mycobiota in chilean chilli capsicum annuum l. used for
TRANSCRIPT
Rodrigo Nicolas Rodriguez Quiroz
Mycobiota in Chilean chilli Capsicum annuum L.
used for Merkén’s production and its mycotoxin
contamination
Master's thesis
Master in Biotechnology
Work performed under the supervision of
Professor Doctor Nelson Lima
&
Co-supervision of
Professor Doctor Cledir Santos
July of 2018
“The best way to predict the future is to invent it.”
Alan Kay.
I
ACKNOWLEDGMENTS
First and foremost, I would like to thank my inspirational sources: my mother Gabriela Quiroz,
my father Luis Rodriguez and my grandfather Alamiro Quiroz for teach me that the key to success is
perseverance, hard work and deeply love what we do day by day.
I would also like to thank my supervisors, Prof. Dr. Nelson Lima and Prof. Dr. Cledir Santos, for
all the continuous support, dedication, patient guidance, constant availability, and for all the scientific,
social and cultural insights.
I would also like to thank my incredible Portuguese friends, Mariana Macedo, Claudio Menses,
Luis Passos, Rui Cardoso and my partner of life Filipa Carvalho for helping me in this long and hard
process away from my homeland and for showing me a different society and a new way of see the world.
I am grateful for the patience, support, help and friendship given by the researchers that daily
worked near me at the Mycology Laboratory, specially, Carla Santos, Ana Guimarães and Tiago Afonso.
I would also like to thank a very special person, Celia Soares, for all the continuous support, for
initiating me in the world of mycology, for his smiles, for her advice and for being an admirable person.
And, to all the people not mentioned above that help me in different occasions to finish this work
I leave to them my gratitude and I hope be able in the future to interchange in some way.
Thank you.
II
The present work was funded by The Advanced Human Capital Program, of the National
Commission for Scientific and Technological Research (CONICYT) Chile, Folio N° 7317076, Application
N° 73170764 and partially funded by the Universidad de La Frontera-UFRO (Temuco, Chile) through the
Project DIUFRO DI16-0135, DIUFRO DI18-0121, PIA17-0006 and EXT18-0043. Moreover, the present
work was partially being funded by the Portuguese Foundation for Science and Technology (FCT) under
the scope of the strategic funding of the UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-
FEDER-006684) and the BioTecNorte operation (NORTE-01-0145-FEDER-000004), funded by the
European Regional Development Fund through Norte2020—Programa Operacional Regional do Norte.
The Author is thankful to Miss Natalia Castillo (Chilean Social Worker) for her indefatigable support on
the development of this work and for the Mapuche Communities from the Region of La Araucanía (Chile)
for all support, contributions and for supplying red pepper and Merkén samples. Thanks are due to
Jéssica Sousa da Costa (PhD. Student of Science of Natural Resources, UFRO) and to Sebastián López
(BSc. Student of Biochemistry, UFRO) for their support in the fungal isolation evaluated in this Master
Thesis.
The experimental work developed herein was developed at the Laboratory of Mycology of the
Centre of Biological Engineering of the University of Minho, Braga, Portugal in collaboration with the Unity
of Metabolomics of the Scientific and Technological Bioresource Nucleus BIOREN-UFRO of the
Universidad de La Frontera, Temuco, Chile. The biological materials used are deposited at CCCT and
MUM culture collections.
III
RESUMO
No Chile, o fruto da planta Capsicum annuum L. cv. "Cacho de Cabra” é usado para a produção dum
piri-piri em pó chamado Merkén. As práticas usadas pelos produtores locais de Merkén são altamente empíricas
e não consideram a prevenção contra fungos micotoxigénicos pelo que, nos últimos anos, contaminações por
micotoxinas têm sido reportadas. Deste modo, o objetivo geral deste estudo é identificar os fungos potencialmente
micotoxigénicos no processo tradicional de produção do piri-piri chileno usado na produção de Merkén e detetar
contaminações por micotoxinas no Merkén produzido e comercializado no Chile. Amostras de frutos maduros de
C. annuum de 3 pontos de amostragem e amostras de Merkén foram obtidas de agricultores e mercados locais
da Zona Sul do Chile. O micobiota de frutos maduros foi isolada em meio de MEA, DRBC e DG18%. Estirpes
fúngicas isoladas foram identificadas usando técnicas clássicas e por biologia molecular. Para a taxonomia
clássica, foram avaliadas pelas características macro- e micro-morfológicas. A identificação molecular das estirpes
fúngicas foi realizada através do sequenciamento de β-tubulina (BenA) e ITS (ITS1-5.8S-ITS2). Além disso, genes
associados à produção de OTA (Otapks, Otanps, Otachl e Otatra) foram testados numa estirpe de Penicillium
crustosum e de P. verrucosum. Na extração e purificação de micotoxinas (ocratoxina A (OTA) e aflatoxinas (AFLs)
B1, B2, G1 e G2) foram usados métodos de extração líquido-líquido e colunas de imunoafinidade. Duzentos e seis
fungos foram isolados e identificados usando macro- e micro-morfologia clássica. Destes fungos, 190 foram
posteriormente identificados por técnicas de biologia molecular ao nível da espécie. Para genes associados à
produção de OTA, P. verrucosum apresenta resultados positivos para o gene Otanps e Otatra, e P. crustosum
apresenta resultados positivos apenas para o gene Otatra. Para contaminação do Merkén por micotoxinas, OTA
foi detetada em 100% amostras com valores de 0,79±0,05 a 19,81±0,70 µg/kg e quanto às aflatoxinas,
unicamente AFB1 foi detetada em 57% amostras, com valores que variaram de 0,29±0,37 a 1,67±0,32 µg/kg.
No computo geral, os resultados mostraram que o micobiota presente em frutos de C. annuum durante o processo
de produção de Merkén é cada vez mais seletiva para ocorrência de espécies de Aspergillus e Penicillium,
principalmente para a ocorrência cada vez mais seletiva de fungos potencialmente micotoxigénicos, como A. niger,
A. flavus, P. expansum e P. verrucosum. Estes resultados demonstram a importância de conhecer ainda mais o
micobiota e potenciais fungos micotoxigénicos presentes em cada etapa de produção do Merkén para estabelecer
pontos críticos de controlo para obtenção de produtos mais seguros e de melhor qualidade.
PALAVRAS-CHAVE: FUNGOS MICOTOXIGÉNICOS, PIMENTA CHILENA, MERKÉN, IDENTIFICAÇÃO DO MICOBIOTA
IV
ABSTRACT
In Chile, Capsicum annuum L. cv. "Cacho de Cabra” berry fruits are used for the manufacture of a
traditional chilli powder known as Merkén. The agricultural practices used by Merkén local producers are empirical
and do not consider the prevention of mycotoxigenic fungi and, in the last years, mycotoxin contaminations have
been reported in Merkén. Therefore, the main goal of this study was to search for the potentially mycotoxigenic
culturable mycobiota on the traditional agriculture production process of the Chilean traditional chilli used for
Merkén’s production and search for mycotoxin contamination in Merkén produced and commercialised in Chile.
Ripened fruit samples of C. annuum from 3 sampling point and Merkén samples were obtained from local farmers
and markets of the South Zone of Chile. Mycobiota from ripened fruit were isolated on MEA, DRBC and DG18
media. Isolated fungal strains were identified using classical morphology and molecular biology techniques. For
the classical taxonomy, macro- and micro-morphology traits where observed. For molecular identification of fungal
strains sequencing of β-tubulin (BenA) and ITS (ITS1-5.8S-ITS2) were performed. In addition, genes associated
with OTA production (Otapks, Otanps, Otachl and Otatra) were tested in one strain of P. crustosum and one strain
of P. verrucosum. For the extraction and clean-up of ochratoxin A (OTA) and aflatoxins (AFLs) B1, B2, G1, G2,
liquid-liquid extraction and immunoaffinity columns were performed. A total of 206 strains were obtained using
classical macro- and micro-morphology. From these fungi, 190 were identified by molecular biology techniques at
the species level. For genes associated with OTA production, P. verrucosum present positive results for Otanps
and Otatra genes, and P. crustosum present positive results for Otatra gene only. For mycotoxins contamination of
Merkén, OTA was presented in 100% samples in a range of 0.79±0.05 and 19.81±0.70 µg/kg and for AFLs, only
AFB1 were detected in 57% samples in a range of 0.29±0.37 and 1.67±0.32 µg/kg. Overall, these results show
that the mycobiota present in C. annuum berry fruits during the process of Merkén production is increasingly
selective for occurrence of Aspergillus and Penicillium species, mainly for the increasingly selective occurrence of
potentially mycotoxigenic fungi such as A. niger, A. flavus, P. expansum and P. verrucosum. These results
demonstrate the importance of knowing even more about the mycobiota and potential mycotoxigenic fungi present
in each stage used for the Merkén production, in order to establish critical control points for a safe and high-quality
product.
KEYWORDS: MICOTOXIGENIC FUNGI, CHILEAN RED PEPPER, MERKÉN, MYCOBIOTA IDENTIFICATION
INDEX
Acknowledgments .............................................................................................................................................. I
Resumo ........................................................................................................................................................... III
Abstract ........................................................................................................................................................... IV
1. Introduction .............................................................................................................................................. 1
1.1 Mycobiota and mycotoxins in chilli products ............................................................................................ 2
1.2 Identification of food-borne fungi .............................................................................................................. 6
1.2.1 Conventional microbiological techniques. ......................................................................................... 6
1.2.2 Genetic identification ........................................................................................................................ 7
1.2.3 Genetic identification of ochratoxigenic fungi ................................................................................... 10
1.3 General objective .................................................................................................................................. 13
1.3.1 Specific objectives.......................................................................................................................... 13
2. Materials and methods ............................................................................................................................ 13
2.1 Samples ............................................................................................................................................... 13
2.2 Fungal strains isolation ......................................................................................................................... 14
2.3 DNA extraction ...................................................................................................................................... 15
2.4 Genotypic identification of fungal strains ................................................................................................ 16
2.5 Genetic characterisation of potentially OTA producer fungal strains ........................................................ 17
2.6 Quantitative analyses of OTA and AFLs contamination in Merkén ........................................................... 17
2.6.1 Mycotoxins extraction and clean-up ................................................................................................ 17
2.6.2 OTA determination ......................................................................................................................... 18
2.6.3 AFLs determination ........................................................................................................................ 18
3. RESULTS ................................................................................................................................................ 19
3.1 Fungal isolation and identification .......................................................................................................... 19
3.2 Molecular Identification ......................................................................................................................... 23
3.3 Preliminary genetic characterisation of potentially OTA producer fungal strains ....................................... 30
3.4 Quantitative analyses of OTA and AFLs contamination in Merkén ........................................................... 31
4. Discussion .............................................................................................................................................. 32
5. Conclusions ............................................................................................................................................ 39
6. References .............................................................................................................................................. 40
Annex I – Isolated strains ................................................................................................................................ 50
Annex II – Standard curves of OTA and AFLs.................................................................................................... 55
1
1. INTRODUCTION
Capsicum annuum L. is the most well-known species, widespread and cultivated plants of the
genus Capsicum within the family Solanaceae. This species encompasses a wide variety of shapes and
sizes of peppers, both mild and hot, ranging from bell peppers to jalapeños to cayenne peppers. Its berry
fruits are a key element for the creation of endless condiments widely used in gastronomy. According to
the FAO the production area for chillies and peppers is over 1.9 million ha, with a production of 32 million
ton of harvested product per year, with 13.3% produced in America (FAOSTAT, 2017).
In Chile, C. annuum L cv. "Cacho de Cabra” berry fruits are used for the manufacture of a
traditional chilli powder known as Merkén. This is a powder obtained from chilli “Cacho de Cabra” with
a smoky flavour, originally produced by ancestral Amerindian Mapuche communities. These communities
live mainly in the central-south zone of Chile. According to data reported by the National Institute of
Statistics of Chile, in 2016 about 1.5 million of habitants identified themselves as members of the
Mapuche ethnicity (INESTAT, 2016). Although the Mapuche communities are currently disappearing at
an accelerated rate, their cultural and gastronomic influence are of great importance for the identity of
Chile.
Merkén is produced by Mapuche families mainly in the Regions Bio-Bio and La Araucanía and
commercialised on the national and international markets. Production of Merkén can be carried out
mainly in artisan or semi-industrial process. At the artisanal level, the production of Merkén begins with
the harvest of the fruit at the beginning of the civil year, which correspond to the summer season. After
that, the fruit is usually dried by sun exposure or in “Rucas” (typical Mapuche houses made of wood and
straw). After drying, the farmers smoke the fruit, and then crush and powder it and mix it with crushed
coriander seeds and table salt, and some of them add also crushed garlic or other spices. It gives a
darker colour and a smoky flavour to the Merkén that, once processed, generates a very characteristic
chilli. The final product has about 70% of chilli, 20% of coriander seeds and garlic, and 10% sodium
chloride (FIA, 2010).
Due to its high demand, nowadays Merkén is start to be processed industrially to promote the
standardisation of the product and obtain a commercial product of higher quality and local identity. In the
year 2015, total Chilean exports of Merkén to the world reached about 4.4 million US dollars, an
increasing of 11.3% compared to 2014 (ProChile, 2016). However, contamination with mycotoxin, mainly
2
ochratoxin A (OTA), have been reported in January 2017 by the Chilean Ministry of Health. It put the
market of this commodity under risk.
The agricultural practices used by Merkén producers are highly empirical, based on ancestral
agriculture practices and do not consider the prevention of fungal growth and further contamination with
mycotoxins. Overall, warm and humid climate in the production areas, the poor hygienic conditions during
transport, packaging and storage, may increase the risk of contamination and fungal proliferation on
peppers (Mandeel, 2005; Schweiggert et al., 2005).
To date, few publications have reported scientific data on chilli’s mycobiota. Only one study by
Ikoma et al. (2015), confirmed the presence of extremely high concentrations of OTA in dried red chilli
peppers from Chile, showing an OTA concentration range of 163.4-1059.2 µg/kg with an average OTA
amount of 355.6 µg/kg. These concentrations are very high and exceed the limits established by the
European Commission for this type of food (15 µg/kg) (EC No. 1137/2015). In addition, a recent risk
profile study published by the Chilean Agency for Quality and Food Safety (by its acronym in Spanish,
ACHIPIA) of the Ministry of Agriculture of Chile, expose the limited information about OTA contamination
and mycobiota in Merkén and Chilli berry fruits from Chile (Di Pillo and Martínez, 2018).
This makes evident the importance of devising strategies to control or reduce the presence of
mycotoxigenic fungi in early and critical stages of the food chain and to establish guidelines to support
farmers in the production of sustainable, safe and high-quality food.
1.1 Mycobiota and mycotoxins in chilli products
Foods are not considered as ecosystems because they contain a large amount of substances
and modifications induced by human, however, foods are systems rich in nutrients and organic matter
that provide an ideal habitat for the proliferation of some microorganisms, such as filamentous fungi
(Santos et al., 1998).
Abiotic factor such as temperature, pH, availability of oxygen and water activity (aw) are perhaps
the most important factors in the fungal growth in food. Temperature is one of the main physical factors
that affect the fungal growth in food and biosynthesis of its secondary metabolites. Although most fungi
are mesophiles (optimal growth between 20-25 °C), some fungi can grow under very high temperatures.
Among the thermotolerant fungi we can find Aspergillus fumigatus with a growth temperatures ranging
about 12 and above 50 °C and with optimal growth temperatures of 40-42 °C. Other thermotolerant
3
species of interest in the food industry are Aspergillus flavus and Aspergillus niger, these fungi can grow
at temperatures that vary between 8 to 45 °C (Santos et al., 1998; Bhabhra et al., 2005).
The concentration of oxygen and carbon dioxide in the system are other important factor to be
considered in the growth of food-borne fungi. Most of the fungi can easily grow and germinate up to 4%
of oxygen. Among the most common cases there is Penicillium expansum that can grow at 2.1% of
oxygen. Another outstanding example is P. roqueforti, which can withstand atmospheres of 80% of carbon
dioxide and 4% of oxygen. Some fungi that are able to survive without oxygen such as P. glabrum, P.
corylophilum, Fusarium oxysporium and F. equiseti (Santos et al., 1998; Pitt and Hocking, 2009a).
On the other hand, aw can be defined as the ratio between the partial pressure of water vapour in
foods and the saturation pressure of pure water vapour under the same assay conditions. These concept
is, certainly, one of the most important parameters to consider in food production (Santos et al., 1998).
aw plays a predominant role on growth of filamentous fungi with importance in food industry such as
Penicillium spp., Aspergillus spp. and Fusarium spp. (Frisvad and Samson, 1991; Santos et al., 1998;
Pitt and Hocking, 2009a).
Although drying process contributes to the decrease of the microbial biomass in chilli powder,
many fungi can survive in spores form and germinate when the moisture value exceeds the safety limits
for the consumer (Martin et al., 2005; Dijksterhuis, 2007). On the other hand, the warm and humid
climate in the production areas and the poor hygienic condition during packaging/storage process and
sale areas, may increase the risk of fungal proliferation and contamination (Mandeel, 2005).
Due to the components present in chilli powder manufacturing processes, fungi can fall into two
possible broad ecological categories: (1) Field and (2) Storage. Some studies have found field species
that are generally associated with plants and soil such as Alternaria spp., Cladosporium spp., Mucor spp.
and Rhizopus spp. (Almela et al., 2007; Fatimoh et al., 2017). On the other hand, storage fungi reported
in the literature are mainly related to the genus Aspergillus, Penicillium and Eurotium (Mandeel, 2005;
Almela et al., 2007; Fatimoh et al., 2017).
Due to the aw of these product, xerophilic fungi are the main spoilage microbiota found in chilli
powder. Xerophilic fungi are defined by their ability to grow under very low aw conditions. It means that
fungi have the ability to carry out their life cycles on dried or concentrated substrates, under high
percentages of soluble solids, such as salts or sugars (Pitt and Hocking, 2009b). Moderate xerophilic
fungi and mycotoxigenic fungi such as Aspergillus spp., Penicillium spp. and Fusarium spp. have been
detected among the mycobiota of Capsicum berry fruits and its processed powders. Moreover, aflatoxins
4
(AFLs) and OTA have also been found as contaminant in these fresh and processed food (Hierro et al.,
2008; Iqbal et al., 2010; Gherbawy et al., 2015).
Mycotoxins are a broad group of chemical substances produced by some species of fungi that
can cause acute or chronic intoxications leading to illness or death in animals and humans. Exposition to
mycotoxins can occur by ingestion of contaminated foodstuffs, but also by skin contact or inhalation
(Magan and Olsen, 2004).
Due to the aw influences the fungal spore germination and fungal growth, it is certainly a decisive
parameter to produce mycotoxins that could contaminate the final product. For example, Aspergillus
flavus has the minimum aw values for its growth of 0.78-0.80, but is a producer of AFLs when aw reaches
values between 0.83 and 0.87; Aspergillus ochraceus has the minimum aw values for its growth of 0.76-
0.83, but it is a producer of OTA when reaches values of aw between 0.83 and 0.87, and Penicillium
expansum has minimum aw values for its growth of 0.82-0.85, but it is a producer of patulin when reaches
aw of 0.99 (Frisvad and Samson, 1991; Pitt and Hocking, 2009a; Mannaa and Kim, 2017).
The mycotoxins of great significance in chilli powder and chilli fruits include AFLs, OTA,
zearalenone (ZEA), trichothecenes and sterigmatocystin (Santos et al., 2011; Gambacorta et al., 2018;
Hossain et al., 2018). Due to high toxicity, the most important mycotoxin are AFLs followed by OTA and
ZEA. Normally AFLs, OTA and ZEA are produced by fungi in growing crops, while AFLs can also be
produced during storage. Poor hygienic conditions and deficient aw control during processing are main
causes for mycotoxin production in chilli (Santos et al., 2008; Mannaa and Kim, 2017).
AFLs are producers by fungi of the genus Aspergillus section Flavi (especially A. flavus, A. nomius,
A. parasiticus and A. tamari) and can be toxic, carcinogenic, mutagenic and teratogenic to most animal
species, being classified as the most dangerous and extremely carcinogenic mycotoxins (Smith et al.,
1994; Paterson and Lima, 2010b).
OTA is mainly produced by Aspergillus sections Circumdati and Nigri and Penicillium verrucosum
(Almela et al., 2007; Gil-Serna et al., 2009b). Species of Aspergillus section Nigri are among the most
important sources of mycotoxins contamination of foods and feeds, mainly OTA. OTA is toxic to animals
and the International Agency for Research on Cancer classified it as a group 2B carcinogen, i.e., a possible
human carcinogen (IARC, 1993).
ZEA is produced by species of the genus Fusarium and can be classified as a weak estrogen,
non-steroidal, that has mechanisms of competitiveness with the estrogens of the human body activating
and deactivating its metabolic pathways (Smith et al., 1994).
5
Mycotoxin determination and quantification in Chilli powder generally consist of five steps: 1)
sampling, 2) sample preparation, 3) extraction, 4) clean-up and 5) quantification (Hu et al., 2006; Santos
et al., 2008; Zhang et al., 2018). The extraction and purification (clean-up) depend on the
physicochemical properties of the sample (Hu et al., 2006; Zhang et al., 2018). The extraction in paprika
and chilli powder is usually done by solvent organic such as methanol, dichloromethane, chloroform,
acetone/hexane, and organic solvent mixed with water or other solvent, such as methanol-water,
acetonitrile-water and benzene-acetic acid (Santos et al., 2010a; Santos et al., 2010b). The extract
purification (clean-up) is necessary because solvents are not selective and can co-extract substances
(pigments and lipids) from the sample (Pal et al., 2005; Hu et al., 2006). Some authors used hexane,
ether or centrifugation to remove the lipids (Pal et al., 2005). However, a more selective treatment can
be selected for clean-up, such as liquid-liquid extraction, solid-phase extraction, strong anion exchange or
immunoaffinity columns (Patel et al., 1996; Pal et al., 2005; Hu et al., 2006; Almela et al., 2007; Zhang
et al., 2018).
The quantification of mycotoxins from paprika and chilli powder include high performance thin-
layer chromatography (HPTLC), high performance liquid chromatography (HPLC), gas chromatography
(GC) and mass spectrometry (MS). Normally AFLs and OTA are analysed by HPLC with fluorescence
detection (Patel et al., 1996; Soares et al., 2010; Abrunhosa et al., 2014). Other methods like Enzyme
Linked Immuno Sorbent Assay (ELISA) are used to detect and quantify mycotoxins and usually do not
need the clean-up step. However, the possibility of false positives because of cross-reaction and
interference in complex matrixes are reported (Hu et al., 2006). Nowadays, due to the low availability of
equipment and high costs associated, the use of HPLC remains the most common.
Mycotoxins contamination of chilli powder has been reported worldwide. The range of
contamination for AFLs has varied between below the limit of detection (<LOD) and 124.6 μg/kg (Santos
et al., 2008), for OTA has varied between 0.4 and 355 μg/kg (El-Kady et al., 1995; Fazekas et al., 2005;
Hierro et al., 2008; Santos et al., 2010c; Ikoma et al., 2015) and for ZEA has varied between <LOD and
131 μg/kg (Patel et al., 1996; Santos et al., 2010b; Motloung et al., 2018).
The European Commission has established regulations for AFLs in Capsicum fruits with
maximum tolerable limits set at 10 µg/kg for total AFLs (AFLB1+AFLB2+AFLG1+AFLG2) and at 5.0 µg/kg
for AFLB1 (EC No. 1881/2006). A new regulation established for the maximum levels of OTA in spices
of 20 μg/kg for Capsicum powder and 15 μg/kg for mixtures of Capsicum with other species (EC No.
1137/2015). According to the European Commission Regulations, the maximum tolerable ZEA
concentration is not yet established for chilli powder.
6
1.2 Identification of food-borne fungi
The identification of fungal species has been carried out mainly by conventional microbiological
techniques, such as macro- and microscopic features on different culture media, and DNA-based
methods, such as the amplification and sequencing of different DNA barcodes.
1.2.1 Conventional microbiological techniques.
Over the past two centuries, the identification of different filamentous fungi were performed by
classical microbiological techniques. These methods allow the differentiation according morphological
criteria and microscopic features in accordance with appropriate dichotomous keys (Pitt et al., 1993).
At the time of performing a conventional identification, a series of parameters must be taken into
consideration, such as the macroscopic characteristics (e.g., colony colour and cultural characteristics)
and microscopic (e.g., spore size and shape) phenotypic characteristics observed from cultures grown
on different media (Pitt et al., 1993; Ryu et al., 2015).
When the identification of filamentous fungi is based on morphological features the use of
standard culture media is important because the morphology and colonies colour vary from medium to
medium. In the case of mycological analyses in foods, the aw is a fundamental parameter for successful
fungal growth. Many of the culture media found in the literature for fungal isolation from chilli powder are
formulated considering its the aw (Pitt et al., 1993; Mandeel, 2005; Martin et al., 2005; Santos et al.,
2008; Santos et al., 2011).
Selective fungal enumeration in chilli powder has been achieved using oxytetracycline-glucose
yeast extract agar (OGYEA) incubated at 25 ºC for 5 days (Almela et al., 2007), yeast extract glucose
chloramphenicol agar (YGC agar) at 25 ºC for 5 days and potato dextrose agar (PDA) at 28 ºC for 2-5
days (Schweiggert et al., 2005). In the case of food-borne fungi, Dicloran Rose Bengal Chloramphenicol
Agar (DRBC Agar) (King et al., 1979; Mandeel, 2005), Dicloran 18% Glycerol Agar (DG18) (Hocking and
Pitt, 1980), Czapex-Dox Agar (CD) (Thom and Church, 1926; Mandeel, 2005), Aspergillus
Flavus/Parasiticus Agar (AFPA) (Pitt et al., 1983), Corn Yeast Agar (CYA) and Malt Extract Agar (MEA)
(Thom and Church, 1926; Mandeel, 2005; Martin et al., 2005) are the most commonly used media.
Usually, for the study of mycobiota, these media are incubated at temperature between 21 and
28 ºC for 5-15 days. In the most of cases, due to it reduces interferences from both bacteria and rapidly
growing fungi, DG18 is the medium of choice for the isolation of moderate xerophilic fungi from food with
low aw (0.955) (Hocking and Pitt, 1980). However, in some cases it is necessary to use culture media
7
with very low aw such as Malt Extract 50% Glucose Agar (MY50G) with aw of 0.89 or Malt Extract Yeast
Extract 70% Glucose Fructose Agar (MY70GF) with aw of 0.75 (Ryu and Wolf-Hall, 2015).
After incubation, the microorganisms are visualised by light field microscopy using different
stains. Most of the dyes used in mycology are slow acting because the fungal cell wall is highly resistant
to staining due to their composition. The most used dyes in mycology include lactofucsins, lactophenol
blue, cotton blue and erythrosine in ammonia (Pitt and Hocking, 1992).
Although the identification by conventional techniques is fundamental for the identification of
filamentous fungi, they are time consuming, subjective and usually requires taxonomic expertise. For this
reason, more efficient and non-subjective methods have been used in recent years, such as DNA-based
methods (Frisvad and Samson, 2004; Geiser et al., 2007; Rodrigues et al., 2009; Simões et al., 2013;
Silva et al., 2015; Decontardi et al., 2018).
1.2.2 Genetic identification
With the advance of molecular biology in the last quarter of XX century, cutting-edge technologies
have been developed for the identification of microorganisms based on their genomic DNA. The DNA-
based techniques permit rapid, sensitive and specific detection, of filamentous fungi in food samples and
are faster than conventional methods (Frisvad and Samson, 2004; Geiser et al., 2007; Rico-Muñoz et al.,
2018).
The identification of filamentous fungi from chilli powder has been achieved using PCR-based
methods (Santos et al., 2010a; Santos et al., 2010b). These methods allow the specific detection of small
amounts of target organisms by amplifying their DNA in a considerably short time frame. The most
common used methods are conventional and real-time PCR assays, but next generation DNA sequencing
is becoming more common (Rodrigues et al., 2011; Silva et al., 2015; Decontardi et al., 2017; Decontardi
et al., 2018).
In this context, the extraction of high quality and quantity DNA from fungi is one of the most
important parameters to consider. The DNA extraction protocol has a pronounced effect on the result of
a molecular study and must be optimised for each food product or in some cases for each kind of fungi.
On the other hand, some studies have shown that different methods of DNA extraction, as well as different
purities or qualities can affect the interpretation of results (Costea et al., 2017).
The DNA extraction of fungi in culture media can present in some cases great difficulties due to
the cellular structure of each fungus. Fungi can produce unicellular structures such as conidia in the case
of Aspergillus and Penicillium or multicellular structures such as Alternaria and Fusarium. These
8
structures can be thin-walled, but can also be thick-walled with large amounts of melanin (melanised
walls). Certain species such as Cladosporium and Alternaria (Dothideomycetes) produce resistant and
melanised cell walls and the rupture of these cells is difficult. It is expected that DNA recovery will be
different for each cell type (Summerbell, 2011).
In the case of DNA purity, this can be very important at the time of using PCR-based methods, if
the DNA is used in a PCR based assay, then compounds in the DNA extract can affect or inhibit the PCR
reaction by binding or denaturing the Taq DNA polymerase (Hayat et al., 2012)
Methods based on PCR techniques have a theoretic detection limit down to 1 to 10 molecules of
DNA. However, the concentration, quality, purity and PCR conditions (melting temperature, hybridization
time and elongation time) substantially affect its performance and its veracity. However, internal
amplification controls can be used to determine the presence of inhibitory substances or failure of a PCR
(Hoorfar et al., 2004).
In the last years, different target housekeeping genes have been used for the identification of
fungi. These are usually divided into two groups: 1) single-copy and 2) multicopy. Single-copy gene
analyses (for example, genes encoding actin, calmodulin, chitin synthetase, heat shock proteins 90 or
superoxide dismutase) are highly species-specific; however, since they are single-copy, they may not be
detected easily. In contrast, the amplification of multicopy genes considerably increases the sensitivity
due to a greater number of copies of the target gene in the sample (Mitchell et al., 1995; Chen et al.,
2002; Cabañes, 2002).
Although the single-copy genes are more species-specific, the most used target genes in the last
years are the multicopy genes found in the complex of ribosomal DNA complex (rDNA). Due to the fact
that ribosomes are responsible for protein synthesis, these genes have been conserved in all life forms,
presenting small alterations generated by evolutionary processes. These genes are excellent genetic
markers for the identification of fungi (Mitchell et al., 1995) and recommended as universal DNA barcode
marker for fungal identification (Schoch et al., 2012).
In fungi, the rDNA is a tandem formation of 40-240 copies per genome in the haploid genome
of these microorganisms (O´Donnel, 1992). It comprises the coding regions of the small subunit of the
ribosome (18S), the 5.8S gene and the large subunit of the ribosome (28S). Separating the subunits 18S-
5.8S and 5.8S-28S are non-coding regions Internal Transcribed Spacer (ITS) (Chen et al., 2002; Iwen et
al., 2002).
The ITS rDNA regions have been accepted as the official DNA barcode for fungi and is used as
universal region to identification of fungal species (Hebert et al., 2003; Geiser et al., 2007; Schoch et al.,
9
2012). In the case of chilli powder the identification of A. flavus, A. niger, A. ochraceus, A. tubingensis
and A. westerdijkiae have been archived amplifying the region ITS1-5.8S-ITS2 (Gil-Serna, 2009b; Santos
et al., 2010a; Santos et al., 2010b). Also, the primers 5.8S1/5.8S2 have been using to amplify the 5.8S
region to identify A. carbonarius (Patiño et al., 2005), A. steynii (Gil-Serna, 2009a), A. flavus and A.
parasiticus (González-Salgado et al., 2008).
Although the ITS region is considered as a universal barcoding, the resolution of this region can
be insufficient for species level identification especially for fungi of food origin as such as Aspergillus,
Cladosporium, Fusarium and Penicillium. For this reason, sequencing of a secondary barcode is
recommended (Peterson, 2008; Roe et al., 2010; Varga et al., 2011; Peterson, 2012; Pérez‐Izquierdo
et al., 2017).
Other barcoding genes such as β-tubulin (BenA), Calmodulin (CaM) and translation elongation
factor-1 alpha (TEF-1α) have been used in many fungal identification and classification studies in recent
years (Perrone et al., 2011; Rodrigues et al., 2011; Varga et al., 2011; Soares et al., 2012; Frisvad et
al., 2013; Visagie et al., 2014; Decontardi et al., 2017; Decontardi et al., 2018).
For Aspergillus and Penicillium species, the most commonly used genes are BenA and CaM. In
the case of the tubulin superfamily, only α-, β- and γ-tubulins have homologues in fungal genomes
(Dutcher, 2001). The β-tubulin paralogs are represented in Aspergillus by two genes called BenA and
tubC. The BenA gene encodes polypeptides designated as β1- and β2-tubulin. This gene is an excellent
molecular marker for these fungi and is currently the third most used gene in fungal multilocus
phylogenies (Feau et al., 2011).
Although the genes to be amplified and sequenced must be sufficiently distinctive to identify
genetically related fungi, the use of the databases is another important point to consider. The results of
sequencing and identification depend strongly on the quality and quantity of information deposited in the
databases. In relation to the GenBank database, it is estimated that about 20% of fungal identifications
deposited have errors or are unreliable (Nilsson et al., 2006; Bidartondo et al., 2008).
Nowadays, different analytical techniques allow the identification of microorganisms with the use
of biomolecules as fingerprint. The Matrix-Assisted Laser Desorption Ionization-Time-Of-Flight Mass
Spectrometry (MALDI-TOF MS) technique can be used for fungal identification. MALDI-TOF MS has a short
turnaround time, is robust and can be reliable. The technique measures the difference in size of
biomolecules such as proteins, peptides, glycoproteins, biomarkers, sugars and also organic molecules.
The use of MALDI-TOF MS for the identification of food spoilage filamentous fungi has successfully been
10
explored (Santos et al., 2010d; Rodrigues et al., 2011; Chaves da Silva et al., 2015; Santos et al., 2015;
Chang et al., 2016; da Cruz Pedrozo Miguel et al., 2017; Lima and Santos, 2017).
Fourier transform infrared spectroscopy (FT-IR) is another powerful analytical technique that can
be applied for characterising the chemical composition of very complex probes such as filamentous fungi.
This technique has been successfully applied for the identification of filamentous fungi. Fungal FT-IR
typing is fast, effective and reagent-free. Moreover, it requires a small quantity of biomass. When FT-IR is
applied on the fungal cell, it generates a unique ‘‘fingerprint’’ representing the total cell chemical
composition such as lipids, proteins, nucleic acids and polysaccharides. FT-IR spectra are highly specific
for each fungal strain and species (Santos et al., 2010e).
For this reason, the most accurate approach to get a precise and reliable identification is the
polyphasic identification, merging the morphological, chemical and genetic trait results in the final
identification (Frisvad and Samson, 2004; Geiser et al., 2007; Rodrigues et al., 2009; Simões et al.,
2013; Silva et al., 2015; Decontardi et al., 2018).
1.2.3 Genetic identification of ochratoxigenic fungi
The early and rapid detection of possible mycotoxin-producing fungi is important to reduce the
negative impacts on food commodity. In recent years, PCR systems have been developed for the detection
and differentiation of the main species and groups of mycotoxigenic fungi, by means of the amplification
of sequences that code for associated enzymes in the metabolic pathways of production of these
mycotoxins. These strategies have been successfully applied to aflatoxin-producing fungi (Geisen, 1996;
Shapira et al., 1996; Rodrigues et al., 2009), OTA (Geisen et al., 2006), fumonisins (Paterson, 2006a;
Gonzalez-Jaen et al., 2004) and also to patulin producers (Paterson, 2006a; Paterson, 2006b).
The ocratoxina A (OTA) is a mycotoxin produced by the secondary metabolism of many
filamentous species. Biosynthetically, it is a pentaketide derived from the dihydrocoumarins family
coupled to β-phenylalanine (El-Khoury and Atoui, 2010). Although there is much information about the
various toxigenic properties of OTA, unlike other important mycotoxins, not much is known about the
pathway of OTA biosynthesis. It is widely believed that the isocoumarin group is a pentaketide formed
from acetate and malonate through a polyketide synthesis route (Niessen et al., 2005).
In 1979 Huff and Hamilton proposed a biosynthetic route based on a mechanistic model
according to the structure of the OTA (Figure 1). In this model, a polyketide synthase (PKS) is considered
as a key enzyme in the biosynthesis of OTA in a similar way to other polyketide mycotoxins such as
fumonisins (Proctor et al., 1999) and aflatoxins (Bhatnagar et al., 2003).
11
According to Huff and Hamilton (1979), OTA biosynthesis can be summarized in three steps: the
first step involves the synthesis of polyketides of ochratoxin α via Mellein using a polyketide synthase.
The second step includes addition of the acyl group to Mellein, oxidizing the molecule to 7-carboxy-mellein
and forming OTβ. Followed by this chlorination by a chloroperoxidase leads to OTα. OTα then transforms
into a mixed anhydride, an activation reaction that uses ATP. The second precursor phenylalanine is
synthesized through the metabolic pathway of shikimic acid, followed by activation of the ethyl ester so
that it can participate in the subsequent acyl shift reaction. In the third step, the binding of these activated
precursors through a synthetase, generating OTC, an ethyl ester of OTA: the de-esterification by an
esterase or transesterification is the last step in this hypothetical biosynthetic route (Figure 1).
Figure 1. Schematic representation of the hypothetical OTA biosynthetic
pathway. Adapted from Huff and Hamilton (1979).
OTβ
OTα
OTC
OTA
12
In 2006, Geisen et al. identified in Penicillium nordicum fragments of genes highly related to the
production of OTA, which are related to the metabolic pathway proposed by Huff and Hamilton (1979).
Two fragments were identified (Figure 2), a fragment of 10 kb DNA, containing three genes: A gene with
high homology with an alkaline serine protease gene (aspPN), representing the upper edge of the cluster.
A fragment carrying a large part (approximately 2 kb) of the 5' end of a polyketide synthase (otapksPN,
≈500 bp for P. nordicum) and a complete non-ribosomal peptide synthetase (otanpsPN, ≈750 bp for P.
nordicum). The second 4.5 kb fragment harboring three open reading frames encoding putative OTA
biosynthetic proteins: an incomplete open reading frames at the 5' end of the fragment demonstrated
homology with an organic anionic transporter of rat kidneys (otatraPN, ≈400 bp for P. nordicum). A
complete open reading frames of 951 nucleotides that has a limited homology with a chloroperoxidase
(otachlPN) of Gluconobacter oxidans. At the 3' end of this DNA fragment there is an incomplete open
reading frame of a possible nitrate transporter (ntraPN) (Geisen et al., 2006).
Figure 2. Map of the characterised OTA biosynthetic genes. aspPN = alkaline serine protease; otapksPN = ochratoxin polyketide synthase; otanpsPN = ochratoxin non-ribosomal peptide synthetase; otachlPN = ochratoxin
chloroperoxidases; otatraPN = ochratoxin transport protein and ntraPN= nitrate transporter. Adapted from Geisen et al. (2006).
13
1.3 General objective
The main aim of the present work is search for the potentially mycotoxigenic culturable mycobiota
on the traditional agriculture production process of the Chilean traditional chilli Capsicum annuum L. cv.
"Cacho de Cabra” used for Merkén’s production and search for mycotoxin contamination in Merkén
produced and commercialised in Chile.
1.3.1 Specific objectives
1. Isolate the culturable mycobiota on three time points of the traditional agriculture production process
of the Chilean traditional chilli Capsicum annuum L. cv. "Cacho de Cabra” used for Merkén’s production;
2. Identify the isolated fungal strains based on classical morphology and molecular biology techniques;
3. Characterise the genes associated with OTA production for potentially ochratoxigenic strains;
4. Search for OTA and AFLs contamination in Merkén produced and commercialised in Chile.
2. MATERIALS AND METHODS
2.1 Samples
Samples of chilli C. annuum were provided by 8 farmers belonging to the follow localities of the
Chilean Region of La Araucanía (Figure 3): Nueva Imperial (2 farmers), Hualacura (1 farmer), Cholchol
(1 farmer) and Purén (4 farmers). Samples were collected at 3 different sampling points of berry fruits
production: (1) just at the day of ripe fruits harvest; (2) after 1 month of harvest (drying process); and (3)
during the smoking process. A total of 240 samples of chilli berry fruits (8 farmers, 10 berry fruits per
farmer distributed in 3 different sampling points of berry fruits production) were collected. Berry fruits
samples were used for mycobiota isolation. In addition, 10 g of Merkén samples from each farmer and
from 13 different regional markets were collected in order to determine OTA and AFLs contamination. All
Merkén samples were stored at 4 °C until extraction and quantification.
14
2.2 Fungal strains isolation
Isolation of fungal strains was carried out at the Laboratory of the Chilean Culture Collection of
Type Strains (CCCT/UFRO, http://ccct.ufro.cl/), which is hosted at the BIOREN-UFRO Scientific and
Technological Bioresource Nucleus (http://bioren.ufro.cl/) of the Universidad de La Frontera, Temuco,
Chile.
In order to isolate the mycobiota, each fruit was cut and placed on MEA (malt extract 20g/L,
mycological peptone 1g/L, agar 20g/L, glucose 20 g/L), DRBC (KH2PO4 1g/L, MgSO4·7H2O 0.5 g/L,
Figure 3. Location of the sampling regions in the Region of La Araucanía, Chile. (Adapted from Biblioteca Nacional del Congreso de Chile, URL:www.bcn.cl/siit/mapas_vectoriales/mapoteca, access on
15/06/2018).
15
mycological peptone 5g/L, dicloran 0.002g/L, chloramphenicol 0.1 g/L, agar 15g/L, glucose 10 g/L,
rose bengal 0.025 g/L) and DG18 (peptone 5g/L, glucose 10 g/L; KH2PO4 1g/L, MgSO4·7H2O 0.5 g/L,
dicloran 0.002 g/L, agar 15g/L) in Petri dishes. Each plate was incubated for 7 days at 27 °C in the
dark. After isolation, the fungal strains were identified at genus level based on macro- and micro-
morphological traits. Classical fungal identification followed the taxonomic keys and guides available for
each specific genus.
All fungal strains were then sent to the Laboratory of Mycology of the Center of Biological
Engineering of the University of Minho (Braga, Portugal, www.ceb.uminho.pt/amg) for molecular biology
identification. All fungal strains were deposited at CCCT/UFRO and at the Micoteca da Universidade do
Minho (MUM, www.micoteca.deb.uminho.pt/). In this latter case, the international regulations were
compiled and a Material Transfer Agreement was established.
2.3 DNA extraction
Genomic DNA of each isolates was extracted using a modified protocol describe by Rodrigues et
al. (2009). Briefly, spores of each strain were transferred from a 7 days old culture into 50 mL falcon
tubes containing 25 mL of Malt Extract-Glucose-Yeast Extract-Peptone Medium (MGYP, malt extract 3
g/L, glucose 10 g/L, yeast extract 3 g/L, peptone 5g/L). Samples were incubated at room temperature
for 5 days in the dark, at 150 rpm in a shaker.
Fungal biomasses were filtrated and stored at -20 °C. For DNA extraction, 100 mg of biomasses
were transferred into a 1.5 mL Eppendorf tube containing 100 µL of lysis buffer (200 mM Tris-HCl pH
8.5, 250 mM NaCl, 25 mM EDTA, 0.5% (w/v) SDS). Cells lyses were performed using a pellet pestle for
3-4 min. After mechanical lysis, 900 µL of lysis buffer was added and the samples were incubated for 1
hour at 65 °C. Samples were centrifuged at 14000 x g for 10 min at room temperature and 800 µL of
upper phase was transferred into a new 2 mL Eppendorf tube.
Polysaccharides and proteins were precipitated by adding 1 mL of cold sodium acetate (3 M, pH
5.5). Samples were gently mixed by inversion, placed at -20 °C for 10 min and centrifuged at 14000 x g
for 10 min at room temperature. Clean supernatant was then transferred to a new tube and precipitated
with one volume of cold isopropanol (-20 °C). Samples were gently mixed by inversion for 2 minutes,
incubated at −20 °C for 2 h and centrifuged at 14000 x g for 10 min. DNA pellets were washed twice
with 1.0 mL of cold 70% ethanol, centrifuged at 14000 x g for 10 min and dried using a SpeedVac
Concentrator. DNA samples were suspended on 50 µL of ultra-pure water and stored at -20 °C. DNA
16
samples were subjected to quality assessment by quantification of total DNA using Thermo Scientific™
NanoDrop™ instrument and by electrophoresis agarose gel 1% (w/v) for 45 min at 80 V. Electrophoresis
agarose gel SYBR® Safe DNA Gel Stain (Invitrogen) was used as a staining element and NZYDNA ladder
III was used as a DNA molecular weight marker.
2.4 Genotypic identification of fungal strains
In order to identify the fungal strains, amplification of BenA and ITS genes were performed.
The BenA gene was amplified using the primers Bt2a (5´-GGT AAC CAA ATC GGT GCT GCT TTC-
3´) and Bt2b (5´-ACC CTC AGT GTA GTG ACC CTT GGC-3´) design by Glass and Donaldson (1995). For
BenA reactions used 25 µL Taq DNA polymerase Master Mix 2x (VWR Life Science), 2 µL primer Bt2a
10 mM, 2 µL primer Bt2b 10 mM, 2 µL of total DNA and 19 µL of distilled water free of proteases to a
final volume of 50 µL were used. PCR parameters used in the thermal cycler were: 95 °C for 3 min, 35
cycles of 95 °C for 1 min, 56 °C for 45 s, 72 °C for 90 s and a final extension at 72 °C for 10 min.
The ITS1-5.8S-ITS2 rDNA region was amplified using the primers ITS1 (5´-TCC GTA GGT GAA
CCT GCG G-3´) and ITS4 (5´-TCC TCC GCT TAT TGA TAT GCC-3´) design by White et al. (1990). For
ITS reaction 25 µL Taq DNA polymerase Master Mix 2x (VWR Life Science), 2 µL primer ITS1 10 mM, 2
µL primer ITS4 10 mM, 2 µL of Total DNA and 19 µL of distilled water free of proteases to a final volume
of 50 µL were used. PCR parameters used in the thermal cycler were: 94 °C for 3 min, 35 cycles of 94
°C for 1 min, 55 °C for 1 min, 72 °C for 1 min and a final extension at 72 °C for 5 min.
Bands were visualized on 1% (w/v) agarose gel supplemented with SYBR® Safe DNA Gel Stain
(Invitrogen) as a staining element and NZYDNA ladder III as a DNA molecular weight marker. PCR
products were cleaned using NZYGelpure kit according to the manufacturer’s instruction and send to Stab
Vida Oporto Laboratories (Centro de Testagem Molecular, Vairão, Portugal;
http://www.stabvida.com/pt), sending 3 µL of the forward primer and 10 µL of genomic DNA. Gene
sequences were provided in AB1 format file by Stabvida Laboratories.
Every sequence was opened into the analysis software package Mega7 (Molecular Evolutionary
Genetics Analysis version 7.0 for bigger datasets; Kumar et al., 2016) and the quality of the sequences
was verified using as reference the quality graphics sent by Stab Vida. Each sequence was compared
with the GenBank database sequences using the Basic Local Alignment Search Tool (BLAST
https://blast.ncbi.nlm.nih.gov/). Sequences were aligned into the analysis software package Mega7
using MUSCLE alignment (Robert, 2004). Dendrograms were then deduced, opening the sequences into
17
the pattern analysis software package Mega7 and using the Neighbour-Joining method (Saitou and Nei,
1987). All positions containing gaps and missing data were eliminated. The evolutionary distances were
computed using the Kimura 2-parameter method (Kimura, 1980). All sequences of the reference strains,
also included for clustering, were obtained by searching on the website of the fungal collection CBS-KNAW
(http://www.westerdijkinstitute.nl/collections/); also GenBank database
(http://www.ncbi.nlm.nih.gov/genbank/).
2.5 Genetic characterisation of potentially OTA producer fungal strains
In order to identify, preliminarily, a putative OTA biosynthetic genes of Penicillium species.
Genomic DNA of P. verrucosum (CCCT 18.33) and P. crustosum (CCCT 18.46) were subjected to PCR
using the primers, Otapks1 (5´-TAC GGC CAT CTT GAG CAA CGG CAC TGC C-3´) Otapks2 (5´-ATG CCT
TTC TGG GTC CGA TA-3´), Otanps_for (5´-AGT CTT CGC TGG GTG CTT CC-3`), Otanps_rev (5´-CAG
CAC TTT TCC CTC CAT CTA TCC- 3`), Otachl_for (5´-CGT GAT GGC CAG ATG GGC GTG-3`), Otachl_rev
(5´-CCG TCT CTC CAT TCT CTT CCT-3`), Otatra_for (5´-GGT CGG GCC GAT GTT TGA TCG-3`) and
Otatra_rev (5´-CCT CGC ATC TTG TAA GGA ACG C-3`) design by Geisen et al. (2006). Such internal
amplification control (IAC), ITS1-5.8S-ITS2 region using ITS1/ITS4 primers were used. Penicillium
nordicum (CBS 112573) was used like positive control and A. carbonarius (MUM 01.08) was used like
negative control.
For PCR reaction 25 µL Taq DNA polymerase Master Mix 2x (VWR Life Science), 2 µL of each
primer 10 mM, 2 µL of Total DNA and 16 µL of distillate water free of proteases to a final volume of 50
µL. The temperature ramps used in the thermal cycler was 95 °C for 3 min, 33 cycles of 95 °C for 30
min, 60 °C for 40 s, 72 °C for 60 s and a final extension at 72 °C for 10 min. The Samples will be
visualized on 1% agarose gel supplemented with Green Safe Premium (Nzytech MB13201) as a DNA
identifier. Five μL Ladder III Molecular Weight Marker and 5 μL of each amplified sample will be loaded
and the gel run at 75 V for 30 min.
2.6 Quantitative analyses of OTA and AFLs contamination in Merkén
2.6.1 Mycotoxins extraction and clean-up
18
Extraction and clean-up of AFLs and OTA from Merkén samples using liquid-liquid extraction and
immunoaffinity columns were performed with two independent replicates. For the extraction, 2 g of
Merkén were mixed with extraction solution (0.2 g of NaCl; 10 ml of methanol:water 8:2; and 5 mL of
hexane) in Erlenmeyer flasks of 100 mL and shake on a mechanical shaker at 150 rpm for 1 hour at
room temperature. The solution was filtered through WhatmanTM Nº 4 filter paper and separate using a
separatory funnel. After phase separation, 5 mL of the aqueous layer were diluted with 30.7 mL of
Phosphate Buffered Saline (PBS) buffer (137 mM NaCl; 2.7 mM KCl; 10 mM Na2HPO4; 2 mM KH2PO4;
pH 7.4) and filtered using glass microfibre filters WhatmanTM. For the clean-up, 10 mL of solution extracted
previously was added to AflaOchra HPLC immunoaffinity columns VICAM column at flow rate of 2
mL/min. the column was washed with 10 mL of PBS buffer at flow rate of 2mL/min and finally the
mycotoxins were eluted with 1.5 mL of methanol. The eluted samples were quantified by HPLC.
2.6.2 OTA determination
Ochratoxin A quantification was performed according to Abrunhosa et al. (2014) using High
Performance Liquid Chromatography (Waters, Milford, MA, USA) with a reverse-phase C18 silica gel
column (250x4.6 mm, 5 μm), equipped with a Varian 9002 pump (Agilent, Palo Alto, CA, USA), a Varian
Prostar 410 autosampler and Jasco FP-920 fluorescence detector (Jasco Europe, Cremella, Italy).
Excitation and emission wavelengths was set at 333 and 460 nm, respectively. An isocratic mobile phase
of acetonitrile/water/acetic acid (99:99:2, v/v/v) was used with a flow rate of 1.0 mL/min. OTA was
identified by comparing the retention time of the peak samples with the standards. OTA determination in
samples was based on the external standard calibration method, using an OTA concentration range of
0.05–100 ppb. Recovery rates was calculated using Merkén samples spiked with 50 and 100 ppb of
OTA.
2.6.3 AFLs determination
Aflatoxins quantification was performed according to Soares et al. (2010) using High
Performance Liquid Chromatography (Waters, Milford, MA, USA) with a reverse-phase C18 silica gel
column (4.6x250 mm, 5 µm), equipped with a Varian 9002 pump (Agilent, Palo Alto, CA, USA), a Varian
Prostar 410 autosampler and Jasco FP-920 fluorescence detector (Jasco Europe, Cremella, Italy).
Excitation and emission wavelengths was set at 365 and 435 nm, respectively. An isocratic mobile phase
of water/acetonitrile/methanol (3:1:1, v/v/v) was used with a flow rate of 1.0 mL/min. AFLs were
19
identified by comparing the retention time of the peak samples with the standards. AFLs determination
in samples was based on the external standard calibration method, using an AFLs concentration range
of 0.1–20 ppb. Recovery rates was calculated using Merkén samples spiked with 50 and 100 ppb of
each AFB1, AFB2, AFG1 and AFG2.
3. RESULTS
3.1 Fungal isolation and identification
A total of 206 fungal strains belonging to 9 fungal genera were identified using classical macro-
and micro-morphology. The strains were isolated from berry fruits of C. annuum used for the manufacture
of Merkén in all sampling points. From the total isolated fungal strains, 86 strains (41.7%) were isolated
on DG18, 80 strains (38.8 %) were isolated on DRBC and 40 strains (19.4 %) were isolated on MEA.
From total fungi, 190 strains (92.2 %) were identified by molecular biology techniques at the species level.
All the fungal identified strains were deposited at both CCCT/UFRO and MUM culture collections with
their respective unique strain identification numbers (Annex I, Table A).
From the sampling point I (just at the day of ripe fruits harvest), 61 fungal strains were identified
(Figure 4). From these, strains were distributed among Penicillium spp. (25), Fusarium spp. (14),
Alternaria spp. (9), Aspergillus spp. (7) and others (6). In the case of Penicillia, the species with greatest
abundance were P. veridicatum (6; 24%), P. brevicompactum (5; 20%) and P. crysogenum (4; 16%).
Whereas for fusaria the species with greatest abundance were F. oxysporum (5; 38%) followed by F.
esquiseti (4; 29%).
For Alternaria, A. alternata (8; 89%) represents the most abundant species. Aspergilli represents
a low amount of fungi in these sampling point being A. versicolor (2; 29%), A. dimorphicus (2; 29%) and
A. fructus (2; 29%) the most representative. The strains that did not belong to these major taxa were
identified and grouped as “Other species”, which were, Cladosporium westerdijkieae (3; 50%),
Trichoderma koningiopsis (2; 33%) and Boeremia exigua (1; 17%).
From the sampling point II (drying process), 46 fungal strains were identified (Figure 5). These
strains were distributed among: Penicillium spp. (24), Aspergillus spp. (12), Alternaria spp. (7) and
“Others species” (3). In this sampling point, Penicillia were the most predominant species, with greatest
abundance for P. crustosum (8; 33%) followed by P. glabrum (6; 25%). For Aspergilli, A. niger (6; 50%)
and A. flavus (3; 25%) were the most representative ones. For Alternaria, A. alternata (5; 71%) represents
20
the most abundant species. Cladosporium westerdijkieae (1; 33%), Trichurus spiralis (1; 33%) and
Colletotrichum coccodes (1; 33%) were also identified and grouped as “Others species”.
Figure 4. Total of fungal strains isolated in the sampling point I (just at the day of ripe fruits harvest). Number and percentage of strains: (A) at genus level; (B) Penicillium; (C)
Aspergillus; (D) Fusarium; (E) Alternaria and (F) Other species.
21
From the sampling point III (during the smoking process), 83 fungal strains were identified (Figure
6). These strains were distributed among: Penicillium spp. (63), Aspergillus spp. (18), and “Others
species” (2). In these case, Penicillium spp. and Aspergillus spp. were the most dominants ones. For
Penicillia, P. glabrum (13; 21%), P. brevicompactum (13; 21%), P. cyclopium (8; 13 %) and P. crustosum
Figure 5. Total of fungal strains isolated in the sampling II (drying process). Number and percentage of strains: (A) at genus level; (B) Penicillium; (C) Aspergillus; (D) Alternaria and (E) Other species.
22
(7; 11%) were the most abundant ones. For Aspergilli, A. niger (5; 28%), A. awamori (3; 17%), A. flavus
(2; 11%), A. fumigatus (2; 11%) and A. pseudoglaucus (2; 11%) were the most abundant ones.
Trichoderma viride (1; 50%) and Microascus cinereus (1; 50%) were identified and grouped as “Others
species”. In this sampling point, Alternaria and Fusarium are not detected.
Figure 6. Total of fungal strains isolated in the sampling III (during the smoking process). Number and
percentage of strains: (A) at genus level; (B) Penicillium; (C) Aspergillus; and (D) Other species.
23
From all sampling points, Alternaria spp., Aspergillus spp., Boeremia spp., Cladosporium spp.,
Colletotrichum spp., Fusarium spp., Microascus spp., Penicillium spp., Trichoderma spp. and Trichulus
spp. were identify. All genera found in the all sampling point were group in a Venn diagram and analysed
using a Heat map (Figure 7).
The most representative genera were Aspergillus and Penicillium, being representatives of both
species in the three sampling points. From a point of view of relative abundance Penicillium, Aspergillus
and Alternaria were the most abundant respectively.
From all sampling points, 49 species were identify using DNA sequence. All species found in
were group in a Venn diagram and analysed using a Heat map (Figure 8).
In the Venn diagram it is observed that A. flavus, P. brevicompactum, P. cyclopium, P. glabrum,
P. polonicum and P. viridicatum were identified in the three sampling points. From a point of view of
relative abundance P. glabrum, P. brevicompactum and P. crustosum were the most abundant
respectively.
Figure 7. Venn diagram of all genus found in all sampling points (A) and Heat map showing the relative abundance of all fungi at genus level found in all sampling points (B)
24
3.2 Molecular Identification
A total of 190 fungi from all sampling point were analysed by molecular biology. From these, 136
strains were sequenced and identified using BenA amplicons and 54 strains using ITS1-5.8S-ITS2. From
BenA sequences, 32 sequences corresponded to Aspergillus species and 93 correspond to Penicillium
species. For the construction of the phylogenetic trees, only BenA sequences from Aspergillus and
Penicillium species were used.
Robustness of Aspergillus species identification was added to the cluster by the addition of BenA
nucleotide sequences of A. awamori (NRRL 35710; NRRL 4760; CBS 55765; GL 125), A. niger (CBS
55465; KACC 46497; KACC 46495; CBS 101699; CBS 101698), A. tubingensis (CBS 13448), A. flavus
(CBS 10092), A. fructus (NRRL 239), A. pseudoglaucus (CBS 379.75), A. fumigatus (CBS 112389; NRRL
A B
Figure 8. Venn diagram of all species identified in all sampling points (A) and Heat map showing the relative abundance of all fungi at species level found in all sampling points (B).
25
132) from GenBank database. All strains coming from this study grouped very well with their respective
reference strains.
In the case of Aspergillus species, the gene sequencing was well supported by its respective
dendrograms (Figure 9). Aspergillus niger (CCCT 18.11, 18.12, 18. 14, 18.15, 18.19, 18.22, 18.26,
18.27, 18.28, 18.30 and 18.89), A. awamori (CCCT 18.16, 18.18, 18.20, 18.50 and 18.53) and A.
brasiliensis (CCCT 18.23) are related in a well-defined cluster forming A. niger clade (bootstrap >80%).
The cluster of A. luchuensis (CCCT 18.70) and A. tubingensis (CCCT 18.21) are associated with poor
values of bootstrap of 64%, however, these fungi belong to the same clade (A. tubingensis clade). The A.
tubingensis and A. niger clade correspond to A. niger aggregate of the section Nigri.
All A. flavus (CCCT 18.35, 18.36, 18.120, 18.126, 18.128 and 18.186) are related forming the
section Flavi with very well bootstrap values of 99%. In the case of A. fructus (CCCT 18.145 and 18.182)
and A. sydowii (CCCT 18.92), a well-defined phylogenetic closeness with bootstrap values of 99% is
observed. Aspergillus sydowii belongs to A. sydowii subclade and A. fructus belongs to A. versicolor
subclade, both fungi are grouped in the clade A. versicolor and are belonging to section Versicolores.
Two isolated strains of A. pseudoglaucus (CCCT 18.85 and 18.97) are related in a cluster with
bootstrap of 99%. Finally, the strains A. fumigatus CCCT 18.94 and 18.103 are related with their
reference strains in a cluster with bootstrap of 99%.
26
Figure 9. Evolutionary relationships of Aspergillus isolates (BenA). The optimal tree with the sum of branch length = 0.59762008 is shown. The analysis involved 48 nucleotide sequences. All positions containing gaps and missing
data were eliminated. There were a total of 179 positions in the final dataset. “T”: type strain. Trichocoma paradoxa CBS 788.83 is used as outgroup.
27
Robustness of Penicillium species identification was added to the cluster by the addition of BenA
nucleotide sequences of P. expansum (CBS 28197), P. crustosum (CBS 101025 and 47184), P.
viridicatum (CBS 101034), P. polonicum (CBS 101479), P. cyclopium (CBS 47784 and 101136), P.
chrysogenum (CBS 306.48), P. brevicompactum (CBS 110067, 110068, 110069 and 257.29), P.
angulare (NRRL 35683), P. glabrum (NRRL 66390 and CBS 125543) from GenBank database. All strains
coming from this study grouped very well with their respective reference strain.
In the case of Penicillium species, the genes sequencing were well supported by its respective
dendrograms (Figure 10). Penicillium expansum (CCCT 18.131 and 18.116) are related in a well-defined
cluster with bootstrap values >90%. For P. polonicum (CCCT 18.29, 18.42, 18.55, 18.72, 18.80 and
18.88), P. viridicatum (CCCT 18.43, 18.44, 18.95, 18.111, 18.192, 18.196 and 18.204), P.
melanoconidium (CCCT 18.37 and 18.54), P. cyclopium (CCCT 18.60, 18.64, 18.66, 18.71, 18.81,
18.83, 18.84, 18.106, 18.124, 18.203 and 18.215) and P. crustosum (CCCT 18.46, 18.52, 18.62,
18.77, 18.82, 18.96, 18.99, 18.102, 18.117, 18.119, 18.123, 18.132, 18.134 and 18.138) the cluster
present bootstrap values >70%.
All these strains belong to the serie Viridicata. Penicillium verrucosum (CCCT 18.33) and P.
discolor (CCCT 18.91) belong to the serie Verrucosa and serie Camemberti, respectively. This large
cluster of series Viridicata, Verrucosa and Camemberti correspond to the section Fasciculata. The three
P. chrysogenum (CCCT 18.170, 18.200 and 18.207) were grouped in a cluster with bootstrap values of
99%.
In the case of P. bialowiezense (CCCT 18.38), P. brevicompactum (CCCT 18.31, 18.39, 18.49,
18.57, 18.58, 18.59, 18.67, 18.68, 18.74, 18.90, 18.104, 18.105, 18.113, 18.129, 18.130, 18.139,
18.168 and 18.194) and P. buchwaldii (CCCT 18.107) were grouped in a very well cluster with bootstrap
value of 99%. These strains belong to the section Brevicompacta. Penicillium paraherquei (CCCT 18.40
and 18.65) are present in one cluster with bootstrap value of 100%. Penicillium waskmanii (CCCT 18.65)
and P. citrinum do not belong to the same cluster, however, they are very close related at genetic level;
both fungal species belong to the section Citrina.
Penicillium angulare (CCCT 18.109 and 18.110) and P. adametzioides (CCCT 18.108) are
associated in a cluster with bootstrap values of 99%. These fungi belong to section Sclerotiora. Finally, P.
glabrum (CCCT 18.32, 18.47, 18.51, 18.61, 18.63, 18.75, 18.76, 18.86, 18.87, 18.93, 18.96, 18.100,
28
18.101, 18.115, 18.118, 18.122, 18.135, 18.137 and 18.144) are associated in a well-defined cluster
(bootstrap value of 100%). these filamentous fungi belong to section Aspergilloides.
Figure 10. Evolutionary relationships of Penicillium isolates (BenA). The optimal tree with the sum of branch length
= 1.25708392 is shown. The analysis involved 109 nucleotide sequences. All positions containing gaps and
missing data were eliminated. There were a total of 276 positions in the final dataset. “T”: type strain. Talaromyces
flavus CBS 310.38 is used as outgroup.
29
Figure 10. Continued.
Talaromyces flavus CBS 310.38
30
3.3 Preliminary genetic characterisation of potentially OTA producer fungal strains
In order to identify, preliminarily, a putative OTA biosynthetic genes, amplification of otapksPN,
otanpsPN, otachlPN and otatraPN were performed (Figure 11). Penicillium verrucosum (CCCT 18.33),
Penicillium crustosum (CCCT 18.46), Penicillium nordicum (CBS 112573) and Aspergillus carbonarius
(MUM 01.08) were used for this analysis.
Considering the four genes under study, our results show that P. nordicum (CBS 112573) was
positive for the otanpsPN (≈700 bp), otapksPN (≈500 bp) and otatraPN (≈420 bp) amplicons,
nevertheless, negative for otachlPN. For P. verrucosum (CCCT 18.33), this was positive for the otanpsPN
(≈700 bp) and otatraPN (≈420 bp) and negative for otachlPN and otapksPN. In the case of P. crustosum
(CCCT 18.46), this was positive only for otatraPN (≈420 bp). A. carbonarius (MUM 01.08) was negative
for all genes tested (present only the IAC).
Figure 11. Agarose gel electrophoretic pattern of amplification of otapksPN, otanpsPN, otachlPN and otatraPN for P. nordicum (P.n.), P. verrucosum (P.v.), P. crustosum (P.c.) and A. carbonarius (A.c.). B= blank.
31
3.4 Quantitative analyses of OTA and AFLs contamination in Merkén
For verifying the co-occurrence of AFLs and OTA, twenty one Merkén samples from 8 farmers
and 13 markets from the Chilean Region of La Araucanía were analysed by HPLC with fluorescence
detection (Table 1-2). The retention time for the mycotoxins analysed was: AFB1, 17.18 min, AFB2, 14.39
min, AFG1, 12.90 min, AFG2, 11.00 min and OTA 11.28 min (For standard curves, see Annex II).
In the case of AFLs, only AFB1 were detected in 12 samples of Merkén (57%). AFB2, AFG1 and
AFG2 were not detected in all Merkén samples. Concentrations of AFB1 were in the range of 0.19±0.26-
1.44±0.10 µg/kg for 6 farmer samples. In markets samples, only 6 samples present AFB1
concentrations in the range of 0.29±0.37-1.67±0.32 µg/kg. The samples present very low concentration
of AFLs (near to the LOD). In relation to recovery rates (%), AFB1, AFB2, AFG1 and AFG2 present
48.91±3.61%, 66.58±0.97%, 46.16±0.65% and 70.47±0.57% respectively.
For OTA, the samples analysed showed a wide range of contamination. OTA was detected in all
Merkén samples in the range of 0.79±0.05-5.99±0.68 µg/kg for farmer samples and 0.83±0.83-
19.81±0.70 µg/kg for markets samples. The recovery rates (%) for OTA was 97.57±19.91%.
Samples OTA
(µg/kg)
AFB1 (µg/kg) AFB2 (µg/kg) AFG1 (µg/kg) AFG2 (µg/kg)
Farmer I 5.71±0.11 0.64±0.30* ND ND ND
Farmer II 3.38±0.24 0.19±0.26* ND ND ND
Farmer III 1.99±1.99 0.72±1.01* ND ND ND
Farmer IV 2.46±0.29 0.45±0.63* ND ND ND
Farmer V 2.39±0.31 0.50±0.11* ND ND ND
Farmer VI 0.91±0.26 1.44±0.10* ND ND ND
Farmer VII 5.99±0.68 ND ND ND ND
Farmer VIII 0.79±0.05 ND ND ND ND
Table 1. AFLs and OTA results from 8 farmers of the La Araucania Region, Chile. Values are the
average of two independent replicates ± standard deviation. Data shown in this table were not
corrected for recoveries.
*= Very close to LOD; ND= Not Detected.
32
4. DISCUSSION
Three different culture media (DG18, DRBC and MEA) were used to isolate the mycobiota on
each step of Capsicum annuum L. cv. "Cacho de Cabra” berry fruits chain production used for Merkén
production. The culture medium DG18 followed by DRBC showed the greatest potential to isolate fungi
from the collected samples (42% and 39%, respectively). This is due to the lower aw of both culture media
Samples OTA (µg/kg) AFB1 (µg/kg) AFB2 (µg/kg) AFG1 (µg/kg) AFG2 (µg/kg)
Market 1 2.89±0.39 ND ND ND ND
Market 2 2.8±0.32 ND ND ND ND
Market 3 13.56±1.56 ND ND ND ND
Market 4 6.49±2.05 0.95±0.23* ND ND ND
Market 5 3.23±0.60 1.62±0.23* ND ND ND
Market 6 3.50±0.12 0.29±0.41* ND ND ND
Market 7 10.36±0.69 ND ND ND ND
Market 8 1.37±0.13 0.29±0.37* ND ND ND
Market 9 1.43±0.08 ND ND ND ND
Market 10 1.70±0.17 0.33±0.47* ND ND ND
Market 11 5.99±2.73 ND ND ND ND
Market 12 19.81±0.70 1.67±0.32* ND ND ND
Market 13 0.83±0.83 ND ND ND ND
Table 2. AFLs and OTA results from 13 markets of the La Araucania Region, Chile. Values are the
average of two independent replicates ± standard deviation. Data shown in this table were not
corrected for recoveries.
*= Very close to LOD; ND= Not Detected.
33
(0.955 and 0.950, respectively) that usually gives good results for the isolation of xerophilic filamentous
fungi.
In this work, the amplification and sequencing of the housekeeping BenA gene was used for the
identification of fungi at the species level. Gene sequencing is reported in the last years as the gold
standard for the fungal identification at species level (Skouboe et al., 1999; Samson et al., 2004a; Geiser
et al., 2007; Houbraken et al., 2011; Rodrigues et al., 2011; Schoch et al., 2012; Soares et al., 2012;
Decontardi et al., 2017; Decontardi et al., 2018).
Our results showed that species from Alternaria, Aspergillus, Fusarium and Penicillium were the
most commonly species isolated from each step of C. annuum berry fruits chain production used for
Merkén production (Figure 7). These filamentous fungi found in chilli berry fruits can fall into two broad
ecological categories, field and storage fungi. Field fungi belonging to Alternaria and Fusarium species
could be of phytopathological and mycotoxicological risk during the field growth and ripening of the fruits.
Meanwhile, storage-related fungi that belong mainly to the genera Aspergillus and Penicillium could cause
a serious problem of spoilage and mycotoxin contamination of these agri-food products.
Previous studies reported mycotoxigenic fungi and mycotoxins in different varieties of C. annuum
(Hierro et al., 2008; Santos et al., 2011; Salari et al., 2012; Ham et al., 2016). In the case of Aspergillus
and Penicillium, some studies have identified a high incidence of these fungi and mycotoxins as AFLs
and OTA (Santos et al., 2008; Santos et al., 2011; Van de Perre et al., 2014; Ikoma et al., 2015;
Gambacorta et al., 2018; Motloung et al., 2018).
Due to the climatic conditions of the central-south region of Chile which is characterised as
temperate oceanic, the fungi that are mostly favoured, at growth and colonise ability, are those belonging
to the Penicillium genus. At ecologically point of view, Penicillium species are mostly saprophytes and the
majority of species show optimal growth at moderate to low temperatures (near of 25 °C) and are capable
to grow at aw below 0.9. (Frisvad and Samson, 1991; Pitt and Hocking, 2009a). According to the results
observed in the present study, Penicillium species was found in all sampling point as the most abundant
filamentous fungus.
In the sampling point I, just at the day of ripe fruits harvest, the main fungal species found belong
to Alternaria, Fusarium and Penicillium genera. According to the results obtained in the present study, a
high incidence of Penicillium followed by Fusarium and Alternaria was found in each step of chain
production.
Among Fusarium species, F. oxysporum was the most abundant (36%). Fusarium oxysporum is
a pathogenic fungus common in soils and the causative agent of Fusarium wilt, a deadly vascular wilting
34
syndrome in plants (Booth, 1971). In the case of Alternaria species, A. Alternata was the most abundant
one (89%). Fusarium oxysporum is a pathogen for plants, mainly affecting leaves and less severely to the
stolons, overall leading to the complete death of the plant. These results are consistent with the conditions
of the fruit and its proximity to the field (Nelson et al., 1981; Silva and Singh, 2012; Bajwa et al., 2010).
In the sampling point II, after 1 month of harvest (drying process), Fusarium was not found. In
contrast, Penicillium and Aspergillus began to be the abundant and unique fungal genera found in the
dry form of the berry fruits, taking into consideration the culture media used for the mycobiota isolation.
Penicillium crustosum and P. glabrum were the most representatives Penicillium species. Aspergillus
niger and A. flavus were found as other important fungal species in the sampling point II. These results
can be explained due to the decreasing in the aw (drying process).
The low aw could contribute to selectivity for moderate xerophilous fungi. Moreover, the ability of
some Aspergillus and Penicillium species to generate ascospores can be an advantage in relation to other
fungi, once it allows their survival to drying process (Dijksterhuis, 2007), e.g., conidia of A. niger, P.
chrysogenum and P. glabrum, exhibit a high resistance and low reduction of viable spores in a range of
56 and 62 °C (Baggerman and Samson, 1988).
In the sampling point III, during the smoking process, results obtained show a total absence of
Alternaria and Fusarium species. In this case, species belonging to Penicillium, Aspergillus, Trichoderma
and Microaascos were found. For Penicillium genus, the section Fasciculata, (P. crustosum, P.
viridicatium, P. verrucosum, P. discolor, P. melanoconidium, P. polonicum and P. cyclopium) was the
most representative. Most species of this section can grow well at 15 to 25 °C (except those species of
the series Verrucosa, i.e. P. verrucosum), and at low aw (Houbraken et al., 2016). Section Fasciculata
contains species that commonly occur on stored or manufactured foods (Houbraken et al., 2016).
Also, in the sampling point III, the follow species belonging to series Viridicata were found: P.
crustosum, P. viridicatium, P. melanoconidium, P. polonicum and P. cyclopium. Penicillium species
belonging to the series Viridicata are typically associated with stored cereal grains and those belonging to
series Verrucosa are associated with stored cereal grains and dried or salted meat products (Frisvad and
Samson, 2004; Houbraken et al., 2016). Species belonging to series Camemberti (i.e., P. discolor)
typically occur on foods with a high protein or lipid content like Capsicum annum berry fruits (Houbraken
et al., 2016).
On the other hand, fungal species belonging to the section Brevicompacta (i.e., P.
brevicompactum) and the section Aspergilloides (i.e., P. glabrum) represent the seconds most
representative groups in all the Peniciillia found at this sampling point. Penicillium brevicompactum grows
35
between -2°C and 30°C, with an optimum growth temperature around 23 °C, and can grow in an
environment with aw below to 0.78. It is one of the most xerophilic species of the Penicillium genus.
Penicillium glabrum can grows under temperature ranging from 0 to 35 °C and has been referred as a
xerophilic fungus (Pitt and Hocking, 1997).
Still about the fungal sample isolated from the sampling point III, the species of Aspergillus
section Nigri (A. niger, A. awamori and A. tubingensis) were the most representative (51%). Aspergillus
species have the capacity to develop in high temperature conditions. Relatively low aw allows them to
adapt and colonise a wide variety of cereals and dried fruits. Temperature ranging from15 to 30 °C and
aw ranging from 0,97 to 0.85 means the best growth conditions for Aspergillus section Nigri (Astoreca et
al., 2007).
On the other hand, the section Flavi (A. flavus, 11%), represented the second most representative
group in all Aspergillus species found at the sampling point III as well as in the sampling point II (25%).
Aspergillus flavus has been reported by some authors as an important fungus in the production of
mycotoxins and food contamination such as almonds, maize, peanuts (Rodrigues et al., 2011; Soares et
al., 2012; Fountain et al., 2015; Fakruddin et al., 2015).
The ecophysiology of the fungi, their natural occurrence on the environmental conditions at the
place of berry fruits of C. annuum production, and the low aw of the studied substrate can explain the
reason these fungal species are the most common found in all sampling points. Interestingly, according
to the fungal species succession observed in the present study, based on the mycobiota isolated from
each step of C. annuum berry fruits chain production, used for Merkén production, a selectively increasing
on the occurrence of Aspergillus and Penicillium species was observed (Figure 7 and 8).
Potential mycotoxigenic fungi found in the present study include 1) for Penicillium species: P.
brevicompactum, potentially producer of Brevianamide A (BrA) and Mycophenolic acid;, P. verrucosum,
potentially producer of BrA, Citrinin (CIT) and OTA; P. viridicatum, potentially producer of BrA and
Xanthomegnin; P. citrinum, potentially producer of CIT; P. chrysogenum, potentially producer of
Cyclopiazonic acid (CPA); P. crustosum, potentially producer CPA and Penitrem A; and P. expansum,
potentially producer of Patulin and CIT); and 2) for Aspergillus species: A. niger, potentially producer of
OTA and Fumonisin B2 and B4; and A. flavus, potentially producer of AFls and CPA.
Overall, in the present study, it was observed that Aspergillus section Nigri (i.e., A. niger) was the
group of potentially mycotoxigenic fungi with the highest incidence. Previous studies reported that
mycotoxigenic strains of Aspergillus section Nigri has a high incidence in samples with similar
characteristics (Martín et al., 2005; Almela et al., 2007; Varga et al., 2011). Taking into consideration
36
the current scenario of climate change and at the point of view of food safety, the results obtained in the
present study can represent a worrying.
Some authors have referred to the impact of climate change on the increasing of mycotoxin
production by mycotoxigenic fungi, which increases the incidence of mycotoxins with carcinogenic effects
in food products (Paterson and Lima, 2010a; Paterson and Lima 2010b; Battilani et al., 2016). For this
reason, contamination problems in food commodity, and particularly Merkén, can be increased leading,
consequently, to a seriously problem of public health in people consuming Merkén in Chile and abroad.
At genetic point of view, BenA region seems suitable. It shown good clustering with bootstrap
values higher than 70% (Hall, 2013), both for the strains obtained from chilli berry fruits in all sampling
points and for the reference strains.
In the case of genetics similarities of Aspergillus species, no atypical grouping values were found.
The overall clustering was good with bootstrap values commonly above 85%. However, closely related
species (i.e., A. niger and A. awamori, that belong to the section Nigri, Clade A. niger) were not
discriminated (Figure 9). These species are very closely related, are morphologically indistinguishable
and in some cases the genetic regions used to differentiate them are not effective, as is the case of ITS
regions.
The status of A. awamori has been revised several times and has been named with various
synonyms (Sakaguci et al., 1951; Samson et al., 2004b; Varga et al., 2011). However, in recent years,
due to their genetic differences, A. awamori has been classified as a cryptic phylogenetic specie of A.
niger (Perrone et al., 2011). Results obtained in the present study show that gene BenA may not be the
most appropriate choice for molecular identification of these species and, in general, sequencing of more
than one gene is required to obtain a robust identification. Some authors show similar results in their
phylogenetic trees using BenA, and in most cases, the use of more than one gene, or multilocus,
phylogenetic trees help to differentiate A. awamori from A. niger (Perrone et al., 2011; Varga et al., 2011).
Phylogenetic analyses of calmodulin sequences (CaM) and the translation elongation factor-1
alpha (TEF-1α) have been reported as good genetic regions for separating A. niger from A. awamori
(Perrone et al., 2011; Varga et al., 2011). In addition, some sequences of genes coding for enzymes,
associated in the production of OTA reported by Geisen (2006), such as chloroperoxidase, show a great
performance to differentiate A. niger from A. awamori at the phylogenetic level (Varga et al., 2011). The
amplification and sequencing of these regions in A. niger and A. awamori could add required information
to separate these fungal species found in chilli berry fruits.
37
The other species grouped in this phylogenetic tree do not present atypical groupings, that is,
they are associated to their respective strains and reference strains with values of bootstrap over 90%.
In the case of genetics similarities of Penicillium species, no atypical grouping values were found
(Figure 10). The overall clustering was good with bootstrap values commonly above 70%. Moreover, some
strains show variability within their clusters. The isolated strains of P. expansum (CCCT 18.131 and
18.116) were not associated to the same cluster with their reference strain (CBS 28197). However, they
present a bootstrap of 91%, which shows its high similarity of the gene BenA. In addition, more reference
strains from GenBank database could provide more robustness to the cluster and associate these species
in the same cluster.
The same trend is found in the clusters generated for P. brevicompactum (Figure 10). Some
intraspecific variability can be found in this section. Although the clusters are well defined, this variability
could indicate that P. brevicompactum could be more diversified than is currently known.
In the case of the cluster generated for P. crustosum, P. viridicatum, P. melanoconidium, P.
polonicus and P. cyclopium, these are genetically very close to each other. Several studies show similar
groupings among Penicillium species, which is not surprising, due to the fact that these fungi are classified
within the Section Fasciculata (Frisvad et al., 2004; Frisvad et al., 2013; Visagie et al., 2014a; Visagie et
al., 2014b).
Concerning all P. chrysogenum strains, these are associated in a well-identified cluster with
Bootstrap values of 99% (Figure 10). It is important to note that in Penicillium, BenA has limitations to
discriminate P. chrysogenum from P. camembertii species complexes. Studies conducted by Houbraken
et al. (2012) on the Penicillium section Chrysogena, showed that a large number of species are
phylogenetically closely related, and showed that different genes suggest different phylogenies. For
example, in some cases, P. chrysogenum cannot be differentiated from P. allii-sativii, even though CaM
distinguishes P. chrysogenum from P. allii-sativi (Houbraken et al., 2012). The results obtained in the
present study show a good grouping of P. chrysogenum, which can due to the low amount of isolates of
P. chrysogenum and species of the series Camemberti (Figure 10).
The fungal strains from chilli berry fruits classified as P. glabrum showed a high intraspecific
variability, which is observed in a high number of clusters within a larger cluster (Figure 10, Section
Aspergilloides). These data are supported by those information found in the literature.
Penicillium glabrum presents a great variability at the genetic level. Several studies show that
different sequence types of BenA within P. glabrum can be found (Basílio et al., 2006, Barreto et al.,
38
2011). Some authors classify the isolates of P. glabrum as "Penicillium glabrum complex" due to the
great variability between these species (Barreto et al., 2011; Houbraken et al., 2014).
In relation with the genetic characterisation of potentially OTA producer fungal strains, our
preliminary results (Figure 11) show, low congruence with the results reported in literature (Geisen et al.,
2006). For gene amplification, P. nordicum (CBS 112573) was positive for otapksPN (≈500 bp),
otanpsPN(≈720 bp), and otatraPN (≈420 bp). P. verrucosum (CCCT 18.33) was positive for otanpsPN
(≈720 bp) and otatraPN (≈420 bp). In P. crustosum (CCCT 18.46) only otatraPN (≈420 bp) was present.
Although the PCR reaction was performed correctly due to the presence of the IAC (ITS1-5.8S-
ITS2 region) it was not possible amplify all expected genes. First, we associate the absence of the
otachlPN gene due to a low temperature of annealing, this is because the primers used to amplify this
gene have a melting temperature of 68.1°C, which is high in comparison to the other primers and at the
alignment temperature used in the reaction (60 °C). However, to clarify this hypothesis, this gene was
separately amplified using the annealing temperature of 60 °C and no band was obtained (data not
shown). The negative results in this experiment can be due to an inefficient amplification protocol.
Although these data are preliminary, it is advisable to optimize the PCR conditions and optimise the
primers and DNA concentration, to obtain a better amplification and associate these genes with the
potential ochratoxigenic fungi in this samples.
In this study, the mycotoxins contamination of Merkén from the farmers and markets of the La
Araucania region are ranged between 0.79±0.05 to 19.81±0.70 µg/kg for OTA and 0.29±0.37 to
1.67±0.32 µg/kg for AFB1.
The Merkén samples showed a 100% OTA contamination however, only the sample from market
12 presents OTA concentration higher (19.8 ±0.70 μg / kg) than those established by the European
Union. Although the concentrations of OTA in the Merkén samples are within the European limit, the high
incidence of OTA is a serious warning. On the other hand, our results show that OTA concentration is in
general higher in the samples collected from markets than those provided by the farmers. This could be
due to poor hygiene conditions and increased humidity in the stores. Many studies show that the
occurrence of OTA contamination is greater in condiments in the process of storage and sale (Mandeel,
2005; Schweiggert et al., 2005; Santos et al., 2008; Santos et al., 2010c).
The high incidence of OTA could be due to the high incidence of black Aspergillus (i.e. A. niger
complex) in the early stages of Merkén production. Our results show that the Merkén production process
promote this type of mycotoxigenic fungi. However, it is necessary to analyse the mycobiota in subsequent
processes (grinding, additives, storage and sale) to verify other possible ochratoxigenic fungi.
39
OTA is classified as a group 2B carcinogen (IARC, 1993) and several studies show its high toxicity
and cumulative effect on the mammal’s body (Smith et al., 1994; Peterson and Lima, 2010b; Paradellsa
et al., 2015). The high incidence of OTA in the Merkén samples, added to the high and continuous
consumption of food condiment by the Chilean population, should be an important point to consider by
the health authorities. Studies conducted by Ikoma et al. (2015) show that the high incidence of OTA in
red chili peppers in specific geographical areas could be associated with a high incidence of Gallbladder
Cancer in the population.
In the case of AFLs, only AFB1 are present in Merkén samples (57%) in a very low concentration.
Our results only show 6 isolates of A. flavus that could not contaminate the samples significantly.
5. CONCLUSIONS
This work provides for the first time information about mycobiota in berry fruits of Capsicum
annuum L. cv. "Cacho de Cabra” used for Merkén’s production. This study confirms Aspergillus and
Penicillium are the two dominant fungal genera that emerge in the early stages of the Merkén production
process. Overall, these results show that the mycobiota present in C. annuum berry fruits during its
production process, before the Merkén production, is increasingly selective for occurrence potentially
mycotoxigenic fungi such as A. niger, A. flavus, P. expansum and P. verrucosum. Based on the identified
fungal species, the raw material used for Merkén production can be at risk of mycotoxin contamination.
The high co-occurrence of potentially mycotoxigenic fungi found in berry fruits of C. annuum used
for Merkén’s production and the high incidence of OTA in the Merkén samples, warning for the
assessment of the mycotoxigenic potential of isolated strains in the 3 points of fungal isolation.
The genetic characterisation of the mycotoxigenic potential of isolated fungal strains, and the
identification of mycobiota and mycotoxins in the milling and Merkén storage, should be continued in
order to stablish possible contamination points, providing input for effective strategies to mitigate
mycotoxin contamination and identification of the critical control points to generate a safe and high-quality
food product.
40
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50
ANNEX I – ISOLATED STRAINS
Table A-I. Sampling point, isolated media, gene sequencing, fungal Identification and CCCT/UFRO code
assigned to all strain identified at specie level using gene sequencing.
Sampling Isolated Media
Gene Fungal ID CCCT/UFRO Code
Point sequencing 3 MEA BenA Aspergillus niger CCCT 18.11
2 DG18 BenA Aspergillus niger CCCT 18.12
3 MEA BenA Aspergillus niger CCCT 18.14
3 DG18 BenA Aspergillus niger CCCT 18.15
3 DRBC BenA Aspergillus awamori CCCT 18.16
2 DG18 BenA Alternaria atra CCCT 18.17
2 MEA BenA Aspergillus awamori CCCT 18.18
2 DG18 BenA Aspergillus niger CCCT 18.19
2 DRBC BenA Aspergillus awamori CCCT 18.20
3 DRBC BenA Aspergillus tubingensis CCCT 18.21
3 MEA BenA Aspergillus niger CCCT 18.22
2 MEA BenA Aspergillus brasiliensis CCCT 18.23
3 DG18 BenA Aspergillus niger CCCT 18.26
2 DG18 BenA Aspergillus niger CCCT 18.27
2 MEA BenA Aspergillus niger CCCT 18.28
3 MEA BenA Penicillium polonicum CCCT 18.29
2 DG18 BenA Aspergillus niger CCCT 18.30
3 DG18 BenA Penicillium brevicompactum CCCT 18.31
3 DRBC BenA Penicillium glabrum CCCT 18.32
3 DRBC BenA Penicillium verrucosum CCCT 18.33
3 DG18 BenA Aspergillus oryzae CCCT 18.35
3 MEA BenA Aspergillus flavus CCCT 18.36
3 DRBC BenA Penicillium melanoconidium CCCT 18.37
3 DRBC BenA Penicillium bialowiezense CCCT 18.38
3 DRBC BenA Penicillium brevicompactum CCCT 18.39
3 DRBC BenA Penicillium paraherquei CCCT 18.40
3 DG18 BenA Penicillium polonicum CCCT 18.42
3 MEA BenA Penicillium viridicatum CCCT 18.43
3 DRBC BenA Penicillium viridicatum CCCT 18.44
3 MEA BenA Penicillium crustosum CCCT 18.46
3 DRBC BenA Penicillium glabrum CCCT 18.47
3 MEA BenA Penicillium brevicompactum CCCT 18.49
3 MEA BenA Aspergillus awamori CCCT 18.50
3 DRBC BenA Penicillium glabrum CCCT 18.51
3 DG18 BenA Penicillium crustosum CCCT 18.52
3 DRBC BenA Aspergillus awamori CCCT 18.53
3 DG18 BenA Penicillium melanoconidium CCCT 18.54
3 MEA BenA Penicillium polonicum CCCT 18.55
3 DRBC BenA Penicillium paraherquei CCCT 18.56
3 MEA BenA Penicillium brevicompactum CCCT 18.57
51
Table A-I. Continued
Sampling Point
Isolated Media Gene
sequencing Fungal ID CCCT/UFRO Code
3 DRBC BenA Penicillium brevicompactum CCCT 18.58
3 DG18 BenA Penicillium brevicompactum CCCT 18.59
3 DG18 BenA Penicillium cyclopium CCCT 18.60
3 MEA BenA Penicillium glabrum CCCT 18.61
3 MEA BenA Penicillium crustosum CCCT 18.62
3 DG18 BenA Penicillium glabrum CCCT 18.63
3 MEA BenA Penicillium cyclopium CCCT 18.64
3 DRBC BenA Penicillium waksmanii CCCT 18.65
3 DG18 BenA Penicillium cyclopium CCCT 18.66
3 DG18 BenA Penicillium brevicompactum CCCT 18.67
3 DRBC BenA Penicillium brevicompactum CCCT 18.68
3 MEA BenA Aspergillus luchuensis CCCT 18.70
3 DRBC BenA Penicillium cyclopium CCCT 18.71
3 DRBC BenA Penicillium polonicum CCCT 18.72
3 DRBC BenA Microascus cinereus CCCT 18.73
3 DG18 BenA Penicillium brevicompactum CCCT 18.74
3 DRBC BenA Penicillium glabrum CCCT 18.75
3 DG18 BenA Penicillium glabrum CCCT 18.76
3 DRBC BenA Penicillium crustosum CCCT 18.77
3 DRBC BenA Penicillium glabrum CCCT 18.78
3 DRBC BenA Penicillium polonicum CCCT 18.80
3 DRBC BenA Penicillium cyclopium CCCT 18.81
3 DRBC BenA Penicillium crustosum CCCT 18.82
3 DG18 BenA Penicillium cyclopium CCCT 18.83
3 DRBC BenA Penicillium cyclopium CCCT 18.84
3 DG18 BenA Aspergillus pseudoglaucus CCCT 18.85
3 DRBC BenA Penicillium glabrum CCCT 18.86
3 MEA BenA Penicillium glabrum CCCT 18.87
3 DG18 BenA Penicillium polonicum CCCT 18.88
2 MEA BenA Aspergillus niger CCCT 18.89
3 MEA BenA Penicillium CCCT 18.90
3 DG18 BenA Penicillium discolor CCCT 18.91
3 DRBC BenA Aspergillus sydowii CCCT 18.92
3 MEA BenA Penicillium glabrum CCCT 18.93
3 MEA BenA Aspergillus fumigatus CCCT 18.94
3 DRBC BenA Penicillium viridicatum CCCT 18.95
2 DRBC BenA Penicillium crustosum CCCT 18.96
3 MEA BenA Aspergillus pseudoglaucus CCCT 18.97
3 DRBC BenA Trichoderma viride CCCT 18.98
3 DRBC BenA Penicillium crustosum CCCT 18.99
3 MEA BenA Penicillium glabrum CCCT 18.100
3 DRBC BenA Penicillium glabrum CCCT 18.101
3 MEA BenA Penicillium crustosum CCCT 18.102
3 DG18 BenA Aspergillus fumigatus CCCT 18.103
3 DRBC BenA Penicillium brevicompactum CCCT 18.104
52
Table A-I. Continued
Sampling Point
Isolated Media Gene
sequencing Fungal ID CCCT/UFRO Code
3 DG18 BenA Penicillium brevicompactum CCCT 18.105
3 DRBC BenA Penicillium cyclopium CCCT 18.106
3 DRBC BenA Penicillium buchwaldii CCCT 18.107
3 MEA BenA Penicillium adametzioides CCCT 18.108
3 MEA BenA Penicillium angulare CCCT 18.109
3 DRBC BenA Penicillium angulare CCCT 18.110
3 DG18 BenA Penicillium viridicatum CCCT 18.111
3 DRBC BenA Penicillium brevicompactum CCCT 18.113
2 DG18 BenA Penicillium glabrum CCCT 18.115
2 DG18 BenA Penicillium expansum CCCT 18.116
2 DG18 BenA Penicillium crustosum CCCT 18.117
2 DRBC BenA Penicillium glabrum CCCT 18.118
2 DRBC BenA Penicillium crustosum CCCT 18.119
3 DRBC BenA Aspergillus flavus CCCT 18.120
2 DRBC BenA Penicillium glabrum CCCT 18.122
2 DG18 BenA Penicillium crustosum CCCT 18.123
2 MEA BenA Penicillium cyclopium CCCT 18.124
2 DRBC BenA Penicillium citrinum CCCT 18.125
2 DG18 BenA Aspergillus flavus CCCT 18.126
2 MEA BenA Aspergillus flavus CCCT 18.128
2 DG18 BenA Penicillium brevicompactum CCCT 18.129
2 DG18 BenA Penicillium brevicompactum CCCT 18.130
2 DG18 BenA Penicillium expansum CCCT 18.131
2 DG18 BenA Penicillium crustosum CCCT 18.132
2 MEA BenA Penicillium crustosum CCCT 18.134
2 MEA BenA Penicillium glabrum CCCT 18.135
1 DG18 BenA Alternaria botrytis CCCT 18.136
2 DG18 BenA Penicillium glabrum CCCT 18.137
2 DG18 BenA Penicillium crustosum CCCT 18.138
1 DG18 BenA Penicillium brevicompactum CCCT 18.139
2 DG18 BenA Alternaria botrytis CCCT 18.140
2 DRBC ITS1-5.8S-ITS2 Penicillium commune CCCT 18.141
2 DRBC ITS1-5.8S-ITS2 Penicillium crustosum CCCT 18.142
2 DG18 ITS1-5.8S-ITS2 Penicillium polonicum CCCT 18.143
1 DRBC BenA Penicillium glabrum CCCT 18.144
1 DRBC BenA Aspergillus fructus CCCT 18.145
1 DRBC ITS1-5.8S-ITS2 Cladosporium westerdijkieae CCCT 18.146
1 DG18 ITS1-5.8S-ITS2 Alternaria alternata CCCT 18.147
1 DG18 ITS1-5.8S-ITS2 Penicillium steckii CCCT 18.148
1 DRBC ITS1-5.8S-ITS2 Penicillium viridicatum CCCT 18.149
2 MEA ITS1-5.8S-ITS2 Penicillium polonicum CCCT 18.150
1 DG18 ITS1-5.8S-ITS2 Cladosporium westerdijkieae CCCT 18.151
1 DG18 ITS1-5.8S-ITS2 Cladosporium westerdijkieae CCCT 18.152
1 DG18 ITS1-5.8S-ITS2 Penicillium brevicompactum CCCT 18.154
1 DRBC ITS1-5.8S-ITS2 Penicillium viridicatum CCCT 18.155
53
Table A-I. Continued
Sampling Point
Isolated Media Gene
sequencing Fungal ID CCCT/UFRO Code
1 DRBC ITS1-5.8S-ITS2 Penicillium polonicum CCCT 18.156
2 DG18 ITS1-5.8S-ITS2 Aspergillus flavus CCCT 18.157
2 DG18 ITS1-5.8S-ITS2 Cladosporium westerdijkieae CCCT 18.158
2 DG18 ITS1-5.8S-ITS2 Penicillium viridicatum CCCT 18.159
2 DG18 ITS1-5.8S-ITS2 Penicillium glabrum CCCT 18.160
1 DRBC ITS1-5.8S-ITS2 Penicillium polonicum CCCT 18.161
1 DRBC ITS1-5.8S-ITS2 Fusarium equiseti CCCT 18.162
1 DRBC ITS1-5.8S-ITS2 Trichoderma koningiopsis CCCT 18.163
1 DRBC ITS1-5.8S-ITS2 Penicillium freii CCCT 18.164
1 DRBC ITS1-5.8S-ITS2 Fusarium oxysporum CCCT 18.165
1 DG18 BenA Fusarium incarnatum CCCT 18.166
1 DRBC ITS1-5.8S-ITS2 Fusarium redolens CCCT 18.167
1 DG18 BenA Penicillium brevicompactum CCCT 18.168
1 DRBC ITS1-5.8S-ITS2 Alternaria alternata CCCT 18.169
1 DG18 BenA Penicillium chrysogenum CCCT 18.170
1 DG18 ITS1-5.8S-ITS2 Alternaria alternata CCCT 18.171
1 DG18 ITS1-5.8S-ITS2 Alternaria alternata CCCT 18.172
1 DG18 BenA Fusarium equiseti CCCT 18.173
1 DRBC ITS1-5.8S-ITS2 Fusarium equiseti CCCT 18.174
1 DRBC ITS1-5.8S-ITS2 Penicillium freii CCCT 18.175
1 DG18 ITS1-5.8S-ITS2 Penicillium dipodomyicola CCCT 18.176
1 DG18 ITS1-5.8S-ITS2 Aspergillus versicolor CCCT 18.177
1 DRBC ITS1-5.8S-ITS2 Trichoderma koningiopsis CCCT 18.178
1 DRBC ITS1-5.8S-ITS2 Penicillium viridicatum CCCT 18.179
1 DG18 BenA Boeremia exigua CCCT 18.180
1 DG18 ITS1-5.8S-ITS2 Alternaria alternata CCCT 18.181
1 DRBC BenA Aspergillus fructus CCCT 18.182
1 DG18 ITS1-5.8S-ITS2 Fusarium redolens CCCT 18.183
1 DRBC ITS1-5.8S-ITS2 Aspergillus versicolor CCCT 18.185
1 DG18 BenA Aspergillus flavus CCCT 18.186
1 DRBC ITS1-5.8S-ITS2 Penicillium glabrum CCCT 18.187
2 DRBC BenA Trichurus spiralis CCCT 18.188
2 MEA ITS1-5.8S-ITS2 Alternaria alternata CCCT 18.189
2 DG18 ITS1-5.8S-ITS2 Alternaria alternata CCCT 18.190
1 DRBC ITS1-5.8S-ITS2 Alternaria alternata CCCT 18.191
1 DRBC BenA Penicillium viridicatum CCCT 18.192
1 DG18 ITS1-5.8S-ITS2 Fusarium oxysporum CCCT 18.193
1 DRBC BenA Penicillium brevicompactum CCCT 18.194
1 DG18 ITS1-5.8S-ITS2 Fusarium oxysporum CCCT 18.195
1 DRBC BenA Penicillium viridicatum CCCT 18.196
1 DG18 ITS1-5.8S-ITS2 Penicillium chrysogenum CCCT 18.197
1 DG18 ITS1-5.8S-ITS2 Fusarium oxysporum CCCT 18.198
1 DRBC ITS1-5.8S-ITS2 Penicillium brevicompactum CCCT 18.199
1 DRBC BenA Penicillium chrysogenum CCCT 18.200
1 DRBC ITS1-5.8S-ITS2 Alternaria alternata CCCT 18.201
54
Table A-I. Continued
Sampling Point
Isolated Media Gene
sequencing Fungal ID CCCT/UFRO Code
1 DRBC ITS1-5.8S-ITS2 Aspergillus dimorphicus CCCT 18.202
1 DG18 BenA Penicillium cyclopium CCCT 18.203
1 DRBC BenA Penicillium viridicatum CCCT 18.204
1 DRBC ITS1-5.8S-ITS2 Aspergillus dimorphicus CCCT 18.205
1 DG18 ITS1-5.8S-ITS2 Fusarium oxysporum CCCT 18.206
1 DRBC BenA Penicillium chrysogenum CCCT 18.207
1 DG18 BenA Fusarium incarnatum CCCT 18.208
1 DG18 ITS1-5.8S-ITS2 Fusarium equiseti CCCT 18.209
1 DG18 ITS1-5.8S-ITS2 Alternaria alternata CCCT 18.210
2 MEA ITS1-5.8S-ITS2 Alternaria alternata CCCT 18.211
2 MEA ITS1-5.8S-ITS2 Alternaria alternata CCCT 18.212
2 MEA ITS1-5.8S-ITS2 Alternaria alternata CCCT 18.213
2 DG18 ITS1-5.8S-ITS2 Colletotrichum coccodes CCCT 18.214
1 DG18 BenA Penicillium cyclopium CCCT 18.215
1 DG18 BenA Fusarium incarnatum CCCT 18.216
55
ANNEX II – STANDARD CURVES OF OTA AND AFLS
Figure A-II. Calibration curve of Ochratoxin A (OTA).
Figure B-II. Calibration curve of Aflatoxin G2.
y = 1075,8x + 736,3R² = 0,9972
0
20000
40000
60000
80000
100000
120000
0 20 40 60 80 100 120
Are
a (m
V/m
in)
ng/mL
OTA
y = 2010,6x + 324,81R² = 0,9993
0,00
5000,00
10000,00
15000,00
20000,00
25000,00
30000,00
35000,00
40000,00
45000,00
0 5 10 15 20 25
Are
a (m
V/m
in)
ng/mL
AFG2
56
Figure C-II. Calibration curve of Aflatoxin B2.
Figure D-II. Calibration curve of Aflatoxin B1.
y = 1887,3x + 320,1R² = 0,9994
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AFB1
y = 2777,1x + 442,73R² = 0,9994
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57
Figure E-II. Calibration curve of Aflatoxin G1.
y = 1038,4x + 211,16R² = 0,999
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