Genetic Diversity of Aspergillus flavus Isolated from Pepper

at North Sumatera

Kiki Nurtjahja

, Cut Fatimah Zuhra

, Helmina Sembiring

, Betriana N. L. Gultom

Department of Biology, Universitas Sumatera Utara, Medan, Sumatera Utara, Indonesia 20155

Department of Chemistry, Universitas Sumatera Utara, Medan, Sumatera Utara, Indonesia 20155

Keywords: Aspergillus flavus, Genetic Diversity, Pepper, RAPD

Abstract: Aspergillus flavus is one of the postharvest fungi that often infects white and black pepper (Piper nigrum

L.). Fungal infection on pepper not only spoiled physically but also produce mycotoxins. The aim was to

study Aspergillus flavus toxigenicity and their genetic diversity on dried-stored white and black pepper sold

at traditional markets in North Sumatera. Fungal population was determined based on colony forming unit

per mililiter (cfu/mL) using serial dilutions and pour plated in dichloran 18% glycerol agar (DG18) medium.

Toxigenicity of A. flavus was determined culturally using coconut agar medium (CAM) 10%. Phylogenetic

between A. flavus strains were carried out by isolating genome of representative toxigenic and non-toxigenic

Aspergillus flavus strains according to the MiniKit Promega protocol and amplified using 10 Random

Amplified Polymorphic DNA (RAPD) primers. The amplified bands were scored and translated to be biner

data, then analyzed using Numerical Taxonomy and Multivariate System (NTSys) and clustered by

Unweighted Pair Group Method with Arithmatic Average Algorithm (UPGMA). Results showed that a total

of thirty one A. flavus strains were isolated. Based on toxigenicity determination found that twelve strains of

A. flavus were toxigenic (aflatoxin producers) and nineteen strains were non-toxigenic (non-aflatoxin

producers). Dendogram of similar bands was constructed and showed the highest similarity coefficient of A.

flavus strains was 0.84 (84%) which means that the strains were similar even though they were isolated

from different traditional markets.

1 INTRODUCTION

Pepper (Piper nigrum L.) i.e black and white

pepper, is one of spices that commoly produced by

tropical countries with high temperature, hummidity.

Most of the commodity is cultivated conventionally

and lack of good agricultural practices (Pickova et

al. (2020). Similar to crops, dried pepper is

susceptible contaminated by moulds. the infection

can cause spoiled physically and chemically, loss of

aroma, taste and contaminated by mycotoxins.

Fungal infection and aflatoxin contamination on

black and white pepper were previously studied. The

infection of pepper by Penicillium sp was reported

by Bokhari (2007) and Pitt and Hocking (2009).

Black and white pepper sold by retailer at

traditional markets were contaminated by

Aspergillus chevalieri, A. flavus, A. niger and A.

sydowi (Nurtjahja et al. (2019). Fungal infection

and mycotoxin contamination might occur during

preharvest and postharvest handling. Inappropriate

drying, storing might increase fungal population. As

a soil fungi, Aspergillus flavus contaminate crops in

field such as leaves, flowers and fruits crops

(Hererra et al. 2014). There are many strains and

genetic variability of Aspergillus flavus in field,

some of the strains are toxigen (aflatoxin producer)

and the other are non-toxigen (non-aflatoxin

produces) (Midorikawa et al. 2008; Ehrlich, 2014).

The genetic variability of Aspergillus flavus strains

on field was studied by Solorzano et al. (2014). The

objectives of the current study was to determine

genetic diversity of A. flavus strains isolated from

dried-stored black and white pepper sold at

traditional markets in Medan, North Sumatera.

Nurtjahja, K., Zuhra, C., Sembiring, H. and Gultom, B.

Genetic Diversity of Aspergillus Flavus Isolated from Pepper at North Sumatera.

DOI: 10.5220/0010766800002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 631-635

ISBN: 978-989-758-556-2

631

2 MATERIALS AND METHODS

2.1 Sample Collection

A total of 250 g samples of dried white and black

pepper were obtained from 5 retailers at five

traditional markets. Only intact seeds used in this

experiment. Each sample then was placed into a

sterile polyethylene bag and stored in a refrigerator

at ± 12°C for further use.

2.2 Determination Fungal Population

The population of A. flavus was determined by a

dilution and pour plated in dichloran 18% glycerol

agar (DG18) medium. Each sample was ground and

25 g were put into a 500 ml flask and suspended

with 250 ml of sterile distilled water and then

homogenized to obtain a 10

-1

suspension. Dilution

was carried out on 10

-2

, 10

-3

and 10

-4

. One ml of

dilution suspension was cultured on DG18 medium.

Each dilution was repeated 3 times. All plates were

incubated for 5 days (29

C). Population of A. flavus

per gram of pepper (cfu/g) was determined using the

formula:

Fungal population = Z (cfu/g)

X = volume of suspension transferred to each petri

dish (1 ml)

Y = dilution which gives the fungus colonies

separately

Z = average number of colonies of each fungal

species from 3 petri dishes

2.3 Fungal Identification

Each colony of A. flavus was cultured on potato

dextrose agar (PDA) medium then all of the isolates

were identified according to Pitt and Hocking

(2009).

2.4 Toxygenicity Determination of

Aspergillus flavus Strains

The toxigenicity of each A. flavus was determined

qualitatively by culturing in 10% coconut agar

medium (CAM) in petri dish (9 cm in diameter)

according to Lin and Dianese (1978). Toxigenic

strains was indicated by the presence of yellow

pigment at the reverse side of the medium.

2.5 Extraction Genome Aspergillus

flavus Strains

As much as 40 mg fungal mycelia in a microtube

containing 600 µl nuclei lytic was homogenised and

extracted using procedure Mini Kit (Promega,

Madison, WI, USA). Deoxyribonucleic acid

concentration obtained was determined using

nanophotometer (IMPLEN, Munich, Germany).

Electrophoresis of the DNA was conducted using

1.2% agarosa gel (SCIE-PLAS, Cambridge,

England) and stained by 1µl ethidium bromide

(EtBr) and visualise using Gel Doc (Uvitecc,

Cambridge, Serial) under UV light (303 nm).

2.6 Amplification of DNA and

PCR-RAPD

Amplification of DNA was conducted using 10

primers (Macrogen, Korea) as follows:

Primers

Nukleotides sequence

(5’ 3’)

OPA04 AATCGGGCTG

OPB10 CTGCTGGGAC

OPD10 GGTCTACACC

OPD20 ACCCGGTCAC

OPF10 GGCTGCAGAA

OPF13 GGAAGCTTGG

OPK20 GTGTCGCGAG

OPO20 ACACACGCTG

OPQ20 TCGCCCAGTC

OPT20 GACCAATGCC

PCR was performed as follows: a preincubation step

at 94

C for 10 minutes followed by 35 cycles of

denaturation at 94

C for 1 minute, annealing at 65

for 2 minutes, extension at 72

C for 2 minutes. The

PCR products were analyzed by electrophoresis

(SCIE-PLAS. Ltd, Cambridge, England) using 1.5%

agarose gels in 1 × TAE [40 mM Tris-acetate, 1 mM

EDTA (pH 8)]. Gels were stained with 0.5 µg µl

-1

ethidium bromide and visualized using Gel Doc

(Uvitec, Cambridge, Serial no. 13200263) under

UV light (303 nm). Polymorphic bands produced

were read and genetic diversity were analysed using

Numerical Taxonomy and Multivariate Analysis

System, version 2.1. (NTSYSpc21). Dendrogram

analysis was described by Unweighted Pair Group

Method with Arithmatic Average Algorithm

(UPGMA).

IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)

632

HPB3 HPR2 HSB1 HSS2 PSL2 PPR2 PSB3 PSS7

M HPB3 HPR2 HSB1 HSS2 PSL2 PPR2 PSB3 PSS7

3 RESULTS AND DISCUSSION

All samples, dried black and white pepper sold by

retailers at traditional markets, were stored in closed

jars.

3.1 Toxygenicity of Aspergillus flavus

Strains

Thirty one strains of A. flavus were successfully

isolated and the strains consisted of 10 strains

isolated from black pepper and 21 strains were

isolated from white pepper (Table 1).

Table 1.Total number of Aspergillus flavus strains isolated

from black and white pepper sold by retailers at traditional

markets

illus

lavus strains

total

toxigen non-toxigen

population

(cfu/ml)

lack

3 7 0.5×10

white

9 12 0.9×10

The population of A. flavus in black pepper was less

than that of white pepper, in addition to toxigenic

strains were less contaminated in black pepper than

that of white pepper. The presence of A. flavus in

black and white pepper indicate that the fungal

species was able to grow in dried pepper during

storage. We assumed that high fungal population

might occur during inappropriate storage or cross

contamination. Previous study by Leger et al. (2000)

stated that no specific host for A. flavus and they are

able to grow at minimum a

0.78 (Pii and Hocking,

2009). For phyllogenetic study, among of the 31

toxigenic and non-toxigenic A. flavus, only 8

genomes of representative traditional markets and

toxigenic and non-toxigenic A. flavus strains were

extracted as shown in Table 2.

Table 2. Representative toxigenic and non-toxigenic A.

flavus strains isolated from black and white pepper

obtained from each traditional market for phylogenetic

study

A. flavus

code

isolate sources toxigenicity

HPB3

lack peppe

toxigen

HPR2

lack

toxi

HSB1

lack peppe

non-toxigen

HSB2

lack

non-toxi

PSL2 white peppe

non-toxigen

PPR2 white

toxi

PSB3 white peppe

non-toxigen

PSS7 white

toxi

Genome extraction of 8 representative toxigenic and

non-toxigenic A. flavus strains is shown in Figure 1.

The purity each of the genome was between 1.6 to

1.8. Previous study by Sambrook et al. (1989)

reported that the purity of genome for molecular

study at ratio A

280

was 1.8 to 2.0.

Figure 1. Electrophoresis of 8 strains of A. flavus

genomes

Amplification A. flavus genomes and PCR-RAPD

Amplification of genomes of 8 A. flavus strains

using 10 RAPD primers (OPA04, OPB10, OPD10,

OPD20, OPF10, OPF13, OPK20, OPO20, OPQ20,

OPT20) showed different bands, however, among 10

primers used, only 3 primers (OPD20, OPF10,

OPT20) were succesfully amplified and produce 7

bands with polymorphism 100%. The

electrophoresis of genome amplification using

primers were OPD20, OPF10, OPT20 is shown in

Figure 2.

Primer OPD20

Primer OPF 10

Genetic Diversity of Aspergillus Flavus Isolated from Pepper at North Sumatera

633

Primer OPT20

Figure 2. Electrophoresis of amplification of 8 genomes of

A. flavus strains using primers: OPD20, OPF10, and

OPT20. M= marker ladder (100 bp)

Random Amplified Polymorphic DNA (RAPD)

amplification in Figure 2 showed that each primer

has different bands and polymorphism percentage.

Primer OPF10 has the smallest band and the highest

on OPT20

3.2 Genetic Diversity Analysis of

A. flavus Strains

Based on matrix of on Table 4 showed that the

highest genetic distance with genetic similarity 0.21

occured on A. flavus strains: HSS2 and HPB3, PSL2

and HSS2, PPR2 and HPB3, PSB3 and HPR2.

Whereas, the lowest genetic distance with genetic

similarity 0.84 occured only in A. flavus strains

PSB3 and HSB1.

Table 4: Genetic similarity of A. flavus strains isolated

from black and white pepper sold by relailers at traditional

markets based on primers OPD20, OPF10 and OPT20

HPB3 HPR2 HSB1 HSS2 PSL2 PPR2 PSB3 PSS7

HPB3 1.00

HPR2 0.63 1.00

HSB1 0.63 0.26 1.00

HSS2

0.21 0.47 0.47 1.00

PSL2 0.78 0.52 0.73 0.21 1.00

PPR2

0.21 0.36 0.57 0.68 0.31 1.00

PSB3 0.57

0,21 0,84 0.52 0.68 0.52 1.00

PSS7 0.52 0.57 0.68 0.57 0.52 0.68 0.63 1.00



Dendrogram of all A. flavus strains are grouped in

one cluster with similarity 44% (Figure 3).

Figure 3. Dendrogram of A. flavus strains isolated from

dried black and white pepper sold by retailers at traditional

markets

Based on dendrogram in Figure 3 showed high

possibility that A. flavus contamination on dried

black and white pepper sold by retailers at 5

traditional markets took place at out of the markets,

it might occure at distribution chains. Even though,

their population may increase due to high relative

humidity during storage.

4 CONCLUSIONS

Black and white pepper sold at traditional markets

are contaminated by toxigenic and non-toxigenic A.

flavus. Cross contamination may increase during

storage, therefore, good hygienic practices

particularly on distribution chain also required to

reduce fungal infection and aflatoxin contamination.

ACKNOWLEDGEMENT

The research was funded by Universitas Sumatera

Utara, contract DRPM Reseach grant no.

152/UN5.2.3.1/PPM/KP-DRPM/2019

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