Food & Feed Research

ALTERNARIA spp. ON SMALL GRAINS

JOURNAL No:
Volume 39, Issue 2
PAGES
79-88
KEYWORDS
Alternaria spp, small grains, identification of species, toxins
TOOLS
Jovana N. Vučković*1, Jovana S. Brkljača1, Marija I. Bodroža-Solarov1 , Ferenc F. Bagi2,
Vera B. Stojšin2, Jelena N. Ćulafić3, Milica G. Aćimović4
1Institute for Food Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
2Faculty of Agriculture, University of Novi Sad, Trg Dostiteja Obradovića 8, 21000 Novi Sad, Serbia
3Faculty of Medicine, University of Novi Sad Hajduk Veljkova 3, 21000 Novi Sad, Serbia
4Scholar of the Ministry of Education, Science and Technological development, Republic of Serbia

ABSTRACT

As a consequence of climate changes and global warming, high emergence of mycobiota on small grains may have negative impact on quality and safety of food and feed. Genus Alternaria is ubiquitous and includes large number of saproprobes and pathogens. Alternaria spp. are one of the major contaminants of small grains causing “black point” disease. Beside yield losses, Alternaria spp. are responsible for spoilage of commodities during transport, storage and in processing, which may lead to the reduction of technological quality and serious economic losses. There is a growing concern of Alternaria spp. due to their ability to produce secondary metabolites with different toxicological properties, which are harmful for human and animal health. Accurate identification of Alternaria spp. and their metabolites is a crucial phase in the implementation of preventive measures and controls in the system from farm to fork. Considering the importance of Alternaria spp. occurrence on small grains and Alternaria toxins risk assessment, additional studies in this area are indispensable.

Introduction

Alternaria spp. are cosmopolitan mould fungi and can be found in soils, plants, food, feed and indoor air. The genus Alternaria includes both saprobes and plant pathogens which have been reported worldwide infecting crops in the field and causing post harvest decay of many plant products (Thomma, 2003). Alternaria species are frequently found on small grains, causing yield losses in production and processing. Due to their growth even at low temperature, Alternaria spp. are well known post harvest pathogens, responsible for spoilage of food during refrigerated transport and storage (Ostry, 2008). Economical losses are mainly related to quality reduction due to decreased nutritive value, discoloration and insipidness (Kosiak et al., 2004).  In addition to losses in food and feed production, many Alternaria species are mycotoxin producers with different toxicological properties. The most important Alternaria toxins are alternariol (AOH), alternariol monomethyl ether (AME), altenuen (ALT), tenuazoic acid (TEA) and altertoxins (ATX-I, II, III) (Logrieco et al., 2009). The occurrence of Alternaria toxins in small grains and cereal-based products is a global issue of high concern, due to their potential health risks for humans and/or livestock. It has been reported that some Alternaria toxins might have even the carcinogenic effect (Liu et al., 1992). Based on the requests from the European Commission (EC) in order to highlight the need for possible follow up actions, the European Food Safety Authority (EFSA) provided a scientific opinion on the risks for animal and human health related to the presence of Alternaria toxins in food and feed (EFSA, 2011). Moreover, Alternaria spores are considered to be one of the most prolific fungal allergens, which has been associated with respiratory allergies and skin infections (Corden et al., 2003; Kilic et al., 2010; Pavon et al., 2010). Since the great importance of the genus Alternaria related to food safety and quality of small grains and cereal-based products, this paper presents the extensive overview of Alternaria species on small grains with focus on mycotoxin risks in food chain.

OCCURANCE OF ALTERNARIA SPP. ON SMALL GRAINS

Since it has been originally described in 1816 by Nees (Nees von Esenbeck 1816-1817) with A.tenuis as the only type member of the genus, Alternaria species are widely distributed infecting a broad range of economically important crops. Cereal grains are frequently infected by species of the genus Alternaria, in particular A.alternata, which can cause disease called “black point”. Discoloration of ears and grains (particularly embryo end of the grain) is due to the presence of mycelia and conidial masses with dark pigment, melanin, which is characteristic for Alternaria genus (Thomma, 2003). High humidity or frequent rainfall from the milky ripe to soft dough stage can often favor infection by these fungi and may cause serious losses (Logrieco et al., 2003). The importance of “black point” disease does not reflect to yield reduction as much as on the quality of milling wheat, barley and oats for processing. The majority of mycobiota in wheat flour are derived from initial infected kernels in the field (Plavšić et al., 2007). Poor flour and bran color due to discoloration of the grain can have a significant economic impact. Decreased nutritive value, discoloration and insidipidness reduce technological quality of cereal products (Kosiak et al., 2004). Besides damages on ears and grains, disease may occur on leaves in the form of leaf blight lesions, usually caused by species A.triticina. Alternaria spp. may occur in storage causing spoilage of small grains and small-grains based products. After harvest, mainly physical factors dictate whether or not fungi will grow and/or produce mycotoxins. The primary factors influencing fungal growth in stored food products are the moisture content (more precisely, the water activity) and the temperature of the commodity. However, Alternaria growth may be favored in grains stored in moisture storage, but infection may also spread from affected plant products to adjacent healthy ones by secondary infections (Barkai-Golan&Nachman, 2008).
There is a wide range of Alternaria spp. distributed worldwide in both humid and semi-arid regions. Besides wheat, the prevalence of Alternaria spp. was recorded on barley (Medina et al., 2006; Hudec, 2007), oat (Kwasna & Kosiak, 2003) and rye (Grabarkiewicz Szczesna et al.,1989). A.alternata and A.tenuissima are the most frequently reported on wheat in the Mediterranean countries (Logrieco et al., 1990, Bensassi et al., 2009), followed by Estonia (Kütt et al., 2010), Slowakia (Maškova et al., 2012), and Argentina (Patriarca et al, 2007), while A.infectoria was predominate in Norway (Kosiak et al., 2004) Alternaria triticina causes significant yield losses in wheat on the Indiansubcontinent, whence it originates (Prasada & Prabhu, 1962) and has been reported in Argentina as well (Perelló and Sisterna, 2006). Since this specieshas been considered as quarantine pathogen in many countries, it will be necessary to investigate the incidence and importance of this disease in wheat areas worldwide. Recently, Toth et al. (2011) reported a new species on the Hungarian wheat, A.hungarica, considering it a minor foliar pathogen with small economical importance. In Serbia the detected species on wheat are A. alternata and A. logipes (Ivanovic et al.), while in researches of mycobiota on spelt wheat predominant species were A. alternata and A. tenuissima (Vučković et al., 2012). The main Alternaria species of phytopathological interest of small grains are listed in Table 1.
Table 1. Distribution of Alternaria species on small grains across the world

Country

Alternaria spp.

References

Mediterranean countries

A.triticina

Logriecco et al., 1990

A.alternata

Norway

A.infectoria

Kosiak  et al., 2004

A.tenuissima

A.alternata

Estonia

A.alternata

Kütt et al., 2010

A.tenuissima

Tunisia

A.alternata

Bensassi et al., 2009

A.tenuissima

A.japonica

Argentina

A.tenuissima

Patriarca et al., 2007; Perelló & Sisterna., 2007

A.alternata

A.longipes

A.arborescens

A.gaisen

A.mali

A.triticimaculans

Serbia

A.alternata

Ivanović et al., 2011; Vučković et al., 2012

A.tenuissima

A.longipes

DETECTION METHODS OF ALTERNARIA SPECIES

The anamorphic genus Alternaria comprises nearly 300 described taxa (Simmons, 2007). Considering a specific mycotoxin profile of different Alternaria spp., an accurate identification is highly important for proper risk assessment. It is, however, problematic to isolate the fungi of interest due to the presence of competing fungi that may grow faster or produce antagonistic compounds. To overcome this problem fully selective media are desirable in detection procedures. According to literature data, most of the authors recommend Potato Carrot Agar (PCA) and V-8 juice agar which are selective for Alternaria species and encourage abundant sporulation (Hoog and Horre, 2002, Chou & Wu, 2002; Kosiak et al., 2004, Vergnes et al., 2006, Hudec, 2007, Pavón et al., 2010, Maškova et al., 2012). Dichloram Chloramphenicol Malt Extract Agar (DCMA) showed to be very effective for detection of Alternaris strains according to Patriarca et al. (2007). Sørensen et al. (2009) proved that modified PCA (PCA-Mn) based on easy available manganese has several advantages compared to previously-developed semi-selective media for isolation of small-spored Alternaria. This medium makes it easier to detect, otherwise overlooked or suppressed, Alternaria isolates in environmental samples. For further detection the majority of authors are following instructions by Simmons (2007).
Molecular techniques, such as polymerase chain reaction (PCR) based system, have been applied as alternative assays replacing troublesome and time spending microbiological and chemical methods for detection and identification the most serious fungal toxin producers (Niessen, 2007). In PCR method ‘universal’ primers are used to isolate specific DNA regions followed by comparison of the sequence of the target species with that of other species within databases. The coding portions of many fungal 18S, 5.8S and 28S rDNA genes are highly conserved and primers to these regions have been generated (White et al., 1990). These allow the isolation of the internal transcribed spacer sequences (ITS-1 and ITS-2), which lie between the coding regions and are generally responsible for polymorphism at the level of species. The ITS region is amplified from the target fungus and sequenced to identify regions of DNA unique to the fungus of interest (Magan & Olsen, 2004). The ITS region was also used to design primers to differentiate large spectrum of Alternaria species (Pryor & Gilbertson, 2000; Pryor & Michailides, 2002; Hoog & Horre, 2002; Choui & Wu, 2002; Zur et al, 2002, Vergnes et al.,2006.; Bensassi et al., 2009; Pavón et al., 2011, Toth et al., 2010)

ALTERNARIA TOXINS

The problem of mould damage and the hazard of consuming damaged grains have been recognized since historical times, but mycotoxins have attracted considerable attention especially over the last three decades (Bhaat and Miller, 1991). Mycotoxins are secondary metabolites, produced by a range of fungal species. Generally, mycotoxins are chemically and thermally stable compounds, surviving storage and the most food processng conditions and therefore, persist to the final products (Matić et al., 2008). Mycotoxins in cereal-based foods and feeds are a global issue of high concern, due to their potential health risks for humans and/or livestock (Köppen et al., 2010). The Alternaria genus produces more than 70 mycotoxins and phytotoxins but only few occur naturally in foodstuffs or are of major toxicological significance. A. alternata is considered as the most important toxin producing species (Battilani et al., 2009).
The most important Alternaria toxins are divided into three mains structural classes according to Ostry (2008), Logriecco et al. (2009) and Battilani et al. (2009):
  • dibenzo-α-pyrone derivatives: alternariol (AOH), alternariol monomethyl ether (AME), altenuen (ALT), altenuisol (AS);
  • tetramic acid derivates: tenuazoic acid (TEA)
  • perylene derivatives: altertoxins I, II, III (ATX-I,-II,-III)
Alternaria toxins have been detected in wide range of cereal grains and small-grains based products such as bread and rolls, musli, fine bakery wares, pasta etc. (EFSA, 2011).
There are reports of AOH, AME and TEA in “black point wheat” on German market (Siegel et al., 2009, Asam, 2011), AOH, AME and ALT in Slovakian (Maškova et al., 2012) and Czech grains (Malachova et al., 2011), AOH and AME in small cereal grains in Poland Grabarkiewicz Szczesna et al.,1989) and AOH was detected in Estonian grains (Kütt et al., 2010). Li &  Yoshizawa (2000) analyzed wheat kernels in China which were significantly invaded by Alternaria spp., mostly A. alternata, with an average infection frequency of 87.3%. AOH was detected in 20 of 22 tested samples between 116-731 μg/kg and AME at a mean level of 443 μg/kg (range= 51-1426 μg/kg) in 21 samples. The presence of TEA, as major Alternaria toxin in terms of quantity, was detected with an average level of 2419 μg/kg and with a maximum quantity of 6432 μg/kg. The toxigenic potential of Alternaria strains isolated from Argentinean wheat, showed that TEA was the toxin produced at the highest concentration, but in lower frequency (72%), compared to AOH (87%) and AME (91%) (Patriarca et al., 2007). A comprehensive overview of the most common Alternaria toxins in small grains is presented in Table 2.
Table 2. Toxigenic  profile of Alternaria spp. in small grains reported across the worlda

Country

Mycotoxin

N

n>LOQ

LOD/LOQ (µg/kg)

Mean  (µg/kg)

Maximum

Method

Reference

Germany

AOH

13

1

1.05

-

4.01

HPLC-MS/MS

Asam et al., 2011

AME

13

1

0.03

-

0.06

TEA

27

2

50

49

851

Siegel et al., 2009

Belgium

AOH

1

0

12/23

n.d.

n.d.

LC-MS/MS

Monbaliu   et al., 2010

AME

1

0

18/39

n.d.

n.d.

Czech Republic

AOH

8

0

12/23

n.d.

n.d.

LC-MS/MS

Monbaliu et al., 2010

AME

8

0

18/39

n.d.

n.d.

Denmark

AOH

14

0

12/23

n.d.

n.d.

LC-MS/MS

Monbaliu et al., 2010

AME

14

0

18/39

n.d.

n.d.

Estonia

AOH

4

3

100

-

340

HPLC-DAD

Kütt et al., 2010

Hungary

AOH

7

0

12/23

n.d.

n.d.

LC-MS/MS

Monbaliu et al., 2010

AME

7

0

18/39

n.d.

n.d.

Sweden

AOH

18

16

35/45

-

335

HPLC-UV

Häggblom et al., 2007

AME

18

7

35/45

-

184

TEA

18

18

100/135

-

4310

Argentina

AOH

64

4

50

1054

1388

HPLC-UV

Azcarate et al., 2008

AME

64

15

50

2118

7451

TEA

64

12

80

2313

8814

Egypt

AOH

15

4

50

-

2320

HPLC-UV

Abd El-Aal et al., 1997

AME

15

2

300

-

1890

ALT

15

2

100

-

1480

ATX-I

15

2

200

-

1678

TEA

15

5

100

-

658

Russia

AOH

28

4

20

98

192

ELISA

Burkin & Kononenko, 2011

China

AOH

22

20

50

335

731

HPLC-FLD

Li & Yoshizawa, 2000

AME

22

21

50

443

1426

ALT

22

0

100

n.d.

n.d.

ATX-I

22

0

200

n.d.

n.d.

HPLC-UV

TEA

22

22

100

2419

6432

Australia

AOH

5

0

10

n.d.

n.d.

HPLC-UV

Webley at al., 1997

AME

5

0

10

n.d.

n.d.

 

TeA

5

1

10

-

15

a Source: European Food Safety Authority (2011); Abbreviations: AOH:alternariol; AME: alternariol monomethyl ether; ALT:altenuen; TEA:tenuazoic acid; ATX:altertoxin I; N: number of tested samples; n: number of samples>LOQ; LOQ: limit of quantification; LOD: limit of detection, n.d: not detected
The database concerning toxicological effects of Alternaria toxins in experimental animals and/or in humans is currently too limited to be used as a basis for detection of reference points for different toxicological effects. Experiments performed in rodents with purified Alternaria toxins indicate that the acute toxicity is in the following order: ALT > TeA > AME and AOH. However, these data are not suitable for the risk assessment of Alternaria toxins since the risk for public health related to these toxins is not expected to result from acute exposures (EFSA, 2011).Mycotoxins such as AOH and AME are found to be mutagenic and genotoxic and in certain areas in China might be responsible for oesophageal cancer (Liu et al., 1992). The mycotoxingenic potential depends on species and strains of the fungus, composition of matrix and environmental factors, such as temperature and moisture, and particularly water activity (aw) (Fernandez-Cruz et al., 2010). According to Magan et al. (1984) A. alternata needs rather high water activity (aw=0.98) to produce mycotoxins on wheat grain. Knowledge of mycotoxin production under marginal or sub-optimal temperature and aw conditions for growth can be important since improper storage conditions accompanied by elevating temperature and moisture content in the grain can favor further mycotoxin production and lead to reduction in grain quality (Oviedo et al., 2011)
Several methods have been reported in the literature for the determination of Alternaria toxins from food commodities. In particular, analytical methods are largely based on procedures, involving clean up by solvent partitioning or solid phase extraction, followed by chromatographic separation techniques, in combination with ultraviolet, fluorescence electrochemical and mass spectroscopic detection (Ostry, 2008). Since none of the mentioned techniques have been validated by interlaboratory studies and because of the lack of certified reference materials or proficiency studies available for the determination of Alternaria toxins, validated analytical methods for the quantification of Alternaria toxins are needed as a prerequisite for a survey on their occurrence in feed and food (Battilani et al., 2009).

PREVENTION AND FUTURE CONCERNS

The occurrence of mycotoxins in the food chain is an unavoidable and serious problem the world is facing with. Once the foodstuffs are contaminated with toxins it is impossible to eliminate them. Certainly, the best protection against mycotoxins is monitoring their presence in feed and food (Matić et al., 2008). Prevention of fungal contamination and thereby toxin production can be achieved either during preharvest stages by good agricultural practice and the use of a HACCP plan, as well as during postharvest stages by the application of proper drying, storage, and transport procedures (FAO, 2001). Application of fungicides at field might reduce fungal infection resulting in the decrease of mycotoxins production. However the modern trends are toward environmentally friendly alternatives at the field level rather than relying on chemicals (Bhaat et al., 2010). Development of resistant cultivars with the application of modern biotechnological methods would prove to be effective way for prevention and control hazardous fungi and their mycotoxins. A proven system of storage management which includes drying, avoiding grain damages and ensuring proper storage conditions is needed. To reduce or prevent production of most mycotoxins, drying should take place as soon as possible after harvest and as rapidly as feasible. Fungi cannot grow (or mycotoxins cannot be produced) in properly dried foods, so efficient drying of commodities and maintenance of the dry state is an effective control measure against fungal growth and mycotoxin production (FAO, 1989). Damaged grain is more prone to fungal invasion and mycotoxin contamination, thus it is important to avoid damage before and during drying, as well as in storage. Insect pests and storage pests may attack grain and due to  their activities accumulated moisture provides ideal condition for fungal growth.

However, besides toxicity and occurrence, the chemical behavior of mycotoxins during food processing needs to be understood when assessing risks associated with the consumption of food made from contaminated raw materials. Siegel et al. (2010) investigated the stability of AOH, AME and ALT upon bread baking using a spiked wholemeal wheat flour. The obtained results indicated that the Alternaria mycotoxins are barely degraded during wet baking, while significant degradation occurs upon dry baking, with the stability decreasing in the following order AME >AOH > ALT.

Further studies are needed to clarify the possible tranfer of the toxins into wheat flour after milling and their fates during food processing and coocking (Li and Yoshizawa, 2000) Additionally, more work is needed towards impact of Alternaria spp. on technological quality of small grains and small-grains based products. There is relatively large number of studies on the impact of fungal infection on food safety and yield parameters (Plavšić et al., 2010), but information on the effect of mycobiota infection on technological parameters is rather scant. A significant decrease in technological wheat quality in kernels attacked by Fusarium spp. and Alternaria spp. was proved in the researches of Šarić et al. (1997). According to these authors, increased enzyme activity of field moulds in samples with severe infection has negative impact on physical dough properties which leads to a complete wheat uselessness for further processing. Bodroža et al. (2012) found out that flour obtained from wheat affected with Fusarium spp. and Alternaria spp. showed less water and less stability during mixing and higher protein weakening during heating in comparison to the flour from wheat treated with fungicide.

Research gaps include also official validated methods for Alternaria metabolites analysis in order to carry out surveys of food and obtain an estimate of human exposure to Alternaria toxins. Additional toxicological work with purified Alternaria toxins, such as subacute toxicity and cancer studies, are also required (Magan and Olsten, 2004). Since there are no specific regulations for any of the Alternaria toxins in food, surveys to check the occurrence of these metabolities in order to ensure that contamination level do not pose a significant hazard to human health are strongly suggested.

CONCLUSION

Contamination of food and agricultural commodities by various types of toxigenic molds is a serious and a widely neglected problem. Alternaria speciesare ubiquitous plant pathogens and saprobes and are often found on small grains and small-grains based products. Alternaria spp. are also well known as post-harvest pathogens causing considerable economic losses to growers and the food-processing industry. They are of particular interest because suitable conditions may lead to production of a number of mycotoxins, such as AOH, AME, TeA, which may be implicated with human and animal health disorders. Most Alternaria mycotoxins exhibit considerable cytotoxic, carcinogenic, foetotoxic, teratogenic, antitumoral, antiviral and antibacterial activity. Prevention of fungal invasion ongrains is by far the most effective method of avoiding mycotoxin problems. It should consider an integrated management program, focusing on the good agricultural practice and food quality from the field to the consumer. Since there is currently no regulations set on Alternaria toxins in food and feed in the Europe nor worldwide, more attention is needed in monitoring of production of food safety products.

АCKNOWLEDGEMENTS

This work was supported by the project III46005 “Novi proizvodi cerealija i pseudocerealija iz organske proizvodnje” (“New products based on cereals and pseudo cereals from organic production”) financed by the Ministry of Education, Science and Technological development of the Republic of Serbia.

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