Research Article
 

Natural Strain Observation of Button Mushroom (Agaricus bisporus) from South Africa Using 18S rDNA



Mehrnoush Aminisarteshnizi
 
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ABSTRACT

Background and Objective: Agaricus bisporus, known as button mushroom, is an edible basidiomycete, which is distributed worldwide. The purpose of the study was to determine the native button mushroom growing in the natural land of South Africa. Besides, the white button mushroom is the most cultivated A. bisporus in South Africa. Materials and Methods: During a survey on the soil and aquatic biology and ecology in South Africa, a natural strain of button mushroom (Agaricus bisporus) was recovered in association with a pine tree in Sovenga Hills in Limpopo Province. Then the DNA was extracted to assess the 18S rDNA as a marker. Results: The NBLAST showed 99% similarity with an American strain of A. bisporus (acc. no. AY787216) and Chinese strain (acc. no. FJ869172). The phylogenetic analysis using the 18S rDNA marker showed that all A. bisporus strains could be differentiated from A. silvaticus. However, the South African strain of A. bisporus stands close to the Chinese strain of A. bisporus. The genetic distance indicated the highest differences between the South African and a Chinese (acc. no. JX268572) strain of A. bisporus. Conclusion: The results suggest that 18S rDNA could be a helpful marker for the natural strain of the South African A. bisporus. This is the first report of 18S rDNA for A. bisporus in South Africa.

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  How to cite this article:

Mehrnoush Aminisarteshnizi , 2022. Natural Strain Observation of Button Mushroom (Agaricus bisporus) from South Africa Using 18S rDNA. Asian Journal of Plant Sciences, 21: 685-689.

DOI: 10.3923/ajps.2022.685.689

URL: https://scialert.net/abstract/?doi=ajps.2022.685.689
 
Copyright: © 2022. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

The genus Agaricus includes the edible and poisonous types of mushrooms. However, Agaricus bisporus (J. Lange), a button mushroom, is globally cultivated1. Fresh A. bisporus contains Vitamins, Thiamine (B1), Riboflavin (B2), Niacin (B3), Pantothenic acid (B5), Vitamin B6, Folate (B9), Vitamin B12, Vitamin C, Vitamin D and minerals such as Iron, Magnesium, Phosphorus, Potassium, Sodium, Zinc. It is the cause of this mushroom becoming very popular and having a significant economic value. In South Africa, the white button mushroom is widely cultivated. McGee2 reported the economic value of A. bisporus was US$ 4.7 billion in 2009. Agaricus bisporus produce mainly in North America, Europe, India and China. China is the largest producer of mushrooms globally and produces over 30 million tons (87%)3. Due to the daily need for protein by humans, this food source is considered a proper alternative that it is towards food security programs worldwide4. Therefore, mushrooms are becoming more popular in various countries, including South Africa.

Morphologically, commercial mushrooms such as A. bisporus are similar and producing them with tissue cultures is easy. However, this is challenging to distinguish different cultivars and detect new varieties. Therefore, the accurate identification of cultivars is required1. However, the lack of morphological features of mushrooms and the environment can affect their features and their identification is challenging. Molecular markers, such as Single Nucleotide Polymorphism (SNP)5, sequence characterized amplified region (SCAR)6,7, Restriction Fragment Length Polymorphism (RFLP)8, Random Amplified Polymorphic DNA (RAPD)9,10 and Inter Simple Sequence Repeat (ISSR)11,12 have been widely used in mushroom identification, phylogenetic analysis and genetic polymorphism analysis1. During a survey on the ecology and biology of the organism in the natural lands in South Africa, a natural button mushroom was recovered in association with the pine tree in the Sovenga Hills, Limpopo Province. Therefore, the study aimed to identify and study the phylogenetic position of the A. bisporus using 18S rDNA.

MATERIALS AND METHODS

Study area: The sample was collected in the natural land in Sovenga Hills (GPS coordinates: S 23°53'41.763''E, 29°44'21.292''), in Limpopo Province, South Africa, under the pine tree. This study was carried out in December, 2020.

Molecular analysis: DNA extraction was done using the Chelex method13. A piece of A. bisporus was picked up with a sterilized needle and then transferred to a 1.5 mL

Eppendorf tube containing 30 μL double distilled water. The mushroom pieces in the tube were crushed with the fine needle and vortexed. Fifty microliters of 5% Chelex® 50 and 5 μL of proteinase K were added to the tube containing crushed mushroom. These separate microcentrifuge tubes with the mushroom lysate were kept at 56°C for 2 hrs and then incubated at 95°C for 10 min to deactivate the proteinase K and finally spin for 3 min at 16000 rpm14. The supernatant was then extracted from each of the tubes and stored at -20°C. Following this step, the forward and reverse primers, 18s (5'-TTGATTACGTCCCTGCCCTTT–3') and 26s (5'-TTTCACTCGCCGTTACTAAGG–3'), was used to amplify the ITS rDNA. PCR was conducted with 5 μL of the DNA template, 12.5 μL of 2X PCR Master Mix Red (Promega, USA), 1 μL of each primer (10 pmol μL1) and nuclease-free water for a final volume of 25 μL. The amplification was processed using a Thermo Fisher Scientific Thermal Cycler (USA), with the following program: Initial denaturation for 3 min at 94°C, 40 cycles of denaturation for 45 sec at 94°C, 53°C annealing temperatures for ITS rDNA, extension for 45 sec to 1 min at 72°C and finally an extension step of 5 min at 72°C followed by a temperature on hold at 4°C. After DNA amplification, 5 μL of PCR product was loaded on a 1.5% agarose gel in TBE buffer (40 mM Tris, 40 mM boric acid and one mM EDTA) to observe the DNA. The bands were stained with RedGel and pictured under a UV illuminator. The PCR product was stored at -20°C. Finally, the PCR product was purified for sequencing by Inqaba Biotech Company (South Africa, Pretoria). The phylogenetic analysis was done using maximum likelihood implemented in Mega-X software15. The 18S rDNA sequence for A. bisporus from South Africa was deposited in the GenBank under the accession number MZ087879.

RESULTS AND DISCUSSION

Molecular characterization: An 860 bp for 18S rDNA was obtained from a strain of A. bisporus from South Africa. The sequence was analyzed and compared with other 18S rDNA sequences of A. bisporus available from GenBank. The NBLAST for 18S rDNA revealed a 99% similarity with the American strain of A. bisporus (acc. no. AY787216) and Chinese strain (acc. no. FJ869172). The genetic distance estimation ranged from 0.011-0.633. The pairwise genetic distance for the strains of A. bisporus revealed the highest distance (0.633) between the South African and a Chinese (acc. no. JX268572) strain of A. bisporus in Table 1. In contrast, the lowest genetic distance (0.011) was observed between the South African and the Chinese (acc. no. FJ869172) and American (acc. no. AY787216) strain of A. bisporus (Table 1).

Image for - Natural Strain Observation of Button Mushroom (Agaricus bisporus) from South Africa Using 18S rDNA
Fig. 1: Maximum likelihood tree for 18S rDNA of the button strain of A. bisporus recovered from South Africa under the pine tree in Sovenga Hills


Table 1: Estimation of genetic pairwise distance for A. bisporus strains using 18S rDNA (the upper side is a standard error)
Accession number Locality
1
2
3
4
5
6
7
8
9
10
11
MZ087879 South Africa
0.013
0.013
0.207
17.116
0.013
0.013
0.013
0.013
0.013
0.018
FJ641895 Germany
0.011
0.000
0.202
5.872
0.000
0.000
0.000
0.000
0.000
0.011
FJ869172 China
0.011
0.000
0.202
5.872
0.000
0.000
0.000
0.000
0.000
0.011
MH751906 Egypt
0.121
0.108
0.108
5.244
0.202
0.202
0.202
0.202
0.202
17.473
JX268572 China
0.633
0.602
0.602
0.740
5.872
5.872
5.872
5.872
5.872
6.518
AY787216 USA
0.011
0.000
0.000
0.108
0.602
0.000
0.000
0.000
0.000
0.011
AJ012404 Taiwan
0.011
0.000
0.000
0.108
0.602
0.000
0.000
0.000
0.000
0.011
AJ012403 Taiwan
0.011
0.000
0.000
0.108
0.602
0.000
0.000
0.000
0.000
0.011
AJ012402 Taiwan
0.011
0.000
0.000
0.108
0.602
0.000
0.000
0.000
0.000
0.011
AJ012394 Taiwan
0.011
0.000
0.000
0.108
0.602
0.000
0.000
0.000
0.000
0.011
U23724 USA
0.021
0.011
0.011
0.121
0.631
0.011
0.011
0.011
0.011
0.011

In addition, the South African A. bisporus, showed a low genetic distance (0.011) with the Taiwanese strains (acc. no. AJ012402, AJ012403, AJ012404) of A. bisporus.

Phylogeny of A. bisporus, as derived from the corresponding tree in Fig.1, reveals a clear relationship with A. bisporus strains. Additionally, the A. bisporus strains were placed separately from the outgroup A. heterocystis (acc. no. KU366695). The phylogenetic analysis revealed that the South African strains placed close to the Taiwanese (acc. no. AJ012402, AJ012403, AJ012404) and American (acc. no. AY787216) strains of the same species with 100 bootstrap value. Besides, the Egyptian strains (acc. no. MH751906) were grouped separately from other A. bisporus. This is in agreement with the previous record by Wang et al.1 using IGS rDNA.

The 18S rDNA tree placed the present South African A. bisporus close to the same strain from China (acc. no. FJ869172), USA (acc. no. AY787216) and Taiwan (acc. no. AJ012404) with 100 bootstrap values (Fig.1).

The small subunit ribosomal DNA (SSU) is useful for the examination of close relationships in fungus16. Recently, domestic breeding has been analyzed for its divergences in the nucleotide sequences of IGS117. Their result indicated that A. bisporus is a monophyletic group, which is in agreement with the present study. Kwon et al.18 showed that the IGS1 rDNA region in A. bisporus is not inheritable with entirely homologous sequences. It seems that it is a quickly shifting region in the process of breeding. Nevertheless, using IGS regions has been suggested for discriminatory analysis among intra-specific individuals of mushroom species19. DNA barcoding is an approach to rapidly identify species using short and standard genetic markers8. The mitochondrial cytochrome oxidase I gene (COI) has been proposed as the universal barcode locus20, but its utility for barcoding in mushrooms has not been established. Determining and realizing the best DNA barcode markers for identifying mushrooms is essential4. Dentinger et al.21 compared COI and ITS as DNA barcode markers for mushrooms their result indicated a similar performance of COI and ITS as a barcode. The 18S rDNA phylogenetic tree indicated that genetic variation exists among the sequenced mushrooms from South Africa and those deposited in the gene bank (https://www.ncbi.nlm.nih.gov/). However, 18S rDNA is a useful marker for the phylogenetic and molecular identification of the natural button mushroom. The result indicated that the chelex was also a suitable method for edible mushroom DNA extraction. This is the first report of A. bisporus using 18S rDNA from South Africa.

CONCLUSION

In molecular studies, we need to know the different methods for extracting DNA. Also, increase the credibility of work by using different primers. This study, used 18S rDNA for A. bisporus in South Africa for the first time. Current results suggest that 18S rDNA could be a helpful marker for the natural strain of the South African A. bisporus. On the other hand, the result indicated that the Chelex method was a good option for extracting DNA in A. bisporus which is cheap and affordable.

SIGNIFICANCE STATEMENT

This study was discovered for identification of the A. bisporus, using 18S rDNA was useful that can be beneficial for molecular analysis of this mushroom. Furthermore, this study will help the researchers uncover the critical areas of molecular research that many researchers could not explore. Thus, a new theory on identifying button mushrooms may be arrived at.

ACKNOWLEDGMENT

I acknowledge the Aquaculture and Genetic Lab of the Faculty of Health for using the laboratory for processing the specimen.

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