Onions (Allium cepa L.) have been used as food for thousands of years and have many health benefits. They are good source of flavonoids, antioxidants and other bioactive compounds1-3. The fungal contamination of onions is a worldwide problem. Orpin et al.4 investigated micro-organisms associated with the spoilage of onions around dutsinma metropolis and found that a large number of fungi deteriorate the onions. These fungi not only grow in onions but also produce mycotoxins5. A large number of fungal genera produce mycotoxins such as Aspergillus sp., Penicillium sp., Alternaria sp. and Fusarium sp. in onions. Fusarium sp. are known to produce mycotoxins such as trichothecenes, zearalenone and fumonisins. Cereal grains contaminated with trichothecenes are associated with outbreaks of human mycotoxicosis characterized by anorexia, nausea, vomiting, headache, abdominal pain, diarrhea, chills and convulsions6. Trichothecenes are a major mycotoxin produced by some toxigenic Fusarium sp. including F. graminearum, F. sambucinum, F. sporotrichioides, F. culmorum, F. acuminatum and F. solani 7. The most recent study by Jonsson et al.8 and Sharma et al.9 have confirmed that heart is the main target tissue of MON toxicity in rats and avians.
These mycotoxins, if ingested are hazardous and can cause severe infection even in minimal concentration. Therefore, it is important to estimate the mycotoxigenic fungi spoiling the onions of Libya.
This research topic was unexplored by earlier researchers in Libyan region. So, keeping this in mind the present study was designed with the main objective of genetic diversity assessment and prevalence of fungal species in spoiled onions of Libya.
MATERIALS AND METHODS
The present study was carried out over a period of 1 year, from September, 2017 to August, 2018, in the Department of Microbiology, International Medical School, MSU, Shah Alam, Selangor, Malaysia. This study included 110 isolated fungal strains from onion samples of different regions of Libya.
Isolation of mycotoxigenic fungi: A total of 110 onion samples from different regions of Libya were collected and transferred in sterilized polybags before processing. These contaminated onion samples were transferred to sterilized potato dextrose agar plates by maintaining all aseptic conditions. After five days of incubation, the fungal hyphae came out from these samples. These fungi transferred into another sterilized potato dextrose agar plates for purification and storage.
Identification of mycotoxigenic fungi: For identification of fungal strains the lactophenol-cotton blue method was used and these fungi were identified microscopically on the basis of type/shape of conidia, size of conidia/spores, septation and metullae.
Isolation of DNA and RAPD primers: The fungal DNA was isolated as per the standardized protocol. About 50 RAPD primers were initially screened for the PCR amplification of the genomic DNA isolated from the F. solani. Out of which 12 primers gave good amplification in terms of number of bands and reproducibility.
PCR amplification conditions: Each 25 μL reaction mixture contained 12.5 μL of 2×PCR buffer, 2 μL each of dATP, dGTP, dTTP and dCTP, 1 U Taq DNA polymerase, 25 pmol random decamer primer and 50 ng of genomic DNA for PCR amplification. The amplified products from RAPD was separated on 2% agarose gel, visualized under UV trans-illuminator and photographed via gel documentation system.
Genetic diversity assessment: Selected Fusarium solani strains were amplified using 50 random decamer primers to ascertain the level of genetic diversity among them. Out of the 50 primers screened, 12 primers produced reproducible results. The genetic diversity among taxa was appraised using the percentage of polymorphism (number of polymorphic bands/number of total bands). The cluster analysis, using the unweighted pair group method with arithmetic mean (UPGMA) based on Jaccard distance.
Fungal taxon: The mycotoxigenic fungi isolated and identified from spoiled onions belong to 4 fungal taxon Fusarium solani (Fig. 1a, b), Aspergillus niger, Penicillium chrysogenum and Alternaria solani.
Prevalence and occurrence: Out of 110 onion samples, 98 onion samples came out with fungal growth. The maximum occurrence and prevalence obtained was of Fusarium solani in comparison with Aspergillus niger, Penicillium chrysogenum and Alternaria solani (Table 1).
||(a) Culture of Fusarium solani after 5 days of incubation at 28°C and (b) Microscopic view of Fusarium solani under 40X magnification showing phialides, micro-conidia and macro-conidia
||Occurrence of mycotoxigenic fungi in onions
||Interpretation of polymorphic and monomorphic bands by RAPD tool
Screening of mycotoxigenic Fusarium solani strains: These 68 Fusarium solani strains grown in shake culture at 28°C for 5 days for the production of trichothecenes mycotoxin and found that, out of 68 Fusarium solani strains, 12 strains were found potentialistic trichothecenes mycotoxin producer, therefore those strains were selected for genetic diversity assessment.
Genetic diversity assessment by RAPD: Most of the PCR products were in size range of 100-2000 base pairs with 4.83 bands per RAPD primer. A total of 58 RAPD loci were amplified from different isolates. Out of the 58 bands scored, 35 (60.34%) were found to be polymorphic (either occurring in or absent in less than 95% of all isolates) and 23 (39.66%) were found to be monomorphic in nature (Table 2). Data obtained from RAPD analysis alienated 12 isolates into 3 large cluster groups (Fig. 2). The first cluster (CL-I) is further divided into 2 sub-groups (A and B). Sub-group A has 2 isolates (Fu1 and Fu12) whereas, sub-group B contain 8 isolates (Fu2, Fu4, Fu5, Fu7, Fu9, Fu11, Fu6 and Fu8). Cluster second and third (CL-II and CL-III) contain only one isolate Fu3 and Fu10, respectively.
The results shown that a large number of mycotoxigenic fungi contaminate and deteriorate the onion samples in Libya. The common fungal strains belong to Aspergillus niger, Alternaria solani, Penicillium chrysogenum and Fusarium solani. Fusarium sp. is cosmopolitan and common mycotoxin producer all over the world, which is documented by various researchers. Youssef10 found similar mycotoxigenic fungal genera in deteriorating corn of Libya. Fusarium solani strains was found prevalent in comparison with other fungal strains.
Azliza et al.7 also found out Fusarium sp. as largest mycotoxin producer in wild grasses in Malaysia. Out of 68 Fusarium solani strains, 12 strains were found potential mycotoxin producer confirmed and screened by shake culture also documented earlier by Sugiura6.
Randomly amplified polymorphic DNA provides significant polymorphic bands with in the same species, which was documented by Tiwari11 for Alternaria alternata strains. Saber et al.12 also assessed the genetic diversity of Fusarium sp. by randomly amplified polymorphic DNA (RAPD-PCR) causing potato dry rot disease.
Furthermore, Tiwari and Chittora13 used this method for the assessment of genetic diversity of dominant Alternaria solani strains isolated from Pongamia pinnata. Therefore, for further investigation of these strains, randomly amplified polymorphic DNA was chosen and found effective in Fusarium solani strains.
Due to health risks and economic losses associated with mycotoxins produced by Fusarium species, there was an urgent need for better understanding of these fungi.
||Dendrogram showing UPGMA clustering of 12 isolates of Fusarium solani strains
|| CL: Cluster, Sub-g: Sub-group
It was concluded from this research work that the Fusarium solani strains were most prevalent mycotoxigenic fungi in spoiled onions of Libya and genetic diversity among these strains can be assessed by randomly amplified polymorphic DNA.
This study discovered that the randomly amplified polymorphic DNA (RAPD) is a powerful tool for the genetic diversity assessment of Fusarium solani strains isolated from onion samples of Libya. This study will help the researchers to uncover the critical areas of genetic diverse Fusarium solani strains.
Many thanks are addressed to the Management and Science University (MSU) as this paper part of the project funded by the university Seed Grant Number: SG-376-0216-MS. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.