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INTRODUCTION
Mate is a popular non-alcoholic hot beverage consumed in South America. Ilex paraguariensis, also called yerba mate, is the main feedstock for this drink. Its consumption is cultural, linked to history and tradition, dating back mid-1500s by native people called Guaranis (Zanin & Meyer, 2018). Its health benefits, such as stimulating effect, anti-inflammatory and antioxidant activity, are well known (Gullón et al., 2018). Brazil is a significant producer and consumer of yerba mate, with 390 tons produced in 2017 (IBGE, 2018). In the state of Rio Grande do Sul, the per capita consumption is 9kg/year (IBRAMATE, 2018). Due to its high consumption, large amounts of waste are generated and its improper disposal may cause environmental issues and harm wildlife (Neves et al., 2022).
Despite being a natural organic compound, the waste generated by the consumption of mate does not have an adequate destination yet. Research has been made exploring its potential applications as biodegradable films (Arrieta et al., 2018), as a source of antioxidant phenolic compounds (Gullón et al., 2018), and also in the reinforcement of epoxy-based composite materials (Neves et al., 2022). However, its large-scale reuse is not yet a common reality, and most of the waste generated goes to landfills or home gardens (Gullón et al., 2018). This is of concern because pathogens may be present in this waste, potentially causing harm to the environment.
Several studies have been done in the last decades exploring the relationship between fungi and yerba mate. Some works describe the pathogenicity of Fusarium to yerba mate as the main causal agent of root rotting disease (Mezzomo et al., 2021; Mezzomo et al., 2019), and besides Fusarium, some authors attribute this disease to Pythium and Rhizoctonia (Poletto et al., 2015; Poletto et al., 2007). Studies evaluating the phytosanitary quality of yerba mate seeds have revealed the presence of several fungi, including pathogens such as Fusarium (Oliveira et al., 2015; Souza et al., 2019; Souza et al., 2020). Surveys analyzing fungal diseases in yerba mate leaves have also been carried out (Bilenki-Junior et al., 2021; Brito et al., 2021; López et al., 2020; Pimentel et al., 2006), as well as in consumer-ready mate samples (Bernardi et al., 2005; Borges et al., 2002). However, in the case of yerba mate waste, there is still no research on which fungi are associated with it.
In this manner, considering the social-economic importance of this industry to South America and especially considering the way this waste is disposed of in the environment, it is important to know which fungi are associated with yerba mate waste. This can help prevent pathogenic fungi, not only from yerba mate but also from other crops, from being mistakenly discarded into the environment. From this, new control protocols for the use of yerba mate substrate can be provided. Thus, the objective of this work is to evaluate which genera of fungi occur on yerba mate waste.
MATERIAL AND METHODS
Collection of yerba mate residue. The samples were collected during the autumn, winter, and spring seasons of 2020. The collection was carried out for a period of 30 days in each season, being collected by five consumers, totaling 150 samples at the end of each period. The total number of samples analyzed in the experiment was 450. After the consumption of yerba mate in the preparation of mate drink, the residue generated was collected in 500 ml canning jars (approximately 160 g of residue per glass). Each glass was partially closed to create an aerobic environment, constituting a humid chamber. There was no addition of water other than the one previously contained in the substrate. The samples were maintained at ambient temperature.
Evaluation of fungi found in yerba mate residue. After 30 days of collection, the samples were evaluated for the occurrence of fungi, and this observation continued for another month. One part of the filamentous fungi, found only on the exposed surface of each glass container, was removed with the aid of tweezers, and a permanent slide was prepared for the microscopic study. This was performed using a Karl Zeiss Axiostar Plus microscope. The fungi were photographed, and pertinent bibliography was used to identify the species found, especially at the genus level using Putzke & Putzke (2008) and specific literature on each genus found. Lists of fungi for yerba mate were prepared by reviewing the literature on the subject.
Statistical analyses. The data were normalized. Afterward, an analysis of variance test (ANOVA) was applied to verify if there was a significant difference between the occurrence and abundance of fungi during the seasons. Then a cluster analysis was applied, with Euclidean distance measure for abundance. Cluster analysis was performed among all seasons and for the abundance and occurrence of fungi within each season.
RESULTS AND DISCUSSION
Twenty-two genera of fungi associated with discarded mate herb residues were found. A literature review on each of the fungal genera found in yerba mate residue was conducted, in order to indicate which associations of these genera have been previously described for yerba mate (Table 1).
Table 1 Filamentous fungi found in yerba mate (Ilex paraguariensis St. Hil) residuals.
| Species | Kind of relationship | Reference |
|---|---|---|
| Allomyces sp. | Saprophytic. First occurrence in association with Ilex paraguariensis. | |
| Alternaria sp. | Found on seeds | Palmucci et al. (2011) |
| Arthrobotrys sp. | Spores found on flowers and fruits | Souza et al. (2019) |
| Aspergillus sp. | Found on seeds, saprophytic and comaninanting of processed mate. | Lin et al. (2018) |
| Aureobasidium sp. | Saprophytic and pathogenic | Bergottini et al. (2017) |
| Bipolaris sp. | Found on seeds | Palmucci et al. (2011); Thines & Choi (2016) |
| Cercospora sp. | Pathogenic of twigs | Oliveira et al. (2015) |
| Chaetomium sp. | Found on seeds, saprophytic | Lin et al. (2018) |
| Colletotrichum sp. | Pathogenic causing anthracnose and black-paint | Souza et al. (2019); Dixon (2021) |
| Curvularia sp. | On seeds, phytopathogenic | Lin et al. (2018) |
| Fusarium sp. | Pathogenic of leaves, twigs and seeds | Lin et al. (2018) |
| Mortierella sp. | Saprophytic. Symbiotic with roots | Poletto et al. (2015) |
| Mucor sp. | Saprophytic. Contaminating of processed mate | Lin et al. (2018) |
| Nigrospora sp. | Found on seeds | Souza et al. (2019) |
| Penicillium sp. | Saprophytic. On seeds and contaminating processed mate | Lin et al. (2018) |
| Peronospora sp. | Pathogenic. First occurrence in association with Ilex paraguariensis.. | Paula et al. (2018) |
| Pilobolus sp. | Saprophytic. First occurrence in association with Ilex paraguariensis. | - |
| Pythium sp. | Pathogenic. Associated to root rot. | Poletto et al. (2015) |
| Rhizoctonia sp. | Pathogenic | Lin et al. (2018) |
| Rhizopus sp. | Saprophytic. Found in seeds and processed mate | Lin et al. (2018) |
| Thanatephorus sp. | Pathogenic to roots | Dixon (2021) |
| Verticillium sp. | Saprophytic | Souza et al. (2019) |
The genera initially present in the residue consisted of the Zygomycota molds, specifically Rhizopus spp. and Mucor spp., which were found to be the primary colonizers and dominant for at least 19 days during the experiment, covering 100% of the exposed surface. These species are non-pathogenic and are mainly associated with fruit rots (Poletto et al., 2015). Subsequently, other filamentous fungi, particularly pathogenic species from the genera Alternaria, Curvularia, Fusarium, Verticillium, and Pythium (Poletto et al., 2015), begin to appear, sometimes still in association with Zygomycota, but in a final stage with reduced mycelial coverage. The initial colonization of Mucor and Rhizopus in the substrate was also observed in studies of fungal succession in litter decomposition, where they were considered as the primary colonizers, while late colonizers such as Aspergillus, Penicillium, and Chaetomium were found in other studies of fungal succession in yerba mate (Kumar et al., 2020).
The last stage of fungal succession in this waste leads to a fully darkened and highly moist residue, indicating the dominance of yeast-like fungi and bacteria, and is characterized by the strong odor emanating from some samples in the final stages of the evaluation. This phase can be termed as the maturation phase, where an increase in bacterial proliferation occurs, leading to a decline in the fungal population, possibly due to microbial antagonisms, antibiosis, high moisture content, and slightly acidic pH of the compound (Zhou et al., 2016). This finding is consistent with other observations of an "obvious succession" of fungal communities during the decomposition process, with the filamentous fungi appearing in the initial stages (Tian et al., 2017).
It is noteworthy that the proliferation of insects and nematodes in the residual material was observed in some glass containers and on microscope slides, limiting the development of filamentous fungi as they can serve as food source on them, reducing the observation time, which is one of the indicators that the substrate is reaching its maturation stage (Steel et al., 2018). Nematodes belonging to the genus Meloydogine spp. are mainly associated with yerba mate seedlings, causing root infestations (galling) and problems with growth, yellowing, wilt, and drought. The root-knot nematode (Meloidogyne incognita) is considered one of the main phytopathogens of the yerba mate, causing economic losses of up to 56% in some plantations (Laurindo & Souza, 2020).
Given that our study observed 12 genera of fungal known to be pathogenic to yerba mate, all of which are generalists capable of parasitizing other plants, it is important to consider that directly using the residue from yerba mate consumption as an organic fertilizer in flower beds, fruit trees or vegetable gardens - a common practice among the general population- is completely misguided (Santos et al., 2019). This practice introduces these viable fungi, as demonstrated here, into new environments and increases the risk of symptom reproduction in new plants, as these fungi have a broad host range. Fusarium oxysporum, for example, is one of the most economically important pathogenic fungi globally, causing root, stem, and fruit rot in numerous cultivated species, resulting in production losses equivalent to one billion dollars (Rampersad, 2020).
New pathogenic fungi of yerba mate are constantly found, as in the case of Ceratocystis fimbriata Ellis & Halst in two southern Brazilian municipalities, located 163 km apart, causing losses of 5% in plants of different ages (Santos et al., 2018). In Argentina, the species Ceratobasidium niltonsouzanum MP Melo, SI Moreira & PC Ceresini 2018 was identified as pathogenic to yerba mate for the first time, causing symptoms such as white leaf rust and mycelium growth on young branches, indicating that the diversity associated with yerba mate still needs further studies (Lima et al., 2019).
An urgent concern arises regarding the proper disposal of yerba mate residues, particularly due to the presence of many viable pathogens, as highlighted in the present study. Even after undergoing heat treatments in farming, industry, and the domestic settings, reproduction structures of pathogens persist, and their deposition in the environment increases the inoculum potential (Silva et al., 2019; Dors, 2017; Medeiros et al., 2019). This can lead to the contamination of new hosts, the capacity to infect other areas of yerba mate production, and the reduction in the production of this important source of income in southern Brazil. The reintroduction of industrial yerba mate residue to replace the exported macronutrients (N, P, and K), for example, was evaluated and considered as an option as no adverse effects on the soil and plant were observed, although the presence of phytopathogenic fungi was not evaluated (Sousa et al., 2015).
When the analysis of variance (ANOVA) was conducted to determine whether there was a significant difference in the occurrence and abundance of fungi among seasons, no significant difference was observed. However, when the clusters were evaluated based on the occurrence and abundance of fungi (using Euclidean distance), it was observed that the fungi found in autumn and spring seasons had a greater proximity in terms of occurrence and abundance (Figure 1).
Figure 1: Cluster analysis showing the relationship of abundance and occurrence of fungi found at the three seasons analyzed.
Among the twenty-two genera of fungi found associated with yerba mate, according to literature review, twelve of them are considered pathogenic not only to yerba mate but also to several other commercial crops. This study contributes to the expansion of knowledge regarding the fungi associated with Ilex paraguariensis, highlighting that the pathogens remain viable in the final waste, even after all the processing and use by the consumer.
Therefore, greater attention is required when disposing the yerba mate byproducts. Alternatives such as separate collection of organic waste generated in the producing regions, proper disposal in landfills, use of the substrate for growing edible mushrooms, or some other additional form of sterilization that still needs evaluation are mentioned here as possible solutions.
CONCLUSIONS
This study contributes to the expansion of our understanding of the fungal communities associated with I. paraguariensis, particularly with regards to the waste generated by the consumption of yerba mate. The presence of numerous fungal genera, known to be pathogenic according to literature, highlights the importance of proper waste disposal practices in the environment. Given that the improper disposal of this waste can lead to contamination of not only yerba mate but also other commercial crops, it is imperative that greater attention be paid to its disposal.
