Gene deletion patterns in non-aflatoxigenic isolates of Aspergillus flavus

INTRODUCTION

Aflatoxins (AFs) are highly toxic and carcinogenic in animals and humans, leading to hepatotoxicity, teratogenicity, immunotoxicity and even death (Motomura et al. 1999, Wen et al. 2005). Contamination of corn (Zea mays L.), peanuts (Arachis hypogaea L.) and pistachio (Pistachia vera L.) by aflatoxins is a severe economic burden for Iranian growers (Cheraghali et al. 2007, Hedayati et al. 2010, Ghiasian et al. 2011).

Aspergillus section Flavi includes three economically important species: A. flavus, A. parasiticusand A. nomius. Even though these species share numerous common features, they differ in a major attribute, their ability to produce AFs. This widely distributed group of aflatoxigenic species is considered a major problem for animal and human health, since those species are able to grow in almost any kind of crop or food.

After the discovery of a gene cluster as the regulator of AF biosynthesis (Brown et al. 1996), the biosynthetic pathway of AFs has been extensively studied, and most of the enzymes and corresponding involved genes have been identified (Ehrlich et al. 2005, Yabe & Nakajima 2004, Yu et al. 2005, Keller et al. 2005, Wen et al. 2005, Cary & Ehrlich 2006). AF biosynthesis requires at least 25 enzymes and two regulatory proteins encoded by contiguous genes in an 80-kb cluster (Scherm et al. 2005, Yu et al. 2005, Bhatnagar et al. 2006).

The functions of most of the genes required for aflatoxin production have been identified by genetic complementation (Motomura et al. 1999, Chang et al. 2000, Yu et al. 2000).

Generally, the AF biosynthesis genes of A. flavus, A. parasiticus and A. nomius are highly homologous, since the order of the genes within the cluster are the same (Ehrlich et al. 2005, Chang et al. 2007). A significant proportion, but not all of the non-aflatoxigenic A. flavus isolates have been found to contain various deletions in the AF gene cluster (Ehrlich & Cotty 2004, Chang et al. 2005, 2006) which are also common in some strains of A. oryzae (Chang et al. 2005, 2006). The loss of the ability to produce AFGs in A. flavus seems to result from a deletion in the terminal region of the cluster corresponding to genes aflF (= norB) and aflU (= cypA) (Ehrlich et al. 2004).

Molecular techniques have been widely applied to distinguish the aflatoxin producing and non-producing strains of A. flavus and related species, through the correlation of presence/absence of one or several genes involved in the AF biosynthetic pathway and the ability/inability to produce AFs. Recently, DNA–based detection systems have been introduced as powerful tools for detecting and identifying the aflatoxin producing fungi (Geisen 1996). The polymerase chain reaction (PCR) is the method of choice for this purpose (Shapira et al. 1996).

Färber et al. (1997) detected the aflatoxigenic strains of A. flavus in contaminated figs by performing a monomeric PCR with three sets of primers specific for three structural genes of the AF biosynthetic pathway, aflD, aflM and aflO. Other attempts to develop PCR-based methods for detection of aflatoxigenic fungi (A. flavus and A. parasiticus) were underway (Chen et al. 2002, Mayer et al. 2003, Scherm et al. 2005, Baird et al. 2006, Lee et al. 2006, Rahimi et al. 2008). PCR-based methods utilize primers for aflatoxin genes not necessarily unique to aflatoxigenic fungi. These methods have not been tested for reproducibility on a number of different contaminated commodities (Bhatnagar et al. 2006).

Reports on deletion patterns of non-aflatoxigenic A. flavus strains are different. For instance, the loss of aflatoxin production in strain A. flavus 649-1 is associated with large deletions in the aflatoxin gene cluster (Prieto et al. 1996). Non-aflatoxigenic A. flavus AF36, widely used in the management of aflatoxin contamination of cotton in Arizona, has a defect in the gene pksA (Ehrlich & Cotty 2004). Rodrigues et al. (2009) reported that gene expression of aflD in Aspergillus section Flavi was a good indicator to distinguish between toxigenic and non-toxigenic strains of A. flavus. Okoth et al. (2012) tested A. flavus and A. parasiticus isolates for the presence of aflD and aflQ genes.

Strains of A. flavus show a great variation in their ability to produce aflatoxins. There are a number of Aspergillus species that produce aflatoxin but are not classified as section Flavi (Cary & Ehrlich 2006). Greatest successes to date in biological control of aflatoxin contamination in both pre- and post-harvest crops have been achieved through application of biocompetitive non-aflatoxigenic strains of A. flavus and/or A. parasiticus (Yin et al. 2008).

In the current study, we identified genomic deletions of non-aflatoxigenic strains of A. flavus based on polymerase chain reaction with the aflatoxin biosynthesis gene cluster analysis.

MATERIALS AND METHODS

Fungal strains

Fifteen non-aflatoxigenic strains of A. flavus from a collection of 52 strains from peanut (IRG075, IRG129 and IRG517), corn (IRM074, IRM193, IRM014, IRM211, IRM031, IRM041 and IRM081) and pistachio (IRP049, IRP107, IRP082, IRP144), representing a wide range of geographic regions of Iran and different vegetative compatibility groups (IR1 to IR15; unpublished data) were used for this study (Tables 1 & 2). Morphological characterization of non-aflatoxigenic strains of A. flavus was based on seriation on Czapek Yeast Agar (CYA)25. The observed characteristics on this medium were as follows: conidia ornamentation, conidia size (μm), colony color and sclerotia. The colony diameter (cm) was measured on CZ42. Conidia of A. flavus species have relatively thin and finely to relatively rough walls. Their shapes vary from spherical to elliptical, and when grown on Czapek-Dox (CZ), colonies of A. flavus are yellowish-green (Klich, 2002, Samson et al. 2004, 2006, Pildain et al. 2008) (Table 1).

Table 1. List of non-aflatoxigenic strains of Aspergillus flavus.

-: not detected   n.d.: not determined   a: size, in μm: average of 15 sclerotia    b: u = uniseriate; b = biseriate; u/b = predominantly uniseriate; b/u = predominantly biseriate     c: average of 3 colonies, in cm.

Table 2. Non-aflatoxigenic strains of Aspergillus flavus used in this study.

+: Sclerotia producer               -: Sclerotia non-producer               L: Sclerotia >400 µm on CYA25

Aflatoxin analysis

All of the isolates were initially screened for aflatoxigenic ability on yeast extract sucrose agar medium (YES: Yeast Extract 20 g/L, Saccharose 150 g/L, Agar 15 g/L) amended with 0.3% (w/v) methylated β-cyclodextrin (mß-CD) (Fente et al. 2001). All of the non-aflatoxigenic strains of A. flavus were re-tested for confirmation of AFs production in AF-inducing YES. Strains were inoculated on Petri dishes containing YES and incubated at 25-27 °C for 7 days in the dark. A Method based on HPLC with fluorescence detection has been suggested to measure aflatoxins B in A. flavus isolates (Bragulat et al. 2001). Briefly, extraction of aflatoxin using MeOH/H2O (80:20, v/v) and purification by an immunoaffinity column cleanup were carried out. HPLC system was equipped with an autosampler; a pump (Sykam 2100) and a reverse phase C18 column (Genesis ODS2, 250×4.6 mm, 5 μm), fitted with a precolumn with the same stationary phase and a fluorescence detector (RF-10AXL).

The injection volume was 100 µL. The fluorescence detection was carried out at excitation and emission wavelengths of 365 nm and 435 nm, respectively (detection limit = 0.5 ng/g). A standard solution of AFB1 and AFB2 (Sigma Co.) was used. HPLC grade solvents (methanol and acetonitrile) were used to prepare the AF standards in the sample extraction, and also to prepare the mobile phase. The A. flavus aflatoxin producing strain SRKC-G1907 which released only aflatoxins of group B was used as the reference strain.

Isolation of genomic DNA

Total DNA was extracted from mycelia of fungal isolates obtained from 7-day-old cultures grown in YES liquid media according to Prabha et al. (2013) with minor modifications.

The DNA concentration was measured with a UV-Vis spectrophotometer (NanoDrop ND-1000, Wilmington, Delaware USA).

Molecular detection of A. flavus isolates

All the non-aflatoxigenic strains of A. flavus were detected based on sequence analysis of ITS2 rDNA by using specific primer pair FVAVIQ1:5'-GTC-GTC-CCC-TCT-CCG-G-3´ and FLAQ2: 5´-CTG-GAA-AAA-GAT-TGA-TTT-GCG-3', as described by Sardiñas et al. (2011). The primer pairs were based on three multicopy ITS2 rDNA target sequences. The primers FLAVIQ1/FLAQ2 yielded an amplicon of 100 bp in A. flavus.

Analysis of aflatoxin biosynthesis gene cluster PCR primers

To design oligonucleotide primers, known sequences for aflT, pksA, afIR, aflJ, ver-1, omtA, omtB, aflD, ordA, verA, norA, hypA,norB-cypA intergenic region (norB and cypA genes), glcA(sugar utilization gene) and C3 (flanking region gene, 5'end) were derived from the aflatoxin biosynthetic pathway genes of A. flavus AF36 (AY 510455), AF70 (AY 510453), AF13 (AY510451), BN008 (AY 510451.1) and A. parasiticus AY371490 were obtained from GenBank (http://www.ncbi.nlm.nih.gov/). Primer pairs designed using OLIGO (version 5.0; National Biosciences) are depicted in Table 3. Some primer sets were based on Chang et al. (2005) and Ehrlich et al. (2005). The housekeeping gene tub1 coding for ß-tubulin (primer pair tub1-F/tub1-R) was chosen as a system control for PCR (internal amplification control). The PCR products were analyzed by electrophoresis in 1.5% agarose gels, which were stained with DNA green viewer dye (green fluorescent stain, 10 mg/ml).

PCR analysis

The fifteen non-aflatoxigenic strains of A. flavus were tested for presence of 14 genes and one intergenic region of aflatoxin biosynthesis gene cluster by PCR amplification in a 25 µl reaction mixture in a Biometra Thermal Cycler (T1 thermocycler; Biometra, Göttingen, Germany).

Table 3. Primers used in this study, target gene, sequence and expected PCR product size.

DNA amplification conditions

Fourteen aflatoxin clustered genes and the intergenic region norb-cypA were amplified using the primers at a concentration of 10 pmol/μl, 50 ng template DNA, 50 μmol of each of the four dNTPs and 5 units of Taq polymerase (BioNeer Inc., Korea) in a total volume of 25 μl containing 10× assay buffer (1× contains 10 mmol/l Tris-HCl, pH 8.8 at 25^°C, 50 mmol/l KCl, 1.5 mmol/l MgCl2). PCR conditions were as follows: denaturation at 95^°C for 2 min; 30 cycles of 94^°C for 60 sec, primer-specific annealing temperature at 45^°C for 60 sec, extension at 72^°C for 90 sec and a final extension at 72^°C for 5 min. The reaction was carried out in the Biometra Thermal Cycler.

Gel electrophoresis

The PCR products were resolved by electrophoresis in a 1.5% agarose gel in 0.5X TBE buffer. The amplified products were visualized under UV transilluminator (UVsolo TS gel documentation system, Biometra Co.) and compared with a standard DNA size marker (Thermo Fisher Scientific Inc., Fermentas, Germany).

RESULTS

Aflatoxin analysis

In our study, absence of fluorescence on mß-CD was correlated with no AFs production (determined by HPLC). This medium did not yield any false-negatives. In the other words, all the A. flavus strains had already been characterized for their aflatoxigenic ability, after mycelium collection YES broth were analyzed by reverse-phase HPLC to confirm the AF production. Since AF production is extremely dependent on growth conditions, it was important to determine aflatoxigenic ability under the current test conditions.

Molecular detection of A. flavus isolates

All the fifteen non-aflatoxigenic strains of A. flavus had the species specific gene, and were therefore confirmed as A. flavus (Fig 1).

Deletions in the aflatoxin gene cluster

Oligonucleotide primer sets (Table 3) targeted PCR products of 0.3–1.8 kb (300-1800 bp). In the present study, additional set of primers specific for the aflR, pksA, aflJ, ver1, omtA, omtB and aflD genes were also used. The PCR results are summarized in Table 4. On the basis of PCR assay, we grouped the fifteen non-aflatoxigenic strains of A. flavus into deletion patterns. The genomic deletions were identified in all the fifteen A. flavus strains examined, resulting in loss of parts of genes from the aflatoxin gene cluster (Table 2). Based on the banding patterns, twelve deletion patterns (I-XII), designated as A to L were detected among the non-aflatoxigenic strains of A. flavus. The deletion patterns of examined non-aflatoxigenic strains of A. flavus are shown in Table 5. The non-aflatoxigenic strains of A. flavus were placed into five groups based on their number of amplified genes (Table 6). Two strains IRP049 and IRP082 from pistachio have identical deletion patterns (C and I, respectively, Table 6). Deletion patterns were mainly in the left side of aflatoxin gene cluster (hypA, about 10-15 kb) and regulatory genes aflR and aflJ (approximately 35 kb).

Three independent deletions as type I, type II and type III were found in the norB-cypA region (Table 4). Only in A. flavus strain IRG75 from peanut the norB and cypA (norB-cypA intergenic region, deletion pattern type III) were not amplified. In addition, in twelve strains of A. flavus from pistachio and maize and two peanut strains (IRG129, IRG517), the fragments 0.8 and 0.3 kbp were amplified by primer pair norB-cypA-F/norB–cypA-R. Individual VCGs contain isolates from different deletion patterns (Table 6).

DISCUSSION

In this study, 15 of the 52 non-aflatoxigenic strains of A. flavus collected from soil and kernel of peanut, maize and pistachio belonging to various geographical regions and distinct VCGs were analyzed for aflatoxin gene cluster deletion patterns. The 15 non-aflatoxigenic strains of A. flavus were selected in this study, because they had proved to produce polymorphic DNA fragments based on microsatellite primed-PCR (MP-PCR) marker and different VCGs in previous studies (Houshyarfard et al. 2014).

                                                            M    1     2    3     4    5    6    7    8     9   10   11  12  13  14  15  M  C⁺

Fig.1. Specific amplification of internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of extracted genomic DNA electrophoresed in 1.5% (w/v) agarose gel from fifteen aflatoxin non-producing strains of Aspergillus flavus. C⁺ = positive control, M = Molecular marker (50 bp DNA Ladder, Fermentas).

Table 4. Prevalence of aflatoxin-associated genes among fifteen Iranian non-aflatoxigenic strains of A. flavus.

*: S : Soil     K: Kernel    P: Pistachio    G: Peanut     M: Maize     +: present     -: absent     Isolate name in bold indicates the aflatoxin producer.

Table 5. Deletion patterns (A-L) in the aflatoxin gene cluster of non-aflatoxigenic strains of A. flavus.

Filled and empty circles indicate positive and negative PCR products in Iranian strains, respectively. Original gene names are above and new names are below (Yu et al. 2004). Types I, II and III deletions in the norB-cypA region indicate 0.3 kb, 0.8 kb and no product, respectively. C3 and glcA genes are in the flanking and sugar utilization regions of the aflatoxin gene cluster.

Table 6. Grouping of aflatoxin non-producing strains of A. flavus based on the number of amplified genes and one intergenic region.