Chapter 1: Review. CHAPTER 1 A review on aflatoxin studies

Transcription

1 CHAPTER 1 A review on aflatoxin studies 1

2 PREFACE Chapter 1: Review 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. 2

3 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). 3

4 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). AFB 1, B 2, G 1, and G 2 are the four major aflatoxins known in which AFB 2 and G 2 are the dihydro-derivatives of the parent compounds B 1 and G 1 (Deshpande, 2002). AFB 1 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 P 450 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 4

5 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). Types of aflatoxin Figure 2. Structure of major aflatoxins Source : (Santini and Ritieni, 2013) Aflatoxins includes aflatoxin B 1, B 2, G 1 and G 2 (AFB 1, AFB 2, AFG 1 and AFG 2, respectively). In addition, aflatoxin M 1 (AFM 1 ) has been identified in the milk of dairy cows consuming AFB 1 -contaminated feeds. These four major aflatoxins, B 1, B 2, G 1 and G 2, 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 5

6 members of the aflatoxin family, M 1 and M 2, are metabolites of AFB 1 & B 2 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. USA, Argentina, Japan, Nigeria B and G aflatoxins, parvisclerotigenus 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. 6

7 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-omethylsterigmatocystin;afb 1, aflatoxin B 1; AFB 2, aflatoxin B 2; AFG 1, aflatoxin G 1; AFG 2, aflatoxin G 2. 7

8 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 welldefined 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). 8

9 In Aspergillus spp., regulation of clustered AF and ST genes involves a pathway specific transcription factor AflR that belongs to the zinc binuclear domain (Zn 2 Cys 6 ) 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 9

10 (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 B 1 has also been implicated as a cause of human hepatic cell carcinoma (HCC). Aflatoxin B 1 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 B 1, B 2, G 1 and G 2, which includes the sum of aflatoxin, as well as regulatory limits for aflatoxin M 1. Table 2. Permitted level of total aflatoxin level in food and feed samples. Product Food for human consumption (including corn, groundnuts, etc) Milk Animal feeds that are not cottonseed meal or corn Corn/grain feed for immature animals, dairy animals, or feed with unknown destination Corn/grain feed for mature poultry, breeding swine, or breeding beef cattle Corn/grain feed for finishing swine at least 100 pounds Corn/grain feed for finishing beef cattle Cottonseed meal for swine, poultry, or beef cattle Action Level 20 ppb 0.5 ppb 20 ppb 20 ppb 100 ppb 200 ppb 300 ppb 300 ppb Source: Adapted from the USDA Aflatoxin Handbook (2002) and FDA Guidance for Industry (2000). 10

11 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 11

12 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). Molecular methods for Aspergillus Section Flavi species differentiation 12 Source : (Rodrigues et al., 2007) 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

13 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, 13

14 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). 14

15 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, 15

16 cottonseed and tree nuts with aflatoxins before harvest and during storage (Diener et al., 1987; Schroeder and Boller, 1973). The species typically produces AFB 1 and B 2 and cyclopiazonic acid (Horn et al., 2001). In contrast, A. parasiticus is most prevalent in groundnuts and synthesizes AFG 1 and G 2 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 16

17 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 17

18 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 o C and at water potentials as low as -35 MPa. The optimum temperature for growth is o C. 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 18

19 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. 19

20 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 AFB 1 in the soil. He observed only a low level (1-8%) mineralization into CO 2 within 120 days. According to Angle and Wagner (1980) AFB 1 was observed to be rapidly reduced to aflatoxin B 2 when added to the soil. The speed of this reaction suggests a chemical mechanism. The resulting AFB 2 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 AFB 1 is rapidly degraded in field soil at 28 C (half-life 5 days). 20

21 SECTION 3: THE PLANT GROUNDNUT Chapter 1: Review 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 , 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 ( Peanut, 2013). Groundnuts are rich in nutrients, providing over 30 essential nutrients and phytonutrients (Table 3). 21

22 Table 3. Nutritional value of groundnut Daily value Nutrient Amount (%) Nutrient density World s Healthiest Foods Rating Manganese 0.71 mg Good Tryptophan 0.09 g Good Vitamin B3 (niacin) 4.40 mg Good Folate µg Good Copper 0.42 mg Good Protein 9.42 g Good Source: Figure 7. A groundnut plant showing the subterranean, seed-bearing, dry fruit (called a pod). Source: 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 22

23 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 23

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, , and PI F 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, 24

25 87094, 87110, 91278, and 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. Preharvest 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 25

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. 26

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 27

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 ZnSO 4 ) added seedlings 28

29 were affecting uptake of aflatoxin. In the work conducted by Walker et al. (1984), reported both the uptake and distribution of aflatoxin B 1 (AFB 1 ) 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 AFB 1 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 AFB 1 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 B 1 and FB 2 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). 29

30 AIM AND OBJECTIVES OF THE STUDY Chapter 1: Review 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. 30