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1 Institute of Sciences of Food Production, CNR, Via Amendola, 122/O 70126
Bari, Italy
2 Southern Regional Research Center/ARS/USDA, New Orleans, LA 70124,
U.S.A.
3 Department of Microbiology, Faculty of Science and Informatics, University
of Szeged, H-6701 Szeged, P.O. Box 533, Hungary
4 Center for Microbial Biotechnology, BioCentrum-DTU, Building 221,
Technical University of Denmark, DK-2800 Kgs Lyngby, Denmark
5 Department of Food Science and Technology, Agro-Industry Faculty,
Kasetsart University, 10900 Bangkok, Thailand
6 CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The
Netherlands
7 Faculty of Technology and Management, Prince of Songkla University,
Suratthani Campus, 84100 Suratthani, Thailand
*
Correspondence: Giancarlo Perrone,
giancarlo.perrone{at}ispa.cnr.it
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Abstract |
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 TOP Abstract INTRODUCTIONCONCLUSIONSReferences |
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Keywords aflatoxins / Aspergillus Sect. Nigri / Sect. Flavi / grapes / ochratoxin A / polyphasic identification coffee beans
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INTRODUCTION |
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TOPAbstractINTRODUCTIONCONCLUSIONSReferences |
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Ochratoxin A (OTA) is a potent nephrotoxin which may contaminate various food and feed products (grains, legumes, coffee, dried fruits, beer and wine, and meat). It also exhibits carcinogenic, teratogenic and immunotoxic properties in rats and possibly in humans (IARC 1993). The genotoxicity of OTA remains controversial (EFSA 2006). OTA is receiving increasing attention worldwide because of its wide distribution in food and feed and human exposure that most likely comes from low level of OTA contamination of a wide range of different foods (Petzinger & Weidenbach 2002). The economically most important OTA producers belong to Aspergillus sections Circumdati and Nigri (Samson et al. 2004; Frisvad et al. 2004).
In this review we briefly analyze the biodiversity and the phylogenetic relationships within two of the most important sections: Flavi and Nigri occurring in some important agricultural products including grapes and derived products, coffee beans and other agricultural products. We find that, while A. flavus is involved in the majority of the agricultural contamination episodes, at least in the United States, the specific role of the S-strain and L-strain A. flavus has not yet been established.
Biodiversity of black aspergilli on grapes from Europe
Black aspergilli, which comprises species belonging to Aspergillus
section Nigri, are worldwide distributed and have a significant
impact on modern society. Many species cause food spoilage, and several are
used in the fermentation industry (Bennett
& Klich 1992), or candidate in the biotechnology industries.
A. niger has even been granted the GRAS (Generally Regarded As Safe)
status in certain industrial production processes by the Food and Drug
Administration of the US government. Although the main source of black
aspergilli is soil, they are among the most common fungi causing food spoilage
and biodeterioration of other material. Various reports evidenced that members
of the A. niger species complex, together with A.
carbonarius and A. japonicus/aculeatus are frequently
responsible for post-harvest decay of fresh fruit (apples, pears, peaches,
citrus, grapes, figs, strawberries, tomatoes, melons, etc.) and some
vegetables (especially onions, garlic, and yams); furthermore it is also among
the commonest fungi isolated from dried fruit, beans, oil seeds and nuts
(peanuts, pecans, pistachios, hazelnuts, almonds, walnuts etc.)
(JECFA 2001). Recently, the
significance of these species has completely changed since some of them, in
particular A. carbonarius, is considered as the main source of OTA in
grape and wine (Cabanes et al.
2002; Da Rocha Rosa et
al. 2002; Battilani &
Pietri, 2002; Magnoli et
al. 2003, Leong et
al. 2007a). Over the past five years several surveys and
reports were published dealing with the epidemiology, ecology and distribution
of black aspergilli occurring in wine grape and dried grape vineyards. Most of
the surveys were from Mediterranean and South American countries and
Australia. These studies clarified that the biseriate species A.
niger "aggregate" and Aspergillus carbonarius, and
the uniseriate species A. aculeatus and A. japonicus are the
prevalent species occurring on grapes (Da
Rocha Rosa et al. 2002;
Battilani et al. 2003;
Serra et al. 2005;
Leong et al. 2006;
Ponsone et al. 2007).
In general species of the A. niger aggregate appear to be the
dominant black Aspergillus species in all the countries studied,
although some vineyards and years showed higher incidence of A.
carbonarius isolates (Cabanes et
al. 2002; Tjamos et
al. 2004). In particular, the occurrence and frequency of
ochratoxigenic strains in A. carbonarius and A. niger
"aggregate" on grape proved to be similar in the Mediterranean
countries and in Australia. On the contrary, A. niger was reported as
the main ochratoxigenic species occurring on grapes in South America, while
A. carbonarius occurred in Argentina mainly on retailed dried vine
fruits with a low capacity to produce OTA
(Chulze et al.
2006).
Ochratoxin A production of black aspergilli occurring on grapes was widely studied in the last years with sometimes ambiguous reports on the toxigenicity and the percentage of toxigenic strains among the species. The OTA producing strains of A. carbonarius ranged between 70 and 100 % when grown in vitro and tested using HPLC, while the range of producing strains was around 2-20 % for A. niger and A. tubingensis (Battilani et al. 2006; Perrone et al. 2006a). Some reports claimed the production of OTA also by A. japonicus but it has not yet been confirmed (Dalcero et al. 2002; Battilani et al. 2003). Recently, Ponsone et al. (2007) studying the occurrence and toxigenicity of Aspergillus species in Argentinean vineyards found that A. niger aggregate was the most frequent species on grapes with 27 % of the isolates producing OTA. The authors also confirmed the production of OTA by A. japonicus and A. aculeatus strains, but this work lacks molecular identification of the strains.
Black aspergilli are one of the more difficult groups concerning classification and identification. The taxonomy of Aspergillus section Nigri has been studied by many taxonomists, leading to various species concepts (Table 1). The difficulties in species recognition within the Aspergillus niger "aggregate" and the fact that most of the studies carried out on black aspergilli occurring on grapes lack molecular characterisation of the strains perplexed the extent of their natural occurrence and species distribution on grapes and food. In this respect, in 2001-2002 a large survey of black aspergilli occurring on grape from 107 vineyards in different European countries was performed within the EU project Wine-Ochra Risk (QLK1-CT-2001-01761) in order to characterise the species diversity and the potential toxigenic strains in the Mediterranean basin. This survey led to the identification of four main populations separated molecularly using AFLP, RFLP and sequence analyses (Bau et al. 2006; Perrone et al. 2006a, 2006b). These populations included A. carbonarius, A. tubingensis, A. niger, and a group of Aspergillus "uniseriate" isolates morphologically indistinguishable from A. japonicus and A. aculeatus but clearly separated by molecular techniques (Fig. 1). The genetic variability of these four populations observed by AFLP polymorphisms ranged from 15 to 35 % in A. carbonarius, A. tubingensis and the Aspergillus "uniseriate" group and 45-55 % in the A. niger group. The higher genetic diversity encountered in A. niger reflect the complexity of this taxon/group and the difficulties of identification at species level. The main OTA producer was A. carbonarius (95-100 % of strains), while the production of OTA was limited to a smaller proportion of strains in A. niger and A. tubingensis (10-15 % of the strains). No OTA production was observed in strains belonging to Aspergillus "uniseriate" group.
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This species diversity was also revealed by sequence analyses of partial calmodulin (660 bp) and β-tubulin (1360 bp) genes which confirmed a significant molecular divergence of Aspergillus "uniseriate" group from other Aspergillus species. The description of a new species named A. uvarum isolated only from grape has been recently submitted (Perrone et al. 2007). Furthermore, during these surveys A. ibericus, a new species closely related to A. carbonarius and unable to produce OTA, was also described (Serra et al. 2006). Recently, a further characterisation of five atypical A. niger strains (Fig. 1) collected from Portugal grapes evidenced their similarity with other black Aspergillus isolates collected worldwide, which did not fit into any species of Aspergillus section Nigri. This new species called A. brasiliensis has recently been described and characterised by a polyphasic taxonomic approach by Varga et al. (2007) using macro- and micromorphology, secondary metabolite profiles, partial sequences of the β-tubulin, calmodulin and ITS genes, and AFLP analysis.
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In conclusion, a different species distribution of black aspergilli may occur in Europe in relation with metereological conditions (Battilani et al. 2006) and geographical areas: A. tubingensis and A. niger proved to be the dominant species in all countries, while A. carbonarius appears to be prevalent in southern Mediterranean areas (south of France, Southern Italy, Portugal and Greece). The distribution of A. ibericus is limited to Spain and Portugal, while A. uvarum occurs more frequently in Italy, France, Greece and Israel.
Biodiversity of black aspergilli on Thai coffee beans
Ochratoxin A contamination of coffee is a worldwide problem. The presence
of OTA in green coffee bean has been reported by several authors in wide
concentration ranging between 0.2 and 360 µg/kg
(Levi et al. 1974;
Taniwaki 2006). Extensive
sampling of green coffee beans of both Arabica and Robusta types worldwide
indicated that although OTA contamination is more frequent in some areas
including mainly African countries, no producing country was found to be free
of contamination (Taniwaki
2006). Although previously A. ochraceus was suggested to
be sole source of OTA contamination on coffee
(Stack et al. 1983),
recent studies indicated that other species, including A. steynii, A.
westerdijkiae, A. carbonarius, A. lacticoffeatus, A. sclerotioniger and
A. niger are also able to produce OTA on coffee
(Téren et al.
1997; Samson et al.
2004; Frisvad et al.
2004). Different types of black aspergilli were reported in coffee
bean from different countries. A. niger and A. carbonarius
occured most frequently. Extensive studies have been carried out on the
mycobiota of Brazilian coffee recently. From the study of arabica coffee beans
by Taniwaki et al.
(2003), the results showed
that A. niger was the species found most commonly (63 % of potential
OTA producers), but only 3 % of them produced OTA. A. ochraceus also
occurred commonly (31 % of isolates), and 75 % of those studied were capable
of OTA production, a much higher percentage than reported elsewhere. A.
carbonarius was found (6 % of isolates) only in the hottest region
sampled, and only from beans in the drying yard or in storage. However, 77 %
of the A.carbonarius isolates were capable of producing OTA. Other
studies reported similar species distribution on Brazilian coffee beans.
Martins et al. (2003)
used a conventional method to identify fungal flora in coffee bean. The
predominant fungal genus was Aspergillus, including A. niger
(83.3 %), A. ochraceus (53.3 %) and A. flavus (25 %). The
incidence of other genera was substantially lower than that of aspergilli.
Magnani et al. (2005)
isolated and identified Aspergillus spp. that contaminate coffee
beans by sequencing the ITS region of the isolates. The incidence of
potentially ochratoxigenic species was 82 % with A. niger being found
most frequently, followed by A. ochraceus and A.
carbonarius. However, the mycobiota of coffee beans in other countries or
different type of coffee beans can be significantly different, e.g. in Ilic
et al. (2007),
Vietnamese Robusta coffee beans were studied, and A. niger was the
only ochratoxigenic species recovered. However, in another study carried out
by Leong et al.
(2007b) A.
carbonarius isolates have also been recovered from Vietnamese Robusta and
Arabica coffee bean samples.
We examined the mycobiota of coffee beans came from Thailand to clarify which species could be responsible for OTA contamination in this region. Different types of coffee varieties are cultivated in Thailand. Coffea arabica is the one grown in the Northern mountain area with elevation of more than 2 500 feet above sea level and average temperature of 18-25 °C. Coffea canephora var. robusta is grown in the Southern region of Thailand characterised by a totally different geography and climate, with elevation of not more than 500 feet above sea level, much more rain fall and average temperature of 25-35 °C.
Molecular identifications have not been carried out in most studies dealing with the mycobiota of coffee beans, which could lead to mis-identification of some closely-related species. In this study we analyzed the black aspergilli isolated from coffee beans using a polyphasic approach including morphological examinations, analysis of extrolite profiles and sequence analysis.
For Arabica coffee bean samples from the North, two types of samples, parchment coffee bean and green coffee beans were examined. Overall results showed that approximately 75 % of the samples were contaminated by black aspergilli, and similar levels of contamination were observed for isolates belonging to Aspergillus section Circumdati. (Fig. 3) A. niger was the predominant species but there were sometimes more than two species colonising the same beans. The related species A. tubingensis and A. foetidus were also common. Discrimination between A. niger and related species could be easily archeived by partial β-tubulin gene sequencing (Fig. 4). All three species were clustered in separate clade. Compared to the molecular method using sequencing of the ITS regions and with RFLP analysis of rRNA by Magnani et al. (2005), β-tubulin gene sequencing is more applicable and proved to be more efficient for species identification. Surprisingly, A. carbonarius was not detected, possibly as a result of climate selection as A. carbonarius occurs more frequently in hot regions. So species belonging to both sections Circumdati and Nigri could be responsible for OTA contamination in this region.
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The diversity of black aspergilli recovered from Thai coffee beans is summarised in Table 3. Comparing the occurrence of black aspergilli from different parts of Thailand, remarkable differences were observed. A. carbonarius and A. sclerotiicarbonarius were found only in Southern Thailand while A. foetidus was found only in the Northern region. These differences could be due to differences in the geography, climate and methods used for coffee processing in the two regions. The so-called wet method is used for Arabica coffee processing while the dry method is used for Robusta coffee processing. Principally, the dry method has three basic steps: cleaning, drying and hulling. In Thailand, the whole Robusta cherry is directly dried with sun drying. Suarez-Quiroz et al. (2004) also reported that the dry method seemed to increase the presence of A. niger on the coffee beans. The wet method involves one more processing step: a fermentation step followed by cleaning to separate the beans from the pulp. This may cause changes in the natural substrate leading to changes in the species composition of the fungi colonising the beans. Differences in contact surfaces during processing may also play an important role in fungal contamination.
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In conclusion, diversity of black aspergilli in coffee beans occurring in Thailand depends on a combination of various factors including coffee variety, geographic region, climate and processing method. Significantly, more Robusta than Arabica beans were infected by black aspergilli, in agreement with the findings of Leong et al. (2007b) and Pardo et al. (2004). A. niger and related species are more important as contaminants of Arabica coffee beans in Northern Thailand, while A. carbonarius is responsible for OTA contamination of Robusta coffee beans in Southern parts of Thailand.
Genetic diversity in A. flavus and implications for agriculture
Aspergillus flavus is the most common species associated with
aflatoxin contamination of agricultural crops
(Cotty et al. 1994,
Cotty 1997)
(Fig. 7). A. flavus
populations are highly diverse and their stability in the soil and on the
plant is not well understood. An atoxigenic relative of A. flavus, A.
oryzae, is widely used in Asian fermentation processes. It is now
increasingly clear that A. oryzae is not a separate species, but
actually is only one many examples of atoxigenic variants of A.
flavus (Geiser et al.
2000). As much as 40 % of the soil isolates of A. flavus
are incapable of producing aflatoxins
(Cotty et al. 1994).
Addition of atoxigenic strains of A. flavus to the soil of
susceptible crops to dilute out toxin-producing strains is being used to
remediate aflatoxin contamination of cotton and peanuts
(Cotty and Bayman 1993,
Horn et al. 2000,
Horn and Dorner 2002).
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A. flavus soil populations also contain isolates from two morphologically distinct sclerotial size variants, termed the L-strain for isolates with average sclerotial size greater than 400 µm and the S-strain for isolates with sclerotial size less that 400 µm (Cotty 1997). On typical laboratory growth media S-strain isolates produce higher levels of aflatoxins, more abundant sclerotia, and generally fewer conidia. Atoxigenic S-strain isolates are very rarely found in natural environments. Another consistent difference between S- and L-strain isolates is the size of deletion of portions of the genes, norB and cypA in the aflatoxin cluster. The size of the deletion in the norB-cypA gene was 1.5 kb for S-strain isolates and 0.8 kb for L-strain isolates. The gene cypA encodes a P450 monooxygenase that is necessary for formation of G aflatoxins. The deletion, therefore, is the reason why A. flavus is incapable of producing G aflatoxins. (Ehrlich et al. 2004). Most interestingly, A. oryzae isolates have an S-strain type deletion even though they morphologically resemble L strain A. flavus and make abundant conidia. When this gap size is included in a phylogenetic dataset that includes polymorphisms in the omtA gene region of the aflatoxin cluster, a clade was distinguished that contained members of both aflatoxin-producing S strain isolates and L strain isolates incapable of AF production. Another clade was distinguished that contained both A. oryzae and L-strain isolates incapable of AF production. From this data we reasoned that the L-strain is the ancestral species and that A. oryzae derived from an atoxigenic L-strain ancestor, whereas S-strain isolates derived from an aflatoxin-producing L-strain ancestor (Chang 2006).
The adaptation of A. flavus to the carbon-rich environment of certain agricultural communities is perhaps conducive to gene loss. Many of the isolates incapable of aflatoxin production have multiple mutations in their aflatoxin cluster genes. A careful study of deletion patterns in different L-strain A. flavus isolates from peanut fields found that, in these isolates, part or most of the aflatoxin biosynthesis gene cluster is missing (Chang et al. 2005). Isolates of A. oryzae also have large deletions of the aflatoxin gene cluster (Lee et al. 2006). In some of these isolates the remaining aflatoxin biosynthesis genes Neighboured the telomere. Proximity to the telomere may make the cluster more unstable. In A. parasiticus when normal development is thwarted, by forced repeated mycelial transfer, the resulting isolate permanently loses some of its normal developmental functions (Kale et al. 2003). It does not form conidia properly or make aflatoxins. The defects in these isolates remain to be determined.
Production of aflatoxin and its precursor metabolites is associated with increased production of conidia (Wilkinson et al. 2004), but so far, unlike the protective role of melanin, no evidence has been found that the conidia are protected by making the aflatoxin cluster metabolites. It is thought that the red pigmented dothistromin may be a virulence factor for D. septosporum responsible for its pathogenicity to pine (Bradshaw et al. 2002). Like dothistromin, most of the aflatoxin precursor metabolites are red or orange. Because of their color, the metabolites could have helped to foster dispersal. In addition, since section Flavi isolates are normally saprophytic, polyketide metabolites may increase fungal survival in soil. Such a benefit may be unnecessary in carbon-rich agricultural environments. In such environments, the ability to make aflatoxins could be a vestigial function. To support this conjecture, when section Flavi isolates are collected from non-agricultural soils, almost all of the isolates examined were capable of producing aflatoxins (Ehrlich et al. 2007a). Furthermore, in some soils, A. flavus was not the most prominent species. Understanding the role of aflatoxin production and in general secondary metabolite production may only be possible if attempts are made to duplicate in the laboratory the conditions of the natural environment in which these aspergilli evolved.
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CONCLUSIONS |
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TOPAbstractINTRODUCTIONCONCLUSIONSReferences |
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Within A. flavus and other Aspergillus species capable of aflatoxin production, considerable diversity is found. Such diversity makes it more difficult to assign firm taxonomic identity to isolates from such populations. For example, should all A. flavus that are incapable of producing aflatoxins be considered to be A. oryzae? Such isolates are routinely found in agricultural fields, but only some are now classified as A. oryzae. We now know that loss of G-aflatoxin formation in A. flavus is a result of deletions in three genes encoding enzymes required for conv. of O-methylsterigmatocystin to aflatoxin G1 and G2, namely the cytochrome P450, cypA, and the reductases, nadA and norB. The aflatoxin clusters of A. parasiticus and the recently described related taxon, A. minisclerotigenes from Australia, West Africa, and Argentina that produces both B and G aflatoxins contain functional v.s of these genes (Pildain et al. 2007). Further studies are neded to clarify if the other newly described species, A. arachidicola, which is closely related to A. parasiticus, also carry these genes. The separation between A. parasiticus and A. flavus is estimated to have occurred more than 8 Mya. The conidia of A. minisclerotigenes resemble those of A. flavus while those of A. parasiticus are distinctly different in appearance. Further studies need to be done to sort out what selective factors, both environmental and genetic affect cluster gene stability in these related organisms. In this regard, we need to know if agricultural interactions play a role in causing gene instability? We expect that comparisons of different fungal genomes and developing a better understanding of regulatory relationships may help in answering some of these questions.
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Acknowledgments |
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References |
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TOPAbstractINTRODUCTIONCONCLUSIONSReferences |
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