Molecular Characterization of Fusarium Oxysporum F. Sp
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American Journal of Applied Sciences 6 (7): 1301-1307, 2009 ISSN 1546-9239 © 2009 Science Publications Molecular Characterization of Fusarium Oxysporum F. Sp. Cubense of Banana S.K. Leong, Z. Latiffah and S. Baharuddin School of Biological Sciences, University Sains Malaysia, 11800, Minden, Penang, Malaysia Abstract: Problem statement: Morphological characterization of Fusarium species which emphasize on microscopic and cultural characteristics are not sufficient to characterize Fusarium Oxysporum F. sp. Cubense (FOC) from banana as these characteristics could easily influence by environmental factors. As an alternative molecular methods were used to characterize and to assess genetic variation of FOC from different banana cultivars. Knowledge on the genetic variation is important to determine the genetic relationship between FOC isolates from different banana cultivars. Approach: Two PCR- based methods, Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) and restriction analysis of the Internal Transcribed Spacer and 5.8S regions (ITS+5.8S regions) were used to characterize Fusarium Oxysporum F. sp. Cubense (FOC) isolates from different banana cultivars. The genetic relationship of the FOC isolates were analyzed using Un-weighted Pair-Group Method with Arithmetic Averages (UPGMA) cluster analysis based on Jaccard Similarity Coefficient. Results: Restriction patterns of the ITS+5.8S regions using nine restriction enzymes namely, Alu I, Eco RI, Eco 88I, Bsu RI, Bsu 15I, Hin fI, Hin 6I, Msp I and Taq I and ERIC-PCR showed low variation among the FOC isolates studied, indicating close relationship among the isolates. Un- weighted Pair-group Method with Arithmetic Averages (UPGMA) cluster analysis based on Jaccard Similarity Coefficient showed that the FOC isolates were grouped into two main clusters with similarity value of 41.4-100% in PCR-RFLP of ITS + 5.8S and 45-100% similarity based on ERIC- PCR analysis, respectively. Cluster analysis of the combined data also showed that the FOC isolates were grouped into two clusters, sharing 42.9-100% similarity. Conclusion/Recommendations: The results of the present study indicate that the FOC isolates were closely related regardless of banana cultivars and location. Key words: PCR-RFLP, ERIC-PCR, Fusarium oxysporum F. sp. Cubense INTRODUCTION Since the Panama disease outbreak in 1950s, the susceptible Gros Michel has been replaced with the Fusarium Oxysporum F. sp. Cubense (FOC) is the more resistant Cavendish cultivar. However, the causal agent of banana wilt or Panama disease. It is a emergence of race 4 in Taiwan, South Africa and South cosmopolitan soil-borne fungus which colonizes the Queensland, Australia [14], followed by the disease vascular system of the host plant. When the fungus incidence in the tropics sparked the urgency in infected the host plant, it triggers the self-defense formulating control methods especially in breeding mechanisms whereby secretion of gel occurs followed resistant banana cultivar. In Malaysia, FOC race 4 was by formation of tylose in the vascular vessels, thus, first reported in Johor in 1992. blocking the movement of water to the upper part of the Due to the inconsistency in some of the control host plant which in turn causes yellowing, wilting and methods used to control banana wilt such as flood eventually death to the host. At present, there are four fallowing and chemical fumigation, planting of resistant identified races of FOC in which only races 1, 2 and 4 banana cultivar remain as the most promising option to attacked banana cultivars. Race 4 is the most virulent solve banana wilt problem. However, breeding process strain which attacks Cavendish cultivar as well as Gros to produce resistant banana cultivar is not an easy task Michel and Bluggoe which is susceptible to race 1 and as understanding of fungal genetics is essential. 2, respectively. Race 3 only attacks Heliconia sp. and Morphological characterization alone is not enough as only has mild effects on banana [13]. genetics information is not provided. Furthermore, Corresponding Author: Latiffah Zakaria, School of Biological Sciences, University Sains Malaysia, 11800, Minden, Penang, Malaysia 1301 Am. J. Applied Sci., 6 (7): 1301-1307, 2009 expertise and experience are important in accurate Table 1: List of banana cultivars of the FOC isolates morphological identification of Fusarium species since Banana cultivar/ the process emphasized on differences of the Code Location common name I2240N Peningsurat, Indonesia Kepok (Abu) morphological features such as the shapes and sizes of I2244N Pekalongan, Indonesia Uter the macro and micro-conidia as well as the A2279N KM 4, Changkat Jering, Perak Emas conidiogenous cells, pigmentation and growth rates A2282N Titi Gantung, Perak Berangan which could readily be altered by environmental and A2281N Padang Ragut, Bota Kanan, Perak Awak [5] A2295N Bukit Nangka, Lenggong, Perak Kepok (Abu) cultural factors . To compensate for the weaknesses, A2296N Changkat Jambu, Kati, Kuala Berangan molecular methods using PCR-based techniques such as Kangsar, Perak Random Amplified Polymorphic DNA (RAPD), PCR- A2306N Sri Iskandar, Perak Awak RFLP of Internal Transcribed Spacer (ITS) and B2286N Kancong Darat, Banting, Selangor Kepok (Abu) D2293N Wakaf Stan, Kota Bharu, Kelantan Kepok (Abu) Intergenic Spacer (IGS), Enterobacterial Repetitive D2294N Kg Berangan, Tumpat, Kelantan Berangan Intergenic Consensus-PCR (ERIC-PCR) and Random M2300N Merlimau, Melaka Nangka Amplified Microsatellites (RAMS) analysis have been P2305N Sg Semambu, Kubang Semang, Penang Awak Masam used in characterization of Fusarium species. These T2290N Lak Luk, Terengganu Awak techniques served as supportive means which provide T2304N Kampung Penjara, Terengganu Awak genetic insight of Fusarium species. Therefore, in this preliminary study, PCR-RFLP of Restriction analysis: About 5-10 µL of PCR products ITS+5.8S regions and ERIC-PCR were conducted to were digested using nine restriction enzymes, namely characterize and assess genetic variation among FOC Alu I, Eco RI, Eco 88I, Bsu RI, Bsu 15I, Hin fI, Hin 6I, isolates from different banana cultivars in Malaysia. Msp I and Taq I (MBI fermentas) in separate reactions according to the manufacturer’s instructions. The MATERIALS AND METHODS restriction fragments were separated by electrophoresis in 1.7% agarose gel (Amresco) using 1X TBE as Fungal isolates and DNA extraction: Thirteen FOC running buffer. The analysis of restriction analysis after isolates from Malaysia and two isolates from Indonesia electrophoresis was the same as those described for were used in this study (Table 1). For DNA extraction, PCR product analysis. cultures derived from single spores were plated on potato sucrose agar and incubated for 7 days at 28°C. Genomic DNA was extracted using DN easy Mini Plant kit Enterobacterial Repetitive Intergenic Consensus (QIAGEN) according to the manufacturer’s instructions. PCR (ERIC-PCR): PCR amplifications were carried out in 25 µL reaction mixtures using ERIC1R (5’-ATG PCR amplification of ITS+5.8S regions: The TAA GCT CCT GGG GAT TCA C-3’) and ERIC2 (5’- [16] ITS+5.8S regions were amplified using ITS1 (5’-TCC AAG TAA GTG ACT GGG GTG AGC G-3’) . The GTA GGT GAA CCT GCG G-3’) and ITS4 (5’-TCC reaction mixture consists of 1X PCR buffer, 4.0 mM [17] TCC GCT TAT TGA TAT GC-3’) primer pair . MgCl 2, 120 mM of each dNTPs, 0.5 µM of each Amplification was carried out in 25 µL reaction primer, 1.3 U of Taq polymerase, 5 ng of genomic DNA mixture containing 1X PCR buffer, 2.5 mM MgCl 2, 0.6 and de-ionized distilled water using a Peltier thermal mM of each dNTPs, 0.25 µM of each primers, 1.25 U cycler (PTC-200, MJ research). PCR amplification Taq polymerase and 4 ng genomic DNA. The reagents conditions were as follow: Initial denaturation at 95°C were obtained from Promega. for 7 min, followed by 30 cycles of denaturation at PCR amplification was performed in a Peltier 95°C for 1 min, annealing at 53°C for 30s and Thermal Cycler (MJ research, PTC-100). The extension at 65°C for 8 min and final extension was amplification starts with initial denaturation at 95°C for conducted at 65°C for 16 min. The analysis of ERIC- 5 min, followed by 35 cycles of 1 min denaturation at PCR banding patterns was the same as those described 94°C, 1 min of annealing at 55°C and 2 min extension for restriction patterns except that the amplification at 72°C with a 10 min final extension at 72°C. The products were separated in 1.5% agarose gel (Amresco) amplification products were separated by and the approximate sizes of the fragments were electrophoresis in 1.0% agarose gel (Amresco) using Tris Borate EDTA (TBE) as running buffer. After estimated by comparing with 1 kb ladder (MBI electrophoresis, the gels were stained with ethidium fermentas). bromide, visualized under UV light and photographed using GeneSnap photo imaging system (SynGene). The Data analysis: The restriction bands and ERIC-PCR approximate size of the amplified product was bands were scored as presence (1) or absence (0) of a estimated using a 100 bp marker (MBI fermentas). particular band to generate a binary matrix. The binary 1302 Am. J. Applied Sci., 6 (7): 1301-1307, 2009 matrix data were then subjected to Un-weighted Pair- Cluster analysis based on Jaccard similarity Group Method with Arithmetic Averages (UPGMA) coefficient separated the FOC isolates into two main cluster analysis and genetic similarity matrices were clusters, sharing 41.4-100% genetic similarity (Fig. 2). constructed. For both analyses, Jaccard Similarity Most of the FOC isolates were clustered in cluster A, Coefficient was applied. UPGMA cluster analysis except isolate P2305N which formed cluster B. Within based on the combined data of restriction analysis and cluster A, the FOC isolates were further divided into ERIC-PCR were also conducted using Jaccard sub-cluster A1 and A2 with 90% similarity. Isolates in Similarity Coefficients.