Biodiversity of mycobiota throughout the Brazil nut supply chain: From rainforest to consumer

doi:10.1016/j.fm.2016.08.002

Food Microbiology

Volume 61, February 2017, Pages 14-22

https://doi.org/10.1016/j.fm.2016.08.002 Get rights and content

Highlights

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Changes of Brazil nut mycobiota supply chain are described.
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Production of tenuazonic acid was found in several species of Aspergillus section Flavi.
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Over 50 species of fungi identified using polyphasic approaches.

Abstract

A total of 172 Brazil nut samples (114 in shell and 58 shelled) from the Amazon rainforest region and São Paulo state, Brazil was collected at different stages of the Brazil nut production chain: rainforest, street markets, processing plants and supermarkets. The mycobiota of the Brazil nut samples were evaluated and also compared in relation to water activity. A huge diversity of Aspergillus and Penicillium species were found, besides Eurotium spp., Zygomycetes and dematiaceous fungi. A polyphasic approach using morphological and physiological characteristics, as well as molecular and extrolite profiles, were studied to distinguish species among the more important toxigenic ones in Aspergillus section Flavi and A. section Nigri. Several metabolites and toxins were found in these two sections. Ochratoxin A (OTA) was found in 3% of A. niger and 100% of A. carbonarius. Production of aflatoxins B and G were found in all isolates of A. arachidicola, A. bombycis, A. nomius, A. pseudocaelatus and A. pseudonomius, while aflatoxin B was found in 38% of A. flavus and all isolates of A. pseudotamarii. Cyclopiazonic acid (CPA) was found in A. bertholletius (94%), A. tamarii (100%), A. caelatus (54%) and A. flavus (41%). Tenuazonic acid, a toxin commonly found in Alternaria species was produced by A. bertholletius (47%), A. caelatus (77%), A. nomius (55%), A. pseudonomius (75%), A. arachidicola (50%) and A. bombycis (100%). This work shows the changes of Brazil nut mycobiota and the potential of mycotoxin production from rainforest to consumer, considering the different environments which exist until the nuts are consumed.

Introduction

Brazil nuts are one of the most important products extracted from the Amazon rainforest region. Trees of Bertholletia excelsa grow wild, reaching up to 60 m, take 12 years to bear fruit and may live up to 500 years. The Amazon rainforest has multiple ecosystems with a huge biodiversity, which plays an important role in the global weather balance. The equatorial climate is hot and humid, with an average temperature of 26 °C and relative humidity of 80–95%. Brazil nut production is considered totally organic and environmentally correct, since no chemical products are used to control pests and weeds and nor is there the need for fertilizers. It also favors a unique biodiversity of fungal species different from those found in cultivated areas.

Studies on the presence of fungi and aflatoxins in Brazil nuts have been investigated elsewhere (Arrus et al., 2005, Baquião et al., 2012, Baquião et al., 2013, Calderari et al., 2013, Gonçalves et al., 2012, Iamanaka et al., 2014, Massi et al., 2014). All of these studies have shown the high occurrence of Aspergillus section Flavi in Brazil nut samples. However, few studies have been carried out on the changes of Brazil nut mycobiota from rainforest to consumer, considering the different environments which exist until the nuts are consumed.

In the studies on Brazil nut mycobiota, the most commonly isolated species were Aspergillus flavus, A. nomius, A. pseudonomius, A. niger, A. tamarii, Penicillium glabrum, P. citrinum, Penicillium spp., Rhizopus spp., Fusarium oxysporum, Fusarium spp., Phialemonium spp., Phaeoacremonium spp, among others (Calderari et al., 2013, Gonçalves et al., 2012, Olsen et al., 2008, Freire et al., 2000, Bayman et al., 2002, Reis et al., 2012) and A. bertholletius a new species described recently, belonging to A. section Flavi (Taniwaki et al., 2012). More recently a new species of Penicillium named P. excelsum was also isolated from Brazil nuts and its ecosystem (Taniwaki et al., 2015).

Over the last few decades, molecular studies have proven to be a valuable tool for identification of fungi. The application of molecular techniques has helped to overcome problems of the traditional methods and have revealed the existence of a much larger number of species than was known previously, many of them not yet identified or formally accepted. Besides that, the molecular identification is often faced with the limitation of sequences deposited in databases such as NCBI and MycoBank, which cannot contemplate all Brazilian fungal biodiversity. Therefore, the aim of this research was to evaluate the mycobiota of Brazil nuts from the Amazon rainforest to consumer, using traditional methods for identification and, when available, molecular techniques and production of extrolites from the isolates as a tool for species identification. Additionally, the potential for mycotoxin and other metabolite production was analyzed for species in A. section Flavi and A. section Nigri.

Section snippets

Materials and methods

Sampling. A total of 172 Brazil nut samples (114 in shell and 58 shelled), each of approximately 2 Kg, was collected at different stages of: rainforest (57 samples in pods), street markets (54 samples, of which 32 were in shell and 22 shelled), processing at the manufacturing plants (40 samples, of which 21 in shell and 19 shelled) and supermarkets (21 samples, of which 4 in shell and 17 shelled). In the Amazon rainforest which corresponds to Amazonas and Pará states, around 5 pods of Brazil

Results

Mycobiota of Brazil nuts. A huge diversity of fungi was found in several samples throughout the Brazil nut production chain, collected in the rainforest, processing plants, street markets and supermarkets. The frequency of occurrence, the average infection rate, the range of infection and the water activity of the kernel and shell samples are shown in Table 1, Table 2, respectively. The water activity average in samples of Brazil nut kernels ranged from 0.956 in the rainforest to 0.482 in the

Discussion

Because Brazil nuts come from the Amazon rainforest, most common good agricultural practices do not apply during harvesting when pods are falling down from the 30–60 m high trees. The pods can remain in contact with soil for long periods in the humid tropical forest until they are collected. During this period microorganism proliferation will take place and fungal infection may occur because of the porosity of the shells and the pods, or through the action of insects, birds and other physical

Acknowledgments

This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico and Ministério da Agricultura, Pecuária e Abastecimento, (MAPA, Process 578485/2008-7) and Fundação de Amparo à Pesquisa do Estado de São Paulo (Process 2011/50136-0).

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For example, A. minisclerotigenes has been found mostly in South America (Pildain et al. 2008), while A. aflatoxiformans, A. cerealis and A. austwickii, reported as A. flavus SBG, are most common in Africa and Thailand (Probst et al. 2007, 2010, 2012, 2014, Mutegi et al. 2012, Guezlane-Tebibel et al. 2013). Interestingly, many of the (recently) described species producing mycotoxins have been found in foods and are quite common: A. aflatoxiformans (also reported under its synonym A. parvisclerotigenus) (in African corn, Perrone et al. 2014a,b; Mexican and Nigerian sesame, Ezekiel et al. 2014, this study; edible mushrooms, Ezekiel et al. 2013b; peanut, Frisvad et al. 2005), A. arachidicola (in wild peanuts, Pildain et al. 2008; in Brazil nuts, Gonçalves et al. 2012a,b, Calderari et al. 2013, Taniwaki et al. 2017; in corn, Viaro et al. 2017), A. austwickii (stored rice grains and sesame kernels, this study), A. caelatus (in Brazil nuts, Gonçalves et al. 2012a,b, Taniwaki et al. 2017; in peanuts, Guezlane-Tebibel et al. 2013, Martins et al. 2017), A. cerealis (rice and maize grains, this study; peanut, Carvajal-Campos et al. 2017), A. luteovirescens (in Brazil nuts, Gonçalves et al. 2012a,b, Calderari et al. 2013, Taniwaki et al. 2017), A. nomius (in Brazil nuts, Olsen et al. 2008, Gonçalves et al. 2012a,b, Calderari et al. 2013, Massi et al. 2014, Taniwaki et al. 2017; in cocoa Copetti et al. 2011), A. pseudonomius (in Brazil nuts, Massi et al. 2014, Taniwaki et al. 2017) and to a lesser extent A. novoparasiticus (in corn, Viaro et al. 2017), A. pseudocaelatus (in corn, Viaro et al. 2017; in Brazil nuts, Taniwaki et al. 2017), and A. pseudotamarii (in Brazil nuts, Calderari et al. 2013, Taniwaki et al. 2017). Originally, A. pseudotamarii was found in tea field soil (Ito et al. 2001), A. luteovirescens in silkworm environments (Peterson et al. 2001), A. nomius in bees and in soil and silkworm excrements (Kurtzman et al. 1987, Ito et al. 1998), and A. novoparasiticus as a clinical isolate (Gonçalves et al. 2012a,b).
Aflatoxins and ochratoxins are among the most important mycotoxins of all and producers of both types of mycotoxins are present in Aspergillus section Flavi, albeit never in the same species. Some of the most efficient producers of aflatoxins and ochratoxins have not been described yet. Using a polyphasic approach combining phenotype, physiology, sequence and extrolite data, we describe here eight new species in section Flavi. Phylogenetically, section Flavi is split in eight clades and the section currently contains 33 species. Two species only produce aflatoxin B₁ and B₂ (A. pseudotamarii and A. togoensis), and 14 species are able to produce aflatoxin B₁, B₂, G₁ and G₂: three newly described species A. aflatoxiformans, A. austwickii and A. cerealis in addition to A. arachidicola, A. minisclerotigenes, A. mottae, A. luteovirescens (formerly A. bombycis), A. nomius, A. novoparasiticus, A. parasiticus, A. pseudocaelatus, A. pseudonomius, A. sergii and A. transmontanensis. It is generally accepted that A. flavus is unable to produce type G aflatoxins, but here we report on Korean strains that also produce aflatoxin G₁ and G₂. One strain of A. bertholletius can produce the immediate aflatoxin precursor 3-O-methylsterigmatocystin, and one strain of Aspergillus sojae and two strains of Aspergillus alliaceus produced versicolorins. Strains of the domesticated forms of A. flavus and A. parasiticus, A. oryzae and A. sojae, respectively, lost their ability to produce aflatoxins, and from the remaining phylogenetically closely related species (belonging to the A. flavus-, A. tamarii-, A. bertholletius- and A. nomius-clades), only A. caelatus, A. subflavus and A. tamarii are unable to produce aflatoxins. With exception of A. togoensis in the A. coremiiformis-clade, all species in the phylogenetically more distant clades (A. alliaceus-, A. coremiiformis-, A. leporis- and A. avenaceus-clade) are unable to produce aflatoxins. Three out of the four species in the A. alliaceus-clade can produce the mycotoxin ochratoxin A: A. alliaceus s. str. and two new species described here as A. neoalliaceus and A. vandermerwei. Eight species produced the mycotoxin tenuazonic acid: A. bertholletius, A. caelatus, A. luteovirescens, A. nomius, A. pseudocaelatus, A. pseudonomius, A. pseudotamarii and A. tamarii while the related mycotoxin cyclopiazonic acid was produced by 13 species: A. aflatoxiformans, A. austwickii, A. bertholletius, A. cerealis, A. flavus, A. minisclerotigenes, A. mottae, A. oryzae, A. pipericola, A. pseudocaelatus, A. pseudotamarii, A. sergii and A. tamarii. Furthermore, A. hancockii produced speradine A, a compound related to cyclopiazonic acid. Selected A. aflatoxiformans, A. austwickii, A. cerealis, A. flavus, A. minisclerotigenes, A. pipericola and A. sergii strains produced small sclerotia containing the mycotoxin aflatrem. Kojic acid has been found in all species in section Flavi, except A. avenaceus and A. coremiiformis. Only six species in the section did not produce any known mycotoxins: A. aspearensis, A. coremiiformis, A. lanosus, A. leporis, A. sojae and A. subflavus. An overview of other small molecule extrolites produced in Aspergillus section Flavi is given.

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Biodiversity of mycobiota throughout the Brazil nut supply chain: From rainforest to consumer

Highlights

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Acknowledgments

J. Food Prot.

Food Control

Food Chem.

Int. J. Food Microbiol.

Int. J. Food Microbiol.

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Int. J. Food Microbiol.

Int. J. Food Microbiol.

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