Sample collection
The study was conducted in the selected major groundnut-growing AEZs of Uganda and selection was based on the groundnut production statistics in Uganda and the degree of variations in abiotic factors between AEZs. Basing on the groundnut production statistics provided by Uganda Bureau of Statistics, 2014, two districts with the highest groundnut production levels were selected from an AEZ and surveyed.
A total of 240 households and groundnut fields combined, were surveyed. From each household / groundnut field surveyed, at least a sample of groundnut was collected (120 field samples and 120 storage samples). A total of 40 samples were collected from each district (20 field samples and 20 storage samples). From each field, a quadrant measuring 1 m x 1 m was thrown randomly at five different sampling points at least 10 m apart. Then three groundnut stands were pulled and handpicked on the same day of sampling. Extra care was taken to sort out pods that were damaged by soil fauna and later clean pods packed in a paper bag. The groundnut pods were sun-dried for a week, disinfected using a 0.5 % (v/v) sodium hypochlorite solution, hand shelled followed by storage at 4 °C until fungal isolation according to (18). For the case of storage samples, sub-samples of shelled groundnuts from each bag or container were taken randomly from the top, middle and bottom using a sampling probe and later mixed to form a uniform mixture. From this mixture, 250 g were drawn and packed in a sterile paper bag for isolating fungi. Unshelled groundnuts were taken only once from each storage bag or container and packed for laboratory analysis following the method of (19).
Isolation of Aspergillus species from groundnut seeds
Aspergillus species were isolated at National Peanut Research Laboratory, Dawson, Georgia, USA. A selective growth medium, modified dichloran Rose Bengal (MDRB) was used for isolation of Aspergillus section Flavi (20). The MDRB medium is composed of 10 g/L dextrose, 2.5 g/L peptone, 1.0 g/L di-potassium phosphate (KH2PO4), 0.5 g/L magnesium sulphate hepta hydrate (MgSO4.7H2O), 0.5 g/L yeast extract, 20 g/L agar, 0.5 mL of 0.05 % (w/v) Rose Bengal stock solution in acetone, adjusted to 1 L with distilled water and later modified with 0.8 mg/L dichloran. After sterilization, 30 mg/L streptomycin and 0.15 mg/L tetracycline were added to the medium. Twenty seeds per sample were separately put into a sterile 50 mL falcon tube and 15 mL sterile distilled water added. The seeds were washed by shaking in a pulverizing machine, KLECO (Visalia, California, USA) for 2 minutes (21). Thereafter, 50 µL of each of the suspensions was separately plated onto MDRB medium (22), followed by incubation at 37 °C for 3 days. The Aspergillus colonies were counted, and contamination levels (%) were deduced by sample type and AEZ. In a biosafety cabinet, a stereo microscope and a flame sterilized needle were used to isolate conidia from a colony of interest. The conidia were then transferred onto freshly prepared plates of MDRB medium and streaked in a clock-wise pattern so as to effectively disperse the spores into single colonies. After three days of incubation at 37 °C, hyphal tips from single colonies were picked using a flamed scalpel and transferred into Czapek Dox agar (OXOID Ltd, Hampshire, England) slants for identification and storage.
Aspergillus species and strain identification
Morphological characterizations were done on 12-day old pure cultures of Aspergillus grown on Czapek Dox agar at 30 oC. This was to identify the different species and strains in accordance to (23) and comparison to reference cultures in the collection at National Peanut Research Laboratory, Dawson, GA, USA.
Genomic DNA extraction and quantification
Genomic DNA from the Aspergillus flavus isolates was extracted at National Peanut Research Laboratory, Georgia, USA, using Qiagen DNeasy Plant kit (QIAGEN, Hilden, Germany). Sterile disposable plastic loops were used to harvest 3 loopfuls of spores from the culture slants and loaded into each sample tube. Following the manufacturer’s instructions, a 500 μl clear lysate were pipetted into a 2 ml eppendorf tube and later loaded into a QIAcube robot (QIAGEN, Hilden, Germany). The concentration of the eluted DNA was determined using a Nanodrop ND 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA)
Genotyping of Aspergillus flavus isolates
Primers that were previously developed to detect insertions-deletions (InDel) within the aflatoxin-biosynthesis cluster of Aspergillus (21) were used in this study for the genetic fingerprinting of the Aspergillus isolates (Table 1). Since deletions/insertions in this gene cluster are associated to aflatoxin production (21), clustering of isolates were deduced from the shared deletions/insertions patterns that correspond to aflatoxigenicity or non-aflatoxigenicity.
Table 1: Primer pairs that were used in amplification of aflatoxin biosynthesis gene (21)
Marker
|
Forward 5'→3'
|
Reverse 5'→3'
|
Amplicon size (bp)
|
AFLC01
|
CCGACCTCACGACGCATTAT
|
CCGGCTAGCTTCAACAGACG
|
140-370
|
AFLC02
|
GGTTGGCGGATTGAGAGGTA
|
GGAGATCAGCCGAGAAGACA
|
100-296
|
AFLC03
|
TCCGCCGAGAGCCATAATAG
|
GGATGCTGACACCTCGATAG
|
120-160
|
AFLC07
|
GTCAGCAAGAGGAGCCTTCA
|
GGTCACGGAGATCCTCCATA
|
159-404
|
AFLC08
|
CGCCAGCACGGAGATCGAAT
|
CGTCTCCTCAGGCGGTCTAT
|
224-399
|
AFLC12
|
CGCAAGGAGCTCGACCAATA
|
TTCAGCTCAGCGACGAGAGT
|
241-360
|
AFLC13
|
TCGGTTCAATGCTCGAACAC
|
TCCAACCTTCGGCCTAGTCT
|
140-410
|
AFLC15
|
GCTCTACAGGCTGATTCAAG
|
TCGACAGTCCGACAATATGC
|
204-370
|
AFLC16
|
ATCGCAGCGGAAGCTTGGAA
|
AGTCTCGGACTCCGGTGACA
|
145-410
|
AFLC17
|
GCACAACTCGTACAGCTATC
|
TCTAAGTGCGAGGCAACGAA
|
125-390
|
AFLC18
|
GGCAGCCAGACCAAGGAATA
|
CCTTCTCGTAGCCGCTCATC
|
130-400
|
AFLC19
|
ACAGGACCGCACGGATCAAT
|
AGGAGCGGATGTCGAAGTCT
|
260-491
|
AFLC20
|
GCCTAGCGCTCCATTCTCAG
|
CCATCGTATCCGGCTCTATC
|
120-370
|
AFLC21
|
TACCTTACTCCGCTAAGCAG
|
GCGGTCACCTACCAATGAAT
|
150-368
|
AFLC22
|
TTCGCAGGAGTGTAGCCAAG
|
GTTGGAACACGCTCCATAGG
|
120-371
|
AFLC24
|
GAACGAGATAACGGCTGCAT
|
ATCAATCCACGGACCGTTGT
|
100-430
|
The forward primers were tailed with a 5’-CAGTTTTCCCAGTCACGAC-3’ sequence and labeled with 6-carboxyfluorescein (6-FAM). The reverse primers were tailed with 5’-GTTT-3’ sequence to promote non-template adenylation (24). Amplifications were performed using 10 ng of DNA and Titanium Taq polymerase (Clontech) in 5 µl reactions as described before (25). The labeled PCR amplicons were analyzed using an ABI 3730XL DNA analyzer and data were processed by GeneMapper v 4.0 (Applied Biosystems, Foster city, California, USA).
Data analysis
The GenStat Discovery Edition 4 (2002) for windows (VSN International Ltd, Rothamsted Experimental station, UK) software was used for data analysis. The Chi-square test and One –Way analysis of variance (ANOVA) were used to compare the frequencies of groundnut contamination with Aspergillus species and to determine the relative abundance of isolated Aspergillus species and strains at pre- and post-harvest stages. Allele sizes observed as relative fluorescence units were converted to binary data, where presence of an amplicon of any size was scored as ‘1’ whereas its absence was scored as ‘0’. Relationships among the isolates based on InDel data were determined through Neighbor Joining with 1000 bootstrap replications (26), using TREECON for Windows 1.3b version. Analysis of Molecular Variance (AMOVA) in Arlequin version 3.5.2.2 software package using 1000 permutations provided genetic structure based on the AEZs from which groundnut samples were collected. The principal co-ordinate analysis (PCoA) and Mantel test were done to identify genetic clusters and their associations with geographical locations using GenAIEx version 6.502.