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Full Research Article

Molecular Characterization of Aspergillus niger Van Tieghem Isolates by Using Rapd Markers

H.C. Chavda1, Saleha Diwan1, B.K. Prajapati2, A.B. Brahmbhatt3
1College of Agriculture, Parul University, Vadodara-391 760, Gujarat, India.
2Department of Plant Protection, College of Horticulture, S.D. Agricultural University, Jagudan-384 460, Gujarat, India.
3Department of Plant Pathology, Anand Agricultural University, Anand-38110, Gujarat, India.

ABSTRACT

Background: Aspergillus niger Van Tiegham is a filamentous, ubiquitous and cosmopolitan fungus, commonly known as “black mould” or black aspergilli. A. niger is responsible for causing diseases in plants, humans and animals. A. niger causes black mould disease during post-harvest stage of garlic bulbs. The potential of RAPD technique for identifying DNA marker related to the intraspecific diversification of the pathogen led us to instigate the genetic diversity within A. niger. 

Methods: A PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), was used for assessing genomic variability among the ten isolates of A. niger isolated from infected garlic bulbs. RAPD- PCR conditions were optimized for eight primers of series OPA and POC out of 40 total primers. 

Result: RAPD results were evaluated by a statistical software Minitab and a phylogenetic tree was prepared. The primer OPA-8, OPA-12, OPC-1, OPC-2, OPA-7 and OPC-18 showed 100 per cent polymorphism, among the A. niger isolates.

INTRODUCTION

Garlic (Allium sativum L.) is the most important bulbous crop after onion, belonging to Amaryllidaceae family (Sharma, 2012). Black mould rot of garlic is caused by A. niger, which is a filamentous, ubiquitous and cosmopolitan fungus, commonly known as “black mould” or black aspergilli. This fungus is among the most common fungi causing food spoilage and biodeterioration of other materials (Samson et al., 2004). Strains of A. niger are used in the manufacture of several acids like citric, gluconic and itaconic acids and enzymes, like glucose oxidase and lysozyme. Over 99% of total global production of citric acid is through fermentation by A. niger (Dube, 2013). Being saprophytic and filamentous in morphology, A. niger resides and perpetuates in soil, forage, organic debris and food products. The most favourable temperature conditions for the growth of the fungus are 28°C-34°C followed by the worm and moist conditions eliciting infection (Tysony and Fullerton, 2004). However, A. niger has been found to be an opportunistic because of various infections of human beings. It can be dangerous to humans if intense spores inhaled, by causing severe lung problems i.e. Aspergillosis. This fungus can also cause ear infections, which ultimately damage ear canal and tympanic membrane (Schuster et al., 2002). A. niger causes black mould disease during post-harvest stage of garlic bulbs (McDonald et al., 2004). The modified polymerase chain reaction (PCR) with single primers of arbitrary nucleotide sequence and requiring no prior sequence information have proved useful in detecting intraspecific polymorphism among organisms (Welsh and Mc Clelland, 1990 and Williams et al., 1990). This amplification technique (arbitrarily primed PCR on RAPD) can generate specific DNA fragments useful for genome mapping, identification of isolates and application in molecular biology (Hadrys et al., 1992). The potential of this technique for identifying DNA marker related to the intraspecific diversification of the pathogen led us to instigate the genetic diversity within A. niger.
       
In the present study we have characterized ten different isolates of A. niger. The aim of the present study was to characterize isolates of the fungus at molecular level that leads to further elucidation of genetic diversity.

MATERIALS AND METHODS

Source of A. niger isolates
 
Ten isolates of A. niger which were isolated from infected garlic bulbs, collected from different regions of India listed below in Table 1.
 

Table 1: Source of A. niger isolates.


 
Genomic DNA extraction from fungal mycelium
 
DNAs of 10 isolates of A. niger were extracting using a CTAB method as described by Leisova et al., (2005) with some modifications.
 
Random amplification of polymorphic DNA (RAPD) analysis
 
For the molecular characterization of A. niger isolates, the eight different 10-mer RAPD primers were screened for the amplification of template DNA of A. niger (Table 2). The primers that gave clear and polymorphic amplification patterns were used for further analysis of all the 10 isolates of Aspergillus niger. Amplified DNA fragments for each accession were scored as present (1) or absent (0). Data generated by eight RAPD primers were used to compile a binary matrix for cluster analysis. Genetics similarity among accessions was calculated according to Jaccard’s coefficient (Jaccard, 1908).  The present study was carried out at B.A. College of agriculture, department of plant pathology, anand agricultural university, anand during 2015-16.
 

Table 2: List of primers used for the RAPD analysis.

RESULTS AND DISCUSSION

Random Amplification of Polymorphic DNA (RAPD) study
 
In recent years, molecular markers have received arable attention and have been used for genetic diversity phylogenetic, evolutionary, mapping and tagging of agronomically important traits in different plant species. It can also be used in the study of molecular diversity in microorganism such as fungus, bacteria, viruses etc. In this study, the present investigation was carried out with the ten different isolates of A. niger to study the molecular variability in these isolates. Therefore, this technique was employed to detect variability present among ten isolates i.e. AN-1, AN-2, AN-3, AN-4, AN-5, AN-6, AN-7, AN-8, AN-9 and AN-10 of A. niger (Plate 1a, 1b and 1c).
 

Plate 1 (a): RAPD amplification patterns of ten isolates of A. niger using primers OPA 8, OPA 12 and OPA 18.


 

Plate 1 (b): RAPD amplification patterns of ten isolates of A. niger using primers OPC 2, OPC 7 and OPC 8.


 

Plate 1 (c): RAPD amplification patterns of ten isolates of A. niger using primers OPC1 and OPC 18.


       
The present study showed a high level of genetic variability between the isolates of A. niger. Total 40 different 10-mer RAPD primers of OPA and OPC series (Eurofins Genomics India Pvt. Ltd.) were screened for the amplification of template DNA of A. niger. Among these, 32 primers did not produced amplification at all, while only eight primers were able to produce clear banding patterns and were subsequently used to analyze the entire set of 10 isolates of A. niger. The fragment size was detected by comparing the amplicons with a 100 bp Ladder. The highest polymorphism (100%) was recorded by the primer OPA-8, OPA-12, OPC-1, OPC-2, OPA-7 and OPC-18. Whereas, the lowest polymorphism was observed in the primer OPC-8 (97.29%) (Table 3).
 

Table 3: Details of amplification obtained with different RAPD primers.


       
Analysis of the DNA of ten isolates with eight primers showed amplification of a total 360 bands, with a range of 34 to 59 bands per primer. Overall, A. niger isolates exhibited a moderate level of genetic diversity. The maximum 59 bands were generated by a single primer (OPA-8); whereas, OPA-12, OPA-18, OPC-1, OPC-2, OPC-7, OPC-8 and OPC-18 generated 54, 40, 34, 43, 43, 37 and 50 bands, respectively. The lowest number of bands was generated by primer OPC-1 (34 bands). These results showed potentiality of RAPD to discriminate among isolates and suggested their application for species identification. The purpose of this study was to identify the specific primers which are likely to be efficient in revealing the diversity among the isolates of A. niger.
       
Dendrogram (Fig 1) based on “Jaccard (1908) unbiased measures of genetic distance (Table 4) by UPGMA method” formed two clusters namely A and B. Cluster A was divided into two sub-clusters A1 and A2. Sub-cluster A1 was further divided into A11 and A12 clusters, respectively. Cluster A11 having five isolates AN-1, AN-7, AN-8, AN-2 and AN-3 collected from Bhavnagar, Dwarka, Amreli, Anand and Vadodara, respectively. Cluster A12 consisted AN-10 isolate collected form Kerala, while, cluster A2 consisted AN-6 and AN-9 isolates which were collected from Sabarkantha and New Delhi.  Cluster B includes AN-4 and AN-5 isolates which were collected from Surat and Maharastra. The similarity coefficient ranged from 0.18 to 0.55 with all the eight primers. Highest similarity (0.55) was observed between AN-2 and AN-3 isolates, which were collected from Anand and Vadodara respectively, while lowest similarity (0.18) was observed between AN-2 and AN-9 isolates which were collected from New Delhi and Anand, respectively.
 

Fig 1: Dendrogram for isolates of A. niger based on Jaccard’s (1908) similarity coefficient using UPGMA as the clustering method for RAPD.


 

Table 4: Jaccard’s similarity coefficient between isolates of A. niger based on RAPD Makers.


       
Results similar to the present findings have been reported by Prajapati et al., (2016). He carried out RAPD analysis among the ten isolates of A. niger with six primers i.e. OPA-5, OPA-8, OPA-12, OPC-2, OPC-7 and OPC-18 showing amplification of 159 bands with a range of 10 to 38 bands per primer. The similarity coefficient ranged from 0.037 to 0.56.
       
Tawfik et al., (2022) reported 78.56 per cent of polymorphism through RAPD-PCR in Mutant Aspergillus niger infection in different plants. Rani et al., (2018) studied genetic relatedness among eight virulent isolates of A. niger from groundnut by using RAPD analysis and reported that the similarity index values among the A. niger isolates varied from 0.571 to 0.229. Abeer et al., (2015) they studied molecular characterization of twelve isolates of two species A. parasiticus and A. terreus by using DNA markers. They found that the RAPD and ISSR analysis revealed a high level of genetic diversity in A. parasiticus and A. terreus population and RAPD fingerprints of A. niger, A. flavus and A. parasiticus revealed polymorphism in 37, 59, 51 per cent of the analyzed Aspergillus sp.
       
The genomic DNA of ten isolates A. niger purified and were subjected to RAPD analysis by using 20 different primers. Out of 20 random primers, primer P2 (5’- ACGGCGTATG-3’) was optimized to screen all ten isolates of A. niger and showed amplifications of different sizes. Based on amplification patterns, the ten fungal isolates were divided into five groups in dendrogram analysis. Group I contained isolates 658 and 880 (100%), group II have isolates 0074, 840, 1005 and 1109 (68.38%), group III has 506 (55.28%) and IV contained isolates 0002 and 744 (36.76%) and group V included 764 (22.54%) based on genomic similarity percentages (Ishfaq et al., 2014). Irshad and Nawab (2012) studied molecular characterization of seven different species of A. niger. RAPD results were evaluated by a statistical software Minitab and a phylogenetic tree was prepared. GL Decamer B-09 showed 38 bands and GL Decamer B-10 gave 46 bands, showing 50 and 57 per cent similarity respectively, among species. Yuan et al., (1995) studied similarity between two Aspergillus species by using Single primers with arbitrary sequences to generate random amplified polymorphic DNA (RAPD) markers from strains of A. parasiticus and A. sojae. Three decamers, OPA-04, OPB-10 and OPR-01, allowed adequate discrimination between strains of A. parasiticus and A. sojae in RAPD analyses. A. sojae was further separated into group I and group II with the three primers. On the other hand, A. parasiticus was divided into group A and group B when amplified with OPA-04 and OPR-10 primers. The previously misidentified strain CCRC 32423 and the misclassified strain CCRC 30227 were identified as A. flavus and A. sojae, respectively, on the basis of RAPD patterns and morphological characteristics. Lavkor (2019) reported molecular analysis of fungal isolates from soil, air and infected peanut plants identified A. flavus using the β-tubulin gene and PCR-RFLP. Out of 325 isolates, 254 were found to contain aflatoxin biosynthesis genes, with 213 producing aflatoxin. The findings highlight A. flavus as the primary species responsible for aflatoxin contamination and suggest that understanding the aflatoxin gene cluster can aid in assessing toxicological risks and selecting biocontrol measures. Tejpal et al., (2022) identified significant cultural and morphological variability among fungal isolates, with isolate ANJP-04 showing the highest virulence. Among ten groundnut varieties tested, RG-644, M-13 and RG-510 were resistant, while others ranged from moderately resistant to highly susceptible, suggesting these resistant varieties should be cultivated to manage collar rot effectively.
 
Potential application of the findings
 
The molecular characterization of Aspergillus niger isolates using RAPD markers offers precise identification and differentiation of strains causing black mold disease in garlic. This knowledge enables targeted disease management strategies, including tailored fungicide applications and crop rotation practices. By understanding genetic diversity, farmers can implement more effective measures to control disease outbreaks and enhance crop health. This research also lays the groundwork for developing resistant garlic varieties through selective breeding, promoting sustainable agricultural practices in the face of fungal pathogens. Singh et al., (2017) standardized a real-time PCR method using TaqMan probes to amplify the aflatoxin/sterigmatocystin biosynthesis gene omt-1 in Aspergillus spp. Isolates from 53 aflatoxin-positive feed samples were successfully identified, demonstrating the technique’s efficiency in distinguishing toxigenic strains from other molds.

CONCLUSION

The RAPD analysis of ten Aspergillus niger isolates revealed significant genetic diversity among the strains responsible for black mould disease in garlic. The study identified eight effective primers that generated a range of polymorphic DNA fragments, enabling the differentiation and classification of isolates into distinct clusters. This genetic variability underscores the potential for targeted disease management strategies and informs the development of resistant garlic varieties. The findings not only enhance our understanding of A. niger genetic diversity but also support more effective control measures and sustainable agricultural practices to combat fungal pathogens.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest regarding the publication of this research. There are no financial or personal relationships with other people or organizations that could inappropriately influence or bias the content of this study.

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