Chapter 1: Review

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CHAPTER 1

A review on aflatoxin studies

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PREFACE

In this part of the thesis, an attempt has been made to review the relevant literature

under four separate sections

In the first section a brief account of current and a general understanding of fungal

toxins i.e. mycotoxins mainly aflatoxins and an overview of physical, chemical and

biological properties has been presented. Further, a brief description of different types of

aflatoxins, its biosynthetic pathway, effect of aflatoxin on living organisms and its

detection methods are given.

The second section is focused on aflatoxigenic fungi, its ecology and population

biology in soil especially rhizosphere, its genetic diversity and persistence of aflatoxin in

soil.

In the third section, a brief description about the plant groundnut, its contamination

assessment and the methods used to prevent contamination.

The fourth section is a comprehensive account on the uptake of various toxins and

chemicals by plants, and the chance of aflatoxin uptake in plants has been evaluated.

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SECTION 1: MYCOTOXINS AND AFLATOXINS

Fungi are widely distributed in nature, grow over an extremely wide range of

nutrients, temperature and pH, and contaminate food products by many ways. Most of the

fungi are toxigenic in nature. Those species may impart a mouldy odour and taste during

a long storage (Sekar and Ponmurugan, 2008). They are considered as a major factor in

spoilage the food stuff, leading to substantial economic loss and a major public health

hazard by producing a wide variety of mycotoxins (Dwivedi and Burns, 1984).

Mycotoxins are extremely toxic chemical substances produced by certain

filamentous fungi growing naturally in many agricultural crops, especially in cereals

including maize, wheat, barley, rye and most oilseeds, both pre and post harvest and also

later when processed into food and animal feed products. The consumption of such

mycotoxin contaminated foodstuffs can produce toxic symptoms in animals and humans

which are known as mycotoxicosis. Because of the relatively high intake of cereals and

oilseeds contaminated with mycotoxins in the diet of intensively farmed animals such as

poultry, pigs and cattle, there has been extensive documentation of the adverse effects on

animal health and productivity (Berry, 1988; Smith and Moss, 1985).

Mycotoxins are in general, low molecular weight, non-antigenic fungal secondary

metabolites formed by way of several metabolic pathways, e.g. the polyketide route

(aflatoxins), the terpene route (trichothecenes), the amino acid route (aflatoxin) and the

tricarboxylic acid route (rubratoxin). Some mycotoxins such as cyclopiazonic acid are

formed from a combination of two or more of the principal pathways (Smith and Moss,

1985).

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a b c d

Figure 1. Important mycotoxigenic fungi

a.Aspergillus flavus (Mycology online, 2013) b. Fusarium proliferatum (Markus Richard

et al., 2013) c. Penicillium chrysogenum (Penicillium, 2012) d. Aspergiluus ochraceus

(Konietzny and Greiner, 2003)

Although a wide variety of fungi was known to produce mycotoxins, only a few

genera, Aspergillus, Penicillium, Fusarium, Alternaria and Claviceps are considered

important in foods. The most important mycotoxins being: Aflatoxins, Deoxynivalenol,

Ochratoxin A, Fumonisins, Zearalenone, Patulin and T-2 Toxin. Among these

mycotoxins, the most important ones are the aflatoxins.

Aflatoxins

Aflatoxins are the most potent naturally occurring chemical liver carcinogen

known. They are a group of approximately 30 related fungal metabolites (Liu and Wu

2010). AFB1, B2, G1, and G2 are the four major aflatoxins known in which AFB2 and

G2 are the dihydro-derivatives of the parent compounds B1 and G1 (Deshpande, 2002).

AFB1 have been classified as a Group 1 human carcinogen by the International Agency

for Research on Cancer (IARC, 2002). Its carcinogenicity has been demonstrated in many

animal species, including some rodents, nonhuman primates, and fishes (International

Programme on Chemical Safety and WHO, 1998). A Specific group of P450 enzymes in

the liver metabolize aflatoxin into aflatoxin-8, 9-epoxide, which may then bind to

proteins and cause acute toxicity (aflatoxicosis) or DNA to cause lesions that over time

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increase the risk of hepatocellular carcinoma (Groopman et al., 2008). Although aflatoxin

exposure in developed countries is low, it continues to be a major issue in developing

countries and a significant contributor to global disease burden (Liu and Wu 2010; Wild

and Gong 2010).

Figure 2. Structure of major aflatoxins

Source : (Santini and Ritieni, 2013)

Types of aflatoxin

Aflatoxins includes aflatoxin B1, B2, G1 and G2 (AFB1, AFB2, AFG1 and AFG2,

respectively). In addition, aflatoxin M1 (AFM1) has been identified in the milk of dairy

cows consuming AFB1-contaminated feeds. These four major aflatoxins, B1, B2, G1 and

G2, were originally isolated from Aspergillus flavus hence the name A-fla-toxin. The B

toxins fluoresces blue under UV light, and the G toxins fluoresce green. Other significant

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members of the aflatoxin family, M1 and M2, are metabolites of AFB1& B2 respectively

are originally isolated from bovine milk. Almost 28 aflatoxins have been identified and

characterized so far (Basappa, 2009).

Table 1. Species of Aspergillus that are known as aflatoxin producers

Species Known Occurrence Mycotoxins

Aspergillus flavus Ubiquitous in tropics and subtropics B aflatoxin (40% of

isolates),

A. parasiticus USA, South America, Australia B and G aflatoxins (nearly

100%)

A. nomius USA, Thailand B and G aflatoxins (usually)

A. bombycis Japan, Indonesia B and G aflatoxins.

A. pseudotamarii Japan, Argentina B aflatoxins,

A. toxicarius USA, Uganda B and G aflatoxins.

A.

parvisclerotigenus

USA, Argentina, Japan, Nigeria B and G aflatoxins,

A. ochraceoroceus Ivory Coast B aflatoxins,

Sterigmatocystin

A. australius The Southern hemisphere B and G aflatoxins, Source :-(IARC, 2002)

Aflatoxin biosynthetic pathway

The completed 70-kb DNA sequence containing the 25 genes or open reading

frames (ORFs) represents a well-defined aflatoxin pathway gene cluster.

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Figure. 3. Clustered genes (A) and the aflatoxin biosynthetic pathway (B).

Source :-(Yu et al., 2004) Abbreviations: NOR, norsolorinic acid; AVN, averantin; HAVN, 5_hydroxyaverantin; OAVN, oxoaverantin;

AVNN, averufanin; AVF, averufin; VHA, versiconal hemiacetal acetate; VAL, versiconal; VERB, versicolorin B;

VERA, versicolorin A; DMST, demethylsterigmatocystin; DHDMST, dihydrodemethylsterigmatocystin;

ST, sterigmatocystin; DHST, dihydrosterigmatocystin; OMST, O-methylsterigmatocystin; DHOMST, dihydro-O-

methylsterigmatocystin;AFB1, aflatoxin B1; AFB2, aflatoxin B2; AFG1, aflatoxin G1; AFG2, aflatoxin G2.

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Figure 3 represents the clustered genes (A) and the aflatoxin biosynthetic pathway

(B). The generally accepted pathway for aflatoxin and sterigmatocystin (ST) biosynthesis

is presented in panel B. The corresponding genes and their enzymes involved in each

bioconversion step are shown in panel A. The vertical line represents the 82-kb aflatoxin

biosynthetic pathway gene cluster and sugar utilization gene cluster in A.parasiticus and

A. flavus. The new gene names are given on the left of the vertical line, and the old gene

names are given on the right. Arrows along the vertical line indicate the direction of gene

transcription. The ruler at far left indicates the relative sizes of these genes in kilobases.

The ST biosynthetic pathway genes in A. nidulans is indicated at the right of panel B.

Arrows in panel B indicate the connections from the genes to the enzymes they encode,

from the enzymes to the bioconversion steps they are involved in, and from the

intermediates to the products in the aflatoxin bioconversion steps.

On average, about 2.8 kb of chromosomal DNA contains one gene. Among these,

genes, which are, large ones of about 5 to 7 kb each, encoding the fatty acid synthase

(FAS) alpha (5.8 kb) and beta (5.1 kb) subunits (FASα and FASβ) and polyketide

synthase (PKS; 6.6kb). Excluding these three large genes, the average size of the other 22

genes are about 2 kb. In the 5’ end of the cluster sequence, an approximately 2-kb DNA

region with no identifiable ORF was located. This sequence presumably marks the end of

this cluster in this orientation. The 3’ end of this gene cluster is delineated by a well-

defined sugar utilization gene cluster consisting of four genes. The 82,081-bp fully

annotated DNA sequence in A. parasiticus containing the aflatoxin pathway gene cluster

and the sugar utilization gene cluster has been submitted to the GenBank database

(nucleotide sequence accession number AY371490) (Yu et al., 2004).

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In Aspergillus spp., regulation of clustered AF and ST genes involves a pathway

specific transcription factor AflR that belongs to the zinc binuclear domain (Zn2 Cys6)

class (Chang et al., 1995; Payne et al., 1993). In A. parasiticus deletion of aflR (DaflR)

abolished the expression of most AF pathway genes (Cary et al., 2000) and prevented

production of aflatoxin. Overexpression of aflR in both A. parasiticus and A. flavus

caused upregulation of AF gene transcription and aflatoxin accumulation (Chang et al.,

1995; Flaherty and Payne, 1997). Detailed analysis of gene transcription using

microarrays identified 23 genes in A. parasiticus more highly expressed in the wild type

than in the aflR mutant. Eighteen of the genes differentially expressed on the microarray

were aflatoxin biosynthetic genes with a putative consensus AflR binding site (50-

TCGN5CGR-30) in their promoters (Price et al., 2006). But more recent studies report

that the promoters of almost all AF cluster genes contain AflR binding sites (Ehrlich,

2009; Ehrlich et al., 2008).

Effects of aflatoxins on living organisms

Although aflatoxins are most often noted for the ability to induce liver cancer at

extremely low doses, they can cause several problems of economic importance during

animal production (Pier, 1992). Once consumed, aflatoxins are also readily converted to

aflatoxin M, which occurs in milk and can thus cause both human exposure and sickness

in animal offspring (Pier, 1992; Robens and Richard, 1992). In many developed

countries, regulations combined with both an enforcement policy and an abundant food

supply can prevent exposure of human populations, in most cases, to significant aflatoxin

ingestion (Stoloff et al., 1991). However, in countries where either food is insufficient or

regulations are not adequately enforced, routine ingestion of aflatoxins may occur

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(Hendrickse and Maxwell, 1989; Zarba et al., 1992). In populations with relatively high

exposure, a role for aflatoxins as a risk factor for primary liver cancer in humans has

repeatedly been suggested (Robens and Richard, 1992). However, aflatoxins cause a

variety of effects on animal development, the immune system and a variety of vital

organs. Exposure to aflatoxins, particularly in staples, where people dependent upon

relatively few nutrient sources, must be considered a serious detriment. Aflatoxin B1 has

also been implicated as a cause of human hepatic cell carcinoma (HCC). Aflatoxin B1

also chemically binds to DNA and caused structural DNA alterations with the result of

genomic mutation (Groopman et al., 2008).

Most countries established regulatory limits for major aflatoxins B1, B2, G1 and G2,

which includes the sum of aflatoxin, as well as regulatory limits for aflatoxin M1.

Table 2. Permitted level of total aflatoxin level in food and feed samples.

Product Action Level

Food for human consumption (including corn,

groundnuts, etc) 20 ppb

Milk 0.5 ppb

Animal feeds that are not cottonseed meal or

corn 20 ppb

Corn/grain feed for immature animals, dairy

animals, or feed with unknown destination 20 ppb

Corn/grain feed for mature poultry, breeding

swine, or breeding beef cattle 100 ppb

Corn/grain feed for finishing swine at least 100

pounds 200 ppb

Corn/grain feed for finishing beef cattle 300 ppb

Cottonseed meal for swine, poultry, or beef

cattle 300 ppb

Source: Adapted from the USDA Aflatoxin Handbook (2002) and FDA Guidance for Industry (2000).

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There are several publications regarding the mechanism by which aflatoxin

produce toxin responses in plants. In case of plants, aflatoxin affects amylase activity in

germinating seeds causing inhibition of starch hydrolysis and consequent unavailability

of sucrose to the embryonic axis during the period of imbibiton (Sinha and Sinha, 1993).

The embryo of aflatoxin contaminated seeds nevertheless remain alive possessing fairly

high dehydrogenase activity and is incapable of growth in culture when supplemented

with sucrose (Chatterjee, 1988). Similar observations of aflatoxin mediated seed quality

deterioration were made by early researchers in various crops like maize, soybean, red

gram, green gram, black gram, lettuce, cotton etc (Crisan, 1973a, 1973b; El-Naghy et al.,

1999; Janardhana et al., 2011; Mahmoud and Abd-Alla, 1994).

Detection methods for aflatoxins and aflatoxigenic fungi

Several studies describe the cultural and analytical methods for the detection and

quantification of aflatoxins in agricultural commodities and cultures of fungi isolated

from them. These methods vary in accuracy and precision, depending on the end goal of

the analysis. Cultural methods include: 1) blue fluorescence, particularly in the presence

of an enhancer in the medium such as p-cyclodextrin (FL) (Fente et al., 2001; Jaimez

Ordaz et al., 2003); 2) yellow pigmentation, particularly on the undersides of colonies

(YP) (Gupta and Gopal, 2002; Lin, 1976); and 3) Colour change of the yellow pigment to

plum-red on exposure of the culture to ammonium hydroxide vapor (AV) (Saito and

Machida, 1999).

Additionally, there are a few selected media, which may be employed to help less

trained mycologist: the main selective media used are (i) (ADA); (ii) coconut cream agar

(CCA) and (iii) Czapek Dox agar (CZ). Aspergillus differentiation agar is a selective

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identification medium for the detection of A. flavus group strains (Pitt et al., 1983). With

this method is possible to distinguish these species from other Aspergillus based on the

development of orange colour on the reverse of the plates (Figure 4a). The CCA is used

to detect aflatoxin producer strains (Figure 4b). The production of aflatoxin is detected by

a blue fluorescence when exposed to a UV-light (Lin, 1976).

a b

Figure 4. Aspergillus flavus in different culture media

(a) A. flavus in AFPA, after 7 days incubation at 25ºC, with the characteristic orange

colour on the reverse side of the plate; (b) aflatoxigenic A. flavus grown on small plates of

CCA under long-wave UV light, after 7 days incubation (large plate = uninoculated CCA

plate).

Source : (Rodrigues et al., 2007)

Molecular methods for Aspergillus Section Flavi species differentiation

Molecular methods have been widely applied in the identification of a large

number of Aspergillus species. DNA amplification followed by DNA sequence analysis

is a powerful tool in taxonomy studies. In fact, Aspergillus is among the best studied

fungi genetically. The complete genome of A. flavus NRRL 3357 is now completely

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sequenced and has been released to the National Centre for Biotechnology Information

(NCBI) in July 2005, and numerous sequences from several strains of A. flavus group

species are available. The most widely used DNA target regions for discriminating

Aspergillus species are the ones in the rDNA complex, mainly the internal transcribed

spacer regions 1 and 2 (ITS1 and ITS2) and the variable regions at the 5’ end of the 28S

rRNA gene (D1-D2 region) (Hinrikson et al., 2005).

Single-copy conserved genes can also be used as targets for taxonomic studies

within the A. flavus group, when multi-copy segments from the rDNA complex lack

variability. Universal β-tubulin, calmodulin and topoisomerase II genes have been used in

fungal species identification but only within distantly related species, since variability is

generaly low (Rai, 2007). Genes involved in secondary metabolism are considered to be

more variable within closely related species (Rodrigues et al., 2007). Several genes

involved in aflatoxin biosynthesis have been identified, cloned and studied. They include

a regulatory gene locus aflR from A. flavus and A. parasiticus, and several structural

genes, e.g. pksA, nor-1, ver-1, uvm8 and omtA (Yu et al., 2004).

For studies within A. flavus, or for comparing A. flavus with other Aspergillus

species, and even for differentiating aflatoxin producers from non-producers, several

rDNA complex regions and structural aflatoxin genes have been tested for use as

molecular markers, with different levels of success. Some of these studies are based on

Polymerase Chain Reaction (PCR) amplification followed by sequencing for variability

analysis (Rai, 2007). But PCR amplification of known DNA target regions or genes

followed by Restriction Fragment Length Polymorphisms (RFLP) (Kumeda and Asao,

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1996), Single-Strand Conformation Polymorphisms (SSCP) (Kumeda and Asao, 1996) or

Heteroduplex Mobility Assay (HMA) (Christensen and Tuthill, 1986)are easier to apply

in most laboratories for the study of numerous test samples, and NCBI information can be

used for generating primers and DNA probes.

Aflatoxins may be produced but not detected because of the inherent detection

limits of the analytical systems. Not surprisingly therefore, none of the previously

described molecular methods have been able to clearly differentiate aflatoxin producers

from non-producers. Multiplex PCR with the aflatoxin pathway genes aflR, ver-1, omt-1

and nor-1 did not produce any clear pattern (Shapira et al., 1996).

Aflatoxin production and aflatoxigenic strains differentiation can be assessed by

monitoring aflatoxin genes expression in the A. flavus group, using the reverse

transcription PCR (RT-PCR) methodology. RT-PCR allows the detection of mRNAs

transcribed by specific genes by PCR amplification of cDNA intermediates synthesised

by reverse transcription. Such a system has been successfully applied to monitor aflatoxin

production and aflatoxin gene expression based on various regulatory and structural

aflatoxin pathway genes in A. parasiticus and/or A. flavus (Criseo et al., 2001), and it was

found to be very rapid and sensitive. Scherm et al [35] studied 13 strains of both species

and found consistency of 3 genes (aflD, aflO [syn. dmtA=omtB] and aflP [syn. omtA]) in

detecting aflatoxin production ability, further indicating them as potential markers

(Priyanka, 2012).

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SECTION 2: EFFECT OF RHIZOSPHERE & RHIZOPLANE

OF PLANTS ON AFLATOXIGENIC FUNGI

Ecology and population biology of aflatoxigenic fungi in soil

Soil serves as a reservoir for A. flavus and A. parasiticus, fungi that produce

carcinogenic aflatoxins in agricultural commodities. Populations in soil are genetically

diverse and individual genotypes show a clustered distribution pattern within fields.

Surveys over large geographic regions suggest that climate and crop composition

influence species density and aflatoxin-producing potential. Species belonging to

Aspergillus section Flavi are among the most intensively studied of all fungi, largely due

to their formation of carcinogenic aflatoxins in agricultural commodities that impact

animal and human health (Hussein and Brasel, 2001; Peraica et al., 1999). Aflatoxigenic

fungi are common components of soil mycobiota and are actively involved in

decomposition and nutrient cycling (Klich, 2002; White and Johnson, 1982). Members of

section Flavi utilize a wide range of carbon and nitrogen sources (Hesseltine et al., 1970;

Reddy et al., 1971) and produce a diversity of enzymes for degrading plant components

such as cellulose, pectin, lignin and lipids (Betts and Dart, 1989; Cotty, 1989; Long et al.,

1998; Olutiola, 1976). These fungi also invade developing seeds of crops, and the

primary inoculum for infection originates from soil.

Diversity of aflatoxigenic fungi

The agriculturally important species producing aflatoxin are Aspergillus flavus

and A. parasiticus. Both are members of Aspergillus section Flavi. Several other

members of this section also produce aflatoxin, including A. nomius, A. pseudotamarii

and A. bombycis (Cary et al., 2005). A. flavus commonly contaminates corn, groundnuts,

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cottonseed and tree nuts with aflatoxins before harvest and during storage (Diener et al.,

1987; Schroeder and Boller, 1973). The species typically produces AFB1 and B2 and

cyclopiazonic acid (Horn et al., 2001). In contrast, A. parasiticus is most prevalent in

groundnuts and synthesizes AFG1 and G2 in addition to the B aflatoxins but not

cyclopiazonic acid (Dorner and Horn, 2007). A. parasiticus generally produces high

levels of aflatoxins and non aflatoxigenic strains are rare (Horn, 2003; Tran-Dinh et al.,

2009).

The high diversity within A. flavus populations as revealed by colony morphology

in the laboratory has long been recognized. Isolates differ in phenotype according to

sclerotium production (nonsclerotial to predominantly sclerotial), conidial head formation

(densely sporulating to mostly mycelial) and conidial color (bright yellow green to dark

green) (Horn et al., 1995; Klich and Pitt, 1988; Raper and Fennell, 1965). The wide range

in the production of aflatoxins and cyclopiazonic acid by A. flavus isolates is equally

reflective of this variability (Horn and Dorner, 2002; Joffe, 1969; Schroeder and Boller,

1973).

Rhizosphere and rhizoplane

In 1904 the German agronomist and plant physiologist Lorenz Hiltner first coined

the term "rhizosphere" to describe the plant-root interface, a word originating in part

from the Greek word "rhiza", meaning root (Hiltner, 1904). He described the rhizosphere

as the area around a plant root that is inhabited by a unique population of microorganisms

influenced, he postulated, by the chemicals released from plant roots. In the years since,

the rhizosphere definition has been refined to include three zones which are defined based

on their relative proximity to, and thus influence from, the root (Figure 5). The

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rhizoplane is the medial zone directly adjacent to the root including the root epidermis

and mucilage. As might be expected because of the inherent complexity and diversity of

plant root systems, the rhizosphere is not a region of definable size or shape, but instead,

consists of a gradient in chemical, biological and physical properties which change both

radially and longitudinally along the root.

Figure 5. Schematic representation of a root section showing the structure of the

rhizosphere.

Predominance of aflatoxigenic fungi in rhizospheric and geocarposphere soil has been

studied by (Joffe, 1969). Fungal communities in soils of Nigerian maize fields were

examined by Donner et al. (2009) to determine distributions of aflatoxin-producing fungi.

According to them, the most common member of Aspergillus section Flavi (85% of

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isolates) was the A. flavus L-strain. According to (Griffin et al., 2001) mean population

densities of A. flavus group in two commercial fields, in Virginia, USA, were higher than

found previously in most Virginia peanut fields and, along with moderate aggregation,

may be related, in part, to the occurrence of aflatoxin in these fields. Since both the fungi

and their metabolites gain access to the plant under field conditions, the potential effect

on seeds is of interest. The presence of aflatoxin and aflatoxigenic fungi in rhizospheric

and non-rhizospheric environment could potentially result in a number of adverse

environmental consequences. (Kloepper and Bowen, 1991) suggested that A. flavus

colonization of geocarposhpere is one of the mechanisms for infection and subsequent

afatoxin levels in seeds.

The effects of root exudates of groundnut roots on aflatoxigenic fungal growth

have been shown in several reports (Griffin et al., 1976; Kloepper and Bowen, 1991;

Pass, 1974). Aspergillus flavus is saprophytic most of its life cycle and grows on a wide

variety of substrates including decaying plant and animal debris. Thus, the populations of

the fungi are dependent on how well this organism competes in the soil with other soil

flora. Two major factors that influence soil populations are soil temperature and soil

moisture. A. flavus can grow at temperatures ranging from 12-48oC and at water

potentials as low as -35 MPa. The optimum temperature for growth is 25-42oC. Thus this

fungi are semithermophylic and semixerophytic. Jofee (1969) pointed out that Aspergillus

species were most prevalent on heavy soil. Aspergillus and Rhizhopus species are

dominant in the rhizosphere soil of sugarcane (Abdel-Rahim et al., 1983). Abdel-Hafez

(1982), in his work on rhizosphere fungi of Triticum vulgare, got Aspergillus sps in 100%

of samples. Out of which A. flavus occurred at a frequency of 91.6%. A. flavus incidence

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increased with temperature and decreased with latitude. Less than 1% of isolates were A.

nomius or A. parasiticus. The observed differences among communities may reflect

geographic isolation and/or adaptation and may cause different vulnerabilities to aflatoxin

contamination among crops planted in diverse locations. The predominance of

aflatoxigenic fungi in rhizosphere soil was also observed by some other researchers like

Oyeyiola (2009).

Aflatoxigenic fungal infection in crops

Soil serves as a reservoir for primary inoculum that is responsible for the infection

of crops susceptible to aflatoxin contamination. The aerial fruiting of crops such as corn,

cotton and tree nuts dictates important differences in the manner of infection compared to

the subterranean fruiting of groundnuts (Payne, 1998). Aerial crops become infected by

A. flavus conidia that are dispersed by wind and vectored by insects. Sporulation on crop

debris deposited on the soil surface is clearly one source of inoculum. This has been

demonstrated experimentally through biological control in which nontoxigenic strains of

A. flavus and A. parasiticus sporulate profusely on inoculated grain that has been

distributed onto the soil surface. Corn and cottonseed become infected with nontoxigenic

strains, which reduce aflatoxin contamination by competing with native aflatoxigenic

strains (Cotty, 1994; Dorner et al., 1999). Olanya et al. (1997) showed that A. flavus

sporulates on waste corn deposited on the soil surface, creating a linear dispersal gradient

of airborne conidia away from the corn deposits. Secondly, windborne dust containing A.

flavus conidia may directly infect crops.

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Finally, insects disperse conidia of aflatoxigenic fungi directly from soil to the

crop. Soil insects in cornfields harbor A. flavus and A.parasiticus both externally and

internally (Lillehoj et al., 1980).

Fate of aflatoxin in soil

Only very few studies are reported about the fate of aflatoxin in soil. Angle (1986)

conducted some radiological assay to estimate the fate of AFB1 in the soil. He observed

only a low level (1-8%) mineralization into CO2 within 120 days. According to Angle and

Wagner (1980) AFB1 was observed to be rapidly reduced to aflatoxin B2 when added to

the soil. The speed of this reaction suggests a chemical mechanism. The resulting AFB2

decomposed at a much slower rate, declining to a level where it could no longer be

detected at 77 days. Goldberg and Angle (1985) carried out a work to determine the

leaching and adsorption potential of aflatoxin in soils. Leaching and adsorption studies

were conducted with a silt loam, clay loam, sandy loam, and silty clay loam soil. No

aflatoxin was found in the leachate from any of the soils.). Flavobacterium aurantiacum

NRRL B-184, a kind of bacteria from soils and water, showed a very high capability of

detoxifying aflatoxins in feeds and foods (Ciegler et al., 1966) with no new formation of

toxic products. But sometimes longer times of incubation with some fungi leads to

formation of new, unidentified blue-fluorescent compound (Detroy and Hesseltine, 1969).

Accinelli et al. (2008) demonstrated that AFB1 is rapidly degraded in field soil at 28 °C

(half-life ≤ 5 days).

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SECTION 3: THE PLANT GROUNDNUT

Figure 6. Groundnut plant

Source:- (Peanut Facts, 2013)

The groundnut/peanut (Arachis hypogaea L.), is a member of the subfamily,

Papilionaceae in the legume bean family (Fabaceae). Arachis hypogaea L.consists of two

subspecies hypogaea and fastigiata. (Rao and Tulpule, 1967).Groundnut is one of the

widely cultivated oilseeds in the world. Historically, the largest producer of groundnuts in

the world was India, but production in China overtook Indian production in the mid-

1990s. As of 2008-2009, China leads in production of groundnuts having a share of about

32.95% of overall world production, followed by India (18%) and the United States of

America (6.8%). India and China together produce almost 2/3rds of the world crop.

According to USDA Foreign Agricultural Service, India produced 6.25 million metric

tons of groundnuts in the year 2008-2009 (“Peanut,” 2013). Groundnuts are rich in

nutrients, providing over 30 essential nutrients and phytonutrients (Table 3).

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Table 3. Nutritional value of groundnut

Nutrient Amount Daily value

(%) Nutrient density

World’s Healthiest

Foods Rating

Manganese 0.71 mg 35.5 3.1 Good

Tryptophan 0.09 g 28.1 2.4 Good

Vitamin B3

(niacin) 4.40 mg 22.0 1.9 Good

Folate 87.53 µg 21.9 1.9 Good

Copper 0.42 mg 21.0 1.8 Good

Protein 9.42 g 18.8 1.6 Good

Source:http://www.whfoods.com

Figure 7. A groundnut plant showing the subterranean, seed-bearing, dry fruit

(called a pod).

Source: http://www.auntrubyspeanuts.com

After fertilization, the flower stalk (pedicel) of the peanut curves downward and

the developing fruit (legume) is forced into the ground by the proliferation and elongation

Chapter 1: Review

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of cells under the ovary. The peanut pod subsequently develops underground. As in other

members of the enormous legume family (Fabaceae), the roots bear nodules containing

nitrogen-fixing bacteria (Singh and Oswalt, 1995).

Aflatoxin and groundnut plants

Aflatoxin contamination of groundnut is one of the most important constraints to

groundnut production in many countries. It is also of significance in relation to public

health and exports (Anjaiah et al., 1989; Ardic et al., 2008; Gangawane and Ade, 2011).

Most countries/institutions give high priority to research on the groundnut aflatoxin

problem. Many national agricultural research systems (NARS) in Asia and Africa are

faced with this problem because of the difficulty in reducing aflatoxin contamination in

groundnuts and groundnut products to an acceptable level for export.

Aspergillus flavus infection of groundnuts occurs under both preharvest and

postharvest conditions (Cole et al., 1982; Diener et al., 1987; Lillehoj et al., 1980).

Preharvest infection by A. flavus and consequent aflatoxin contamination are important in

the semi-arid tropics (SAT), especially when end-of-season drought occurs. Drought

stress may increase susceptibility to fungal invasion by decreasing the moisture content of

the pod and seed, or by greatly lowering the physiological activity of the groundnut plant

(Cotty, 1994; Mehan et al., 1987).

Research on the aflatoxin problem is not regularly carried out by all groundnut

producing countries. This is because of the lack of qualified personnel. Nevertheless

some countries have been regularly monitoring groundnuts and groundnut products for

aflatoxin at different stages (farm, storage etc.). Before the 1980s, the aflatoxin problem

was considered postharvest issues. Therefore, research was focussed only on postharvest

Chapter 1: Review

24

problems. However, severe preharvest aflatoxin contamination was reported in Australia,

and in several countries in Asia and Africa. Since the early 1980s, several national and

international institutes have carried out research on preharvest aflatoxin contamination. It

is now well established that aflatoxin contamination is also a preharvest problem in the

semi arid tropics, particularly in areas where late-season drought is common. In the more

humid tropics, it is largely a postharvest problem. Investigations on the effects of climate,

edaphic factors, and their interactions in the field and under controlled conditions have

provided considerable information on pre and postharvest infect ion by A. flavus and

consequent aflatoxin production. Accordingly, a number of important recommendations

were formulated for use by farmers and those concerned with purchase, storage, and

processing of groundnuts and groundnut products (Abbas et al., 2011; Mehan et al.,

1987).

One of the possible means of reducing aflatoxin contamination of groundnut is the

use of resistant cultivars. Several studies have established the presence of field resistance

to seed infection by A. flavus in some cultivars. Resistance to pre harvest field infection is

particularly important in areas where late-season drought stress is a common occurrence

(Abbas et al., 2004; Kloepper and Bowen, 1991; Mehan et al., 1987). Some cultivars such

as J 11, 55-437, and PI 337394F have shown stable resistance to A. flavus across

locations. These sources among others have been used in breeding programs, and several

lines have been reported to possess resistance and produce high yield. Several breeding

lines from International crop research institute for semiarid tropics (ICRISAT) have been

reported to be resistant to seed infect ion and colonization; these are ICGVs 87084,

Chapter 1: Review

25

87094, 87110, 91278, and 91284. More resistant cultivars adapted to different product ion

systems need to be developed to meet the requirements of producers and users.

Efforts have been made to develop aflatoxin-resistant transgenic groundnut plants.

This can be an effective long-term genetic approach to the problem.

Several recommendations have been made for the control of aflatoxin by adopting

certain cultural practices. Some cultural practices, such as adjustments of sowing and

harvesting dates, and application of gypsum, are effective in preventing aflatoxin

contamination. The relationship between drought stress, termite population and seed

contamination has been established. A period of drought at the end of the rainy season

also favors aflatoxin contamination and increases the termite population. Among the

oilseeds, aflatoxins pose the most serious problem in groundnut, but they can occur in all

of them. Thus, at least 60 countries have proposed or established limits for the aflatoxin

level in food/feed (Nollet, 2004). Aspergillus flavus causes variety of diseases in

groundnut includes “pod rots” which results in decreased yield and reduced quality. Pre-

harvest aflatoxin contamination of groundnuts was reported to occur commonly in several

parts of the world (Joffe, 1969). The ways and means under which the production of

aflatoxin takes place on the standing crops have been the subject matter of investigation

by many researchers. Soil and plant residues are major reservoirs for sustaining the

Aspergillus spp. This is responsible for contamination of soil and crops with aflatoxins.

The farmed soil samples of groundnut were found to contain A. flavus (Accinelli et al.,

2008; Tran-Dinh et al., 2009). Several biocontrol agents have been reported to control

aflatoxin in groundnut. Cotty (1989) has done considerable research on the use of

nontoxigenic strains of A. flavus to control aflatoxin contamination. This approach is

Chapter 1: Review

26

based on the substitution of aflatoxin-producing strains of A. flavus with nontoxigenic

strains. As high levels of the inoculum of nontoxigenic strains are required, this may

result in the increased incidence of aflaroot in the field, and increased seed infection can

lead to the production of free fatty acids and the loss of seed quality for commercial

processing.

Large-scale detoxification procedures, using ammonia under high pressure, have

been developed; these are now operational in Senegal and Sudan (Waliyar, 1997).

Detoxification techniques suitable for small groundnut processors are needed. In India,

some simple approaches for the detoxification of groundnut oil have been developed.

Detoxification of crude oil in binding aflatoxin in groundnut oil and cake was studied

(Mishra and Das, 2003). Some of these procedures can be used at the small-scale industry

or the household level. The use of red clays in West African countries has been found to

be very effective in binding aflatoxin in contaminated groundnut cake (Okello et al.,

2010). In Senegal, it was found that exposure to sunlight for 18 to 24 h destroyed 100%

of the toxin in contaminated oil (Rustom, 1997). The contaminated soil is kept in sunlight

in transparent and translucent containers. This simple method is a very useful way of

reducing aflatoxin levels, and can be used by oil processors at the village level. Other

methods such as use of electronic devices to remove infected seed from groundnut lots

have been used (Waliyar, 1997). Besides other factors, aflatoxin contamination is

suspected through uptake of aflatoxin present in soil plants.

Chapter 1: Review

27

SECTION 4: UPTAKE OF VARIOUS COMPOUNDS BY

PLANTS

Plants can be exposed to contaminants in different ways. Organic contaminants

enter through passive transport, which proceeds in the direction of decreasing chemical

potential, consists of a series of partitions between plant water and plant organic matter

within various plant components (Chiou et al., 2001). Accounts of the concentration of

non-ionic contaminants in plant in relation to the external concentration in water (or soil

solution) from extensive sources reveal that most of these contaminants enter plants

largely via passive process (Vanier et al., 2001, 1999). Active transport, which may

proceed against the electro-chemical potential gradient, occur for certain nutrients and

other (inorganic and organic) ions. The magnitude and effieciency of uptake depends on

source contaminant, concentration, contaminant properties, plant species/composition,

exposure time, and other system variables (Briggs et al., 1982; King et al., 1966; Li et al.,

2005; Lichtenstein, 1960; Walker, 1972).

The symplastic pathway in fresh roots dominated the transport of polar organic

compounds, while apoplastic pathway dominated the transport of non polar organic

compounds (Su and Zhu, 2007). As evidence for these concepts, several plant uptake

studies have been carried out in different plants in the last few decades found out that the

difference exist in the accumulation of trace elements in cucumber through uptake study.

Saxena and Stotzky (2001) found that maize plants do not take up Bt toxin either

from natural soil or sterile hydroponic solution. It was demonstrated that non-Bt corn and

other species do not take up toxin released to soil in root exudates of Bt corn, from the

degradation of the biomass of Bt corn or even as purified Bt toxin. Intawongse and Dean

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28

(2006) studied heavy metal uptake by vegetable plants grown on contaminated soil and

reported their accumulation. Soongsombat et al. (2009) made a survey of terrestrial plants

growing on lead mine area in Thailand and found out the uptake and accumulation of lead

in those plants. Uptake of veterinary medicines from soils into plants was studied by

Boxall et al. (2006).

The crop plant, which could come into contact with cyanobacterial toxin via spray

irrigation, has the ability of toxin uptake. Plant will be damaged by the toxins and growth

inhibition could occur, which will then lead to decrease in crop yield. The toxin taken up

could have a possible biotransformation of toxins in agriculturally important plants

should be investigated in greater detail (Peuthert et al., 2007). In recent years, much

emphasis has been devoted to the analysis of the toxins in infected plants by means of

chemical and immunological approaches (Neagu et al., 2009).

Jayakumar and Jaleel (2009) studied the uptake of cobalt in soyabean plants and

reported its accumulation in all parts of plant. Su et al. (2010) analysed that rice is more

efficient in arsenite uptake and translocation than wheat and barley through examining

the xylem sap of arsenic exposed samples.

Mycotoxin uptake by plants

A number of mycotoxins like patulin, citrinin, gliotoxin and griseofulvin have

been reported to be present in soils (Goldberg and Angle, 1985; Mertz et al., 1980; Rao et

al., 1982). Studies proved that maize and lettuce seedlings absorb aflatoxins from

contaminated water or soils respectively (Mertz et al., 1981, 1980). Llewellyn et al (1982)

studied the effect of Zn++

in uptake of aflatoxin from perlite and liquid cultured by Zea

mays seedlings, and they observed that the Zn++

(in the form of ZnSO4) added seedlings

Chapter 1: Review

29

were affecting uptake of aflatoxin. In the work conducted by Walker et al. (1984),

reported both the uptake and distribution of aflatoxin B1 (AFB1) by and within the roots

as well as the effects of the toxin upon time-dependent changes in root dry weight. They

showed that, most of the AFB1 which taken-up by roots, remains in a presumed

ribosome-cytosol fraction.

According to Jarvis et al. (1981), feeding experiments with fungus-produced

trichothecenes, show that Baccharis megapotamica absorbs, translocates, and chemically

alters these compounds to ones with structures analogous to those found in the plant in its

native habitat. The mycotoxins, which have no apparent ill effect in Baccharis

megapotamica, kill tomatoes, peppers, and artichokes and this may indicate the potential

for uptake in soils.

According to Rao et al. (1982) mycotoxins citrinin, patulin and terreic acid were

absorbed by rice seedling roots and translocated to shoots. McLean (1994) confirmed the

uptake of AFB1 from the culture medium by immune-cytochemical localization using

gold probe.

Similarly, Mantle (2000) proved that occurrence of ochratoxin A in some green

coffees might arise in the field directly from fungal activity in soil rather than from fungal

infection of cherries or processed green coffee. Both fumonisin B1 and FB2 were taken up

by roots of wheat plants, but both did not move up the plant when given via watering, but

accumulated in root tissues (Zitomer et al., 2010).

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30

AIM AND OBJECTIVES OF THE STUDY

In spite of several previous reports on mycotoxin uptake by plant roots, efforts to

investigate the chances of aflatoxin absorption by groundnut plants through roots and

accumulation in aerial plant parts including seeds were not undertaken. Without this

information, the various techniques used till now, to prevent aflatoxin contamination in

groundnuts, are open to question.

This study hypothesized that groundnut seedlings can uptake aflatoxin from the

soil in which they grow and translocate and accumulate in aerial plant parts. The

objectives include

1. Characterization of aflatoxigenic fungi in the rhizosphere and rhizoplane

of groundnut plants.

2. Identification of aflatoxins in field soils and plant parts such as roots,

stems, and pods of groundnut.

3. Quantification of aflatoxins in field soils and plant parts such as roots,

stems, and pods of groundnut.

4. Studies on direct uptake of aflatoxin by roots of groundnut seedlings and

its subcellular localisation.

5. Mechanism of aflatoxin uptake by groundnut plants and its accumulation

in seeds.