and CLC Genomics Workbench assemblers for Windows. Assembly evaluation was done by the QUAST tool. The parameters evaluated were the number of contigs ≥ 500 bp and ≥ 1000 bp, the length of the longest contig, and the N50 value. For genome annotation PROKKA, RAST, and KAAS tools were used. The best assembly for both genomes was obtained using Velvet. The B. altitudinis 19RS3 genome was assembled into 15 contigs with an N50 value of 1,943,801 bp. The B. altitudinis T5S-T4 genome was assembled into 24 contigs with an N50 of 344,151 bp. Both genomes comprise several genes related to PGP mechanisms, such as those for nitrogen fixation, iron metabolism, phosphate metabolism, and auxin biosynthesis. The results obtained offer the basis for a better understanding of B. altitudinis 19RS3 and T5S-T4 and make them promissory for bioinoculant development.

Occurrence and diversity of Aspergillus in commercial yerba mate elaborated for the Brazilian beverage 'chimarrão'.

Dried leaves and stems of Ilex paraguariensis St. Hil. (yerba mate) are used to make a popular beverage in some countries of South America, commonly known as "chimarrão". The present study was designed to evaluate the occurrence of toxigenic Aspergillus in yerba mate in order to define the mycotoxin risk associated with this foodstuff. All samples tested were positive for fungal contamination, and the fungal load per sample ranged from 2.0 × 102 to 1.6 × 104 CFU/g. Aspergillus section Nigri was found in all samples and represented 76.5% of the total fungi isolated. Aspergillus section Circumdati, Aspergillus section Flavi and Aspergillus section Cremei were found at low frequencies. Thirteen different Aspergillus species were identified. The most common species found was A. luchuensis, which does not produce any harmful toxin for humans. A. niger, A. welwitschiae, A. flavus and A. novoparasiticus, all potentially toxigenic species, were found only in small quantities. The A. niger and A. welwitschiae strains were cultured to test for ochratoxin A and fumonisin B2 production. Only one strain producing ochratoxin A was found, but approximately 29% of the strains were positive for fumonisin B2. The A. flavus and A. novoparasiticus strains were tested for aflatoxins production, and 63% were positive. A. pallidofulvus, recently assigned to A. section Circumdati, was reported for the first time in herbs. All A. pallidofulvus strains analyzed in this study were negative for ochratoxin A production. In conclusion, A. section Nigri occurs with high frequency in yerba mate, and A. luchuensis is the predominant species. Although toxigenic species were found in this herb, the incidence was low.

Avaliação microbiológica de cuias de chimarrão na cidade de Palmares do Sul, RS / Microbiological evaluation of cuimors of chimarrão in the city of Palmares do Sul, RS

O chimarrão é uma bebida típica da região sul do Brasil, que é feita a partir da infusão de água quente com a erva mate (Ilex paraguariensis), necessitando de dois utensílios para ser ingerido: a cuia e a bomba. A cuia é um utensílio feito de porongo (Lagenaria vulgaris) que tem uma textura porosa semelhante à madeira, podendo acomodar micro-organismos e favorecer o seu crescimento. O objetivo deste trabalho foi verificar a presença de microorganismos em cuias após a sua utilização em rodas de chimarrão. Foram coletadas 16 (dezesseis) amostras utilizando um swab estéril, para contagem total de mesófilos. Também foi aplicado um questionário sobre a higienização da cuia. Todas as amostras analisadas apresentaram crescimento microbiano elevado indicando que os procedimentos de higiene adotados não são suficientes para evitar contaminação.

Diversity of endophytic fungal and bacterial communities in Ilex paraguariensis grown under field conditions.

The composition and diversity of the endophytic community associated with yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. Fungal and bacterial diversity did not show significant differences between organ age. The highest fungal diversity was registered during fall season and the lowest in winter. Bacterial diversity was higher in stems and increased from summer to winter, in contrast with leaves, which decreased. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Actinobacteria represented 57.5 % of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. Other bacteria frequently found were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. Nitrogen fixation and phosphate solubilization activity, ACC deaminase production and antagonism against plant fungal pathogens were assayed in endophytic bacterial strains. In the case of fungi, strains of Trichoderma, Penicillium and Aspergillus were assayed for antagonism against pathogenic Fusarium sp. All microbial isolates assayed showed at least one growth promoting activity. Strains of Bacillus, Pantoea, Curtobacterium, Methylobacterium, Brevundimonas and Paenibacillus had at least two growth-promoting activities, and Bacillus, Paenibacillus and the three endophytic fungi showed high antagonistic activity against Fusarium sp. In this work we have made a wide study of the culturable endophytic community within yerba mate plants and found that several microbial isolates could be considered as potential inoculants useful for improving yerba mate production.

Ocurrencia natural de hongos entomopatógenos en suelos cultivados con yerba mate (Ilex paraguariensis St. Hil.) en Misiones, Argentina / Natural ocurrence of entomopathogenic fungi in soils cultivated with Paraguay tea (Ilex paraguariensis St. Hil.) in Misiones, Argentina

Este trabajo tuvo por objetivo aislar, identificar y caracterizar morfológicamente hongos entomopatógenos presentes en suelos cultivados con yerba mate (Ilex paraguariensis St. Hil.). Se realizó una prospección de hongos entomopatógenos nativos a partir de 40 muestras de suelos cultivados con yerba mate ubicados en la provincia de Misiones, República Argentina, desde mayo de 2008 hasta junio de 2010. Las muestras de suelo se obtuvieron en cercanías de plantas de yerba mate, se aplicó la técnica de dilución seriada en medios de cultivos selectivos. Después de obtenidos los cultivos puros, los hongos fueron caracterizados macroscópica y microscópicamente, y se los identificó mediante el uso de claves taxonómicas específicas. Se aislaron 29 cepas fúngicas pertenecientes a las especies Beauveria bassiana (n = 17), Metarhizium anisopliae (n = 2) y Purpureocillium lilacinum (n = 10)

This study aimed to morphologically isolate, identify and characterize entomopathogenic fungi present in soils cultivated with Paraguay tea (Ilex paraguariensis). A survey of native entomopathogenic fungi was conducted from 40 soil samples grown with Paraguay tea in the province of Misiones, Argentina, from May 2008 to June 2010. The soil dilution plate methodology on selective culture media was used to isolate microorganisms. Taxonomic identification was performed using macroscopic and microscopic characters and specific keys. Twenty nine strains, belonging to the species Beauveria bassiana (n = 17), Metarhizium anisopliae (n = 2) and Purpureocillium lilacinum (n = 10) were isolated and identified

[Isolation of Aspergillus section Nigri strains in yerba mate in Posadas (Misiones, Argentina) and evaluation of their ochratoxigenic potential]. / Aislamiento de cepas de Aspergillus sección Nigri en la yerba mate comercializada en Posadas (Misiones, Argentina) y evaluación de su potencial ocratoxigénico.

The objectives of the present work were to investigate the isolation frequency of genus Aspergillus in canchada yerba mate (YMCH) and elaborated yerba mate (YME) (Ilex paraguariensis) and the proportion of section Nigri isolates, as well as to determine ochratoxin A production by Aspergillus species section Nigri. Three hundred twenty eight Aspergillus strains from 20 samples of YMCH and 1306 Aspergillus strains from 36 samples of YME were isolated; of the total, 279 from the first group of strains and 1215 from the latter group, belonged to section Nigri. For the detection of ochratoxin A production, the strains were cultivated on Czapeck yeast extract agar and the toxin was detected by thin layer chromatography under UV light. Uniserate species predominance was observed in the 1494 strains of Aspergillus section Nigri obtained (Aspergillus japonicus var. japonicus and Aspergillus japonicus var. aculeatus), whereas none of the strains analysed showed ochratoxin A production in vitro at the detection level of the methodology employed.

Aislamiento de cepas de Aspergillus sección Nigri en la yerba mate comercializada en Posadas (Misiones, Argentina) y evaluación de su potencial ocratoxigénico / [Isolation of Aspergillus section Nigri strains in yerba mate in Posadas (Misiones, Argentina) and evaluation of their ochratoxigenic potential].

The objectives of the present work were to investigate the isolation frequency of genus Aspergillus in canchada yerba mate (YMCH) and elaborated yerba mate (YME) (Ilex paraguariensis) and the proportion of section Nigri isolates, as well as to determine ochratoxin A production by Aspergillus species section Nigri. Three hundred twenty eight Aspergillus strains from 20 samples of YMCH and 1306 Aspergillus strains from 36 samples of YME were isolated; of the total, 279 from the first group of strains and 1215 from the latter group, belonged to section Nigri. For the detection of ochratoxin A production, the strains were cultivated on Czapeck yeast extract agar and the toxin was detected by thin layer chromatography under UV light. Uniserate species predominance was observed in the 1494 strains of Aspergillus section Nigri obtained (Aspergillus japonicus var. japonicus and Aspergillus japonicus var. aculeatus), whereas none of the strains analysed showed ochratoxin A production in vitro at the detection level of the methodology employed.

Evaluación del Perfil Micológico de la Yerba Mate durante el proceso productivo y producto final en establecimientos yerbateros. Paraguay 2012 / Mycological Profile Evaluation of Mate for the production process and final product establishments herbalists. Paraguay 2012

Introducción: La yerba mate se utiliza para la preparación de distintos tipos de bebidas de consumo masivo en el Paraguay, tales como el mate, tereré, cocido, etc. La presencia de agentes micóticos del género aflatoxigénico en la misma no siempre se relaciona con niveles dañinos de aflatoxinas, capaces de provocar hepatopatías pero sí implica un riesgo alimentario significativo al consumir los productos contaminados. Objetivo: Evaluar el perfil micológico de la yerba mate durante el proceso productivo y el producto final. Materiales y Métodos: Diseño observacional descriptivo de corte trasversal que incluyo 68 muestras recolectadas en diferentes etapas del proceso de producción de la yerba mate elaborada y compuesta con el agregado de hierbas medicinales: hojas in natura de yerba mate (n=7), yerba mate después de la molienda (n=22), yerba mate elaborada (n=22) y yerba mate compuesta (n=17); durante los meses de setiembre a diciembre del 2011 en 6 establecimientos yerbateros seleccionados dentro del territorio nacional. Se utilizaron los valores de referencia de las Normas Paraguayas NP 3500193 (1800 UFC/g) para yerba mate elaborada y NP 3500201 (5000 UFC/g) para yerba mate compuesta. Resultados: En la yerba mate elaborada, 10/22 de las muestras presentan valores superiores a los criterios establecidos en la Norma Paraguaya; 11/17 de las muestras de yerba mate compuesta no cumplen con los criterios establecidos en la Norma Paraguaya para yerba mate compuesta. Conclusión: En la yerba mate elaborada y en la compuesta se encontraron elevados niveles de recuento de mohos y levaduras. El consumo de yerba mate contaminada implica un riesgo alimentario convirtiéndose en un problema potencial de salud pública. Palabras claves: yerba mate, mohos, levaduras, recuento fúngico, salud pública.

Introduction: The Ilex paraguariensis (yerba mate) is used for the preparation of different kind beverages of high consumption in Paraguay such as mate, tereré, cocido, etc. (typical beverages of the country). The presence of fungal agents of aflatoxigenic gender in the beverage is not always related to harmful levels of aflatoxin, capable of causing liver diseases, but involves a significant food related risk through the consumption of contaminated products. Objective: To assess the mycological profile of the Ilex paraguariensis (yerba mate) during the production process and final product. Materials and Methods: observational crosssectional descriptive design that included 68 samples collected at different stages of the production process of Ilex paraguariensis ("yerba mate elaborada" and "yerba mate compuesta" with the addition of medicinal herbs) : leaves of yerba mate in nature (n = 7), yerba mate after grinding (n = 22), "yerba mate elaborada" (n =22) and "yerba mate compuesta" (n = 17); during the months of September to December of the year 2011 in 6 selected herbalist establishments within the national territory. Standard values of Paraguayan Normative NP 3500193 (1800 CFU / g) for "yerba mate elaborada" and NP 3,500,201 (5000 CFU / g) for "yerba mate compuesta" were used as reference. Results: In the "Yerba mate elaborada", 10 out of 22 samples show higher values than that of the Paraguayan Normative , 11 out of 17 of the "Yerba mate compuesta" samples do not meet the Paraguayan Normative criteria. Conclusion: In the "Yerba mate elaborada" and "Yerba mate compuesta" high levels of yeast and mold counts were found. The consumption of contaminated Ilex paraguariensis (yerba mate) implies a high food related risk, becoming a potential problem for human health. Keywords: yerba mate, molds, yeasts, fungal counts, health

Aislamiento de cepas de Aspergillus sección Nigri en la yerba mate comercializada en Posadas (Misiones, Argentina) y evaluación de su potencial ocratoxigénico. / [Isolation of Aspergillus section Nigri strains in yerba mate in Posadas (Misiones, Argentina) and evaluation of their ochratoxigenic potential].

The objectives of the present work were to investigate the isolation frequency of genus Aspergillus in canchada yerba mate (YMCH) and elaborated yerba mate (YME) (Ilex paraguariensis) and the proportion of section Nigri isolates, as well as to determine ochratoxin A production by Aspergillus species section Nigri. Three hundred twenty eight Aspergillus strains from 20 samples of YMCH and 1306 Aspergillus strains from 36 samples of YME were isolated; of the total, 279 from the first group of strains and 1215 from the latter group, belonged to section Nigri. For the detection of ochratoxin A production, the strains were cultivated on Czapeck yeast extract agar and the toxin was detected by thin layer chromatography under UV light. Uniserate species predominance was observed in the 1494 strains of Aspergillus section Nigri obtained (Aspergillus japonicus var. japonicus and Aspergillus japonicus var. aculeatus), whereas none of the strains analysed showed ochratoxin A production in vitro at the detection level of the methodology employed.

Drinking yerba mate infusion: a potential risk factor for invasive fungal diseases?

Yerba mate (Ilex paraguariensis) infusion is a very popular drink in South America. Although several studies have evaluated the potential for fungal contamination in foodstuff, very few investigations have been conducted with yerba mate samples. In order to evaluate for the presence of potentially pathogenic fungi, here we studied 8 brands of yerba mate commercially available in Southern Brazil. Fungal survival in adverse conditions such as gastric pH was determined by incubating samples at pH 1.5. Because hot water is generally used to prepare yerba mate infusion, the effect of several temperatures on fungal growth was also investigated. All but 1 yerba mate brand showed substantial fungal growth, in the range of <10­4900 colony-forming units per gram. Some of these fungi were able to survive extreme variations in pH and temperature. Because of the potential for yerba mate to carry pathogenic fungi, immunocompromised patients may be at risk of acquiring invasive fungal diseases by drinking yerba mate infusion.

Avaliação das características microbiológicas e físico-químicas da erva-mate produzida na região do Vale do Taquari, RS / Evaluation of microbiological and physical-chemical properties of yerba mate produced in the Vale of Taquari, RS

A erva-mate é um produto de grande importância econômica e social para a região Sul do Brasil. Atualmente, visando atingir novos mercados, cada vez mais exigentes, as indústrias têm buscado a padronização da qualidade do seu produto ao longo de todo o processo. Com base nisso, o objetivo desse trabalho foi determinar as características microbiológicas e físico-químicas de uma erva-mate, em diferentes etapas do processamento. Foram realizadas 10 coletas de cinco etapas diferentes do processo produtivo de uma ervateira da região do Vale do Taquari, Rio Grande do Sul (Brasil) e as amostras analisadas quanto aos teores de umidade, cinzas insolúveis, extrato aquoso, cafeína e as contagens microbiológicas de coliformes totais e termotolerantes, bolores e leveduras e Salmonella sp. Os resultados obtidos demonstraram que as características físico-químicas e microbiológicas variaram significamente ao longo das etapas do processamento industrial, principalmente, após sapeco ou secagem. Os dados microbiológicos e físico-químicos gerados nessa pesquisa servirão como ponto de partida para a padronização da qualidade em todo o processamento da indústria ervateira.

Plano de amostragem presença-ausência para Oligonychus yothersi (McGregor) (Acari: Tetranychidae) na cultura da erva-mate / Presence-absence sampling plan for Oligonychus yothersi (McGregor) (Acari: Tetranychidae) on mate-tea orchard

O ataque de ácaros na cultura da erva-mate tem freqüentemente provocado perdas pela queda precoce das folhas. Assim, há necessidade de se monitorar a população desses artrópodes e adotar estratégias de manejo. O objetivo deste trabalho foi avaliar a confiabilidade da amostragem presença-ausência para Oligonychus yothersi (McGregor) na erva-mate. O trabalho foi realizado em um erval comercial, em Cascavel, PR, de abril de 1999 a julho de 2000. Coletas quinzenais de 240 folhas foram feitas em diferentes partes de 10 plantas escolhidas ao acaso. A contagem dos ácaros nas folhas foi realizada em laboratório com auxílio de microscópio estereoscópico. Foi determinado o padrão de agregação através dos coeficientes a e b da lei da potência de Taylor; a proporção de folhas infestadas e o número de amostras requeridas foram estimados através de modelo matemático. O. yothersi apresentou distribuição agregada. A proporção de folhas infestadas calculada pelo modelo matemático mostrou ser um parâmetro confiável para estimar a densidade populacional do ácaro. O número de amostras requeridas foi pequeno, viabilizando a aplicação prática do método de amostragem presença-ausência para O. yothersi na cultura da erva-mate.

Mite infestations to the culture of mate-tea frequently causes losses by the premature fall of the leaves. So, it is necessary to monitor the population of these arthropods, and to adopt management strategies for their control. The objective of this research was to evaluate the trustworthiness of presence-absence sampling for Oligonychus yothersi (McGregor) on mate-tea orchard. This study was conducted in Cascavel, Paraná State, from April 2001 to July 2000, in a mate-tea tree commercial plantation. Biweekly sampling of 240 leaves were collected in different parts of 10 plants, and the number of mites was counted. The aggregation pattern was determined through the coefficients a and b of Taylor's power law. The proportion of infested leaves and the number of required samples were estimated through mathematical model. The mite O. yothersi presented aggregate distribution. The proportion of infested leaves calculated by means of mathematical model showed to be a trustworthy parameter to estimate the population density of the mite. The number of required samples was small, making feasible the practical application of the presence-absence sampling method for O. yothersi in the culture of mate-tea.

[Detection of fungal metabolites showing toxic activity through Artemia salina bioassay]. / Detección de metabolitos fúngicos con actividad tóxica mediante bioensayo sobre Artemia salina.

The aim of this study was to detect toxic metabolites from fungi contaminating food and medicinal herbs by applying the toxicity assay to Artemia salina. According to toxicity percentages, the extracts were classified as nontoxic (NT), slightly toxic (ST), toxic (T) and highly toxic (HT). Those classified as T and HT were assayed for mycotoxins. Only 6 out of 71 strains were found to be T (8.5%) for A. salina. Penicillium brevicompactum Dierckx, isolated from sausages, was found to be HT, mainly due to the presence of ochratoxin A and two other unidentified metabolites.

Avaliação microbiológica e microscópica de erva-mate (Ilex paraguariensis St. Hil.) / Microbiological and microscopical evaluation of Ilex paraguariensis

O objetivo deste trabalho foi determinar os parâmetros microbiológicos e microscópicos de amostras de erva-mate (Ilex paraguariensis St. Hil.) . A matéria-prima utilizada foi a erva-mate cancheada folha verde e folha tostada. Os parâmetros microbiológicos analisados foram os exigidos pela Organização Mundial da Saúde e pela Resolução-RDC n.12, de 2 de janeiro de 2001, do Ministério da Saúde, e constituídos pela contagem de bolores e leveduras (UFC/g), contagem de bactérias mesófilas (UFC/g), contagem de coliformes a 35°C (NMP/g), contagem de coliformes a 45°C (Escherichia coli) (NMP/g) e pesquisa de Salmonella sp/25g. Os parâmetros microscópicos foram realizados conforme a Association of Official Analytical Chemists. Os resultados obtidos demonstraram a genuidade das amostras analisadas e sua conformidade com a legislação vigente, revelando, também, o manejo correto da erva-mate na colheita, durante o transporte, processamento e armazenamento, mantendo a qualidade do produto para o consumidor.