3. Imam (A Species).cdr BIOTROPIA Vol. 19 No. 1, 2012: 19 - 29 A SPECIES-SPECIFIC PCR ASSAY BASED ON THE INTERNAL TRANSCRIBED SPACER (ITS) REGIONS FOR IDENTIFICATION OF , AND ON BANANA Mycosphaerella eumusae M. fijiensis M. musicola IMAN HIDAYAT Recipient of BIOTROP Research Grant 2010/Accepted 28 June 2012 A study on development of a rapid PCR-based detection method based on ITS region of , , and on banana was carried out. The main objecive of this study was to develop a fast and species-specific PCR-based detection method for the presence of species on banana. The methods include collection of specimens, morphological identification supported by molecular phylogenetic analysis, RFLP analysis, species-specific primers development, and validation. Two species of , namely, and , and one unidentified species were found in Java Island. Three restriction enzymes used in the RFLP analysis, viz, AluI, HaeIII, and TaqI were capable to discriminate , , and . Two species-specific primer pairs, viz, MfijF/MfijR and MmusF/MmusR have been successfully developed to detect the presence of and , respectively. banana, detection, fungi, leaf spot, phytopathology Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences (LIPI), Cibinong 16911, West Java, Indonesia M. eumusae M. fijiensis M. musicola Mycosphaerella Mycosphaerella M. fijiensis M. musicola Pseudocercospora M. eumusae M. fijiensis M. musicola M. fijiensis M. musicola Mycosphaerella ABSTRACT INTRODUCTION Key words: Indonesia is one of banana production zones in Southeast Asia. However, crop losses from global climate change and fungal pathogens pose a serious threat not only to Indonesia, but also to global food security. Therefore, these threats should not be underestimated. Among the banana pathogens, three morphologically similar species, viz, (black leaf streak disease/black Sigatoka), (yellow Sigatoka disease), and (Eumusae leaf spot) are well known as important plant pathogens (Crous & Mourichon 2002). In Indonesia, these pathogens are considered as quarantine organisms (http://www.karantina.deptan.go.id/optk/ Mycosphaerella fijiensis M. musicola M. eumusae * Corresponding author : imanhidayat@yahoo.com 19 detail.php?id=731). Therefore, it is important to prevent introduction (entry and establishment) and to limit dissemination of these pathogens in many Indonesian banana-producing regions. Correct and rapid identification is a fundamental step for limiting the dissemination of the plant pathogens (Arzanlou 2007). Failure to manage the pathogens would have far reaching effects on the industry. The 10-14 days incubation and classical isolation of the pathogens by culturing on appropriate media followed by morphological characters examination is a standard method currently used in Indonesia for the imported crops inspection. However, an accurate detection and diagnosis of the and based on the conventional method are complicated due to the similarity in morphological characters (Arzanlou 2007). Consequently, this problem yield difficulties for Indonesian quarantine in inspecting imported banana seeds or crops. Many PCR-detection methods for fungi have shown to be accurate and sensitive in detection various plant pathogens (Bonants 1997; Mumford 2006). ITS sequence analysis has shown that , and are only distantly related in terms of phylogeny (Crous 2002). However, the phylogeny method was still time consuming and lacked specificity to differentiate among the , and (Arzanlou 2007). The lack of specificity was possibly due to the high variability among those three pathogens. Therefore, it is necessary to develop a fast and specific PCR-based detection method with the aim of improving the specificity of the diagnostic procedure and increasing throughout readiness for outbreaks of the disease. Fungal materials were collected from several locations in Bogor and Cibinong (West Java), and one specimen was collected from Wonosobo (Central Java). Specimens with black leaf streak diseases symptoms of were collected during the course of field trips by using a 10×/20× magnifying lens. Specimens were kept in resealable plastic bag. The bags were labelled by adding all necessary information such as location, collector/s, collection date, host name, etc. Microscopic examination of materials was referred to Hidayat . (2007). Ascomata appearances of spp. and caespituli of anamorphic states ( spp.) on the host surface were observed by using stereo microscope (OLYMPUS SZX7). Detailed observations of morphological characters was carried out by means of an OLYMPUS CX31 light microscope using oil immersion (1000×). Water and lactophenol were used as mounting media. Measurements of all important characters and photographing/line drawings were conducted at a magnification of 1000×. Single spore isolation was referred to Choi . (1999). Voucher specimens were deposited at the Herbarium Bogoriense, Research Center for Biology, Indonesian Institute of Sciences-LIPI, Cibinong, West Java, Indonesia. Living cultures were deposited at the et al. M. fijiensis, M. musicola, M. eumusae et al. et al. et al. M. fijiensis, M. musicola M. eumusae et al. M. fijiensis, M. musicola M. eumusae et al. M. fijiensis et al Mycosphaerella Pseudocercospora et al MATERIALS AND METHOD Fungal materials BIOTROPIA Vol. 19 No. 1, 2012 20 LIPIMC microbial culture collection, Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences-LIPI, Cibinong, West Java, Indonesia. Fungal species found in this study were compared to isolate of the , , and obtained from CBS culture collection (Table 1). DNA from fungal cultures was extracted using cetyltrimethylammonium bromide (CTAB) protocols (Rogers & Bendich 1994). The primers ITS1 (5'-GAAGTAAAAG TCGTAACAAG-3') and ITS4 (5'- CCTCCGCTTATTGATATGC-3') (White . 1990) were used to amplify the ITS area. The PCR of 10×concentrated reaction buffer containing 1.5mM MgCl Taq DNA polymerase (5 water . The PCR reaction was performed as follows: 1 cycle of 5 min at 94ºC followed by 40 cycles of 30s at 94ºC, 30s at 52ºC, and 30s at 72ºC. One cycle of 7 min at 72ºC was conducted. After amplification, 5 L of the reaction mixture was loaded onto a 1.0% agarose gel in 0.5×TBE buffer, separated by electrophoresis, stained with ethidium bromide, and viewed and photographed under UV light. A negative control (no DNA target) was included in every experiment to test for contamination, as well as a positive control (DNA from a reference strain of the pathogen). The amplicons was sequenced in both directions using the PCR primers and a DYEnamic ET Terminator Cycle Sequencing kit (Amersham, Biosciences) according to the manufacturer's recommendations. The products were analyzed on an ABI Prism 3700 DNA Sequencer (Perkin-Elmer, Foster City, CA). A consensus sequences were computed from the forward and reverse sequences with SeqMan from the Lasergene package (DNAstar, Madison, WI). The sequences obtained from the respective primers (ITS5 and ITS4) were aligned in Clustal X (Thomson . 1997) and Bioedit (Hall 1999). Phylogenetic analysis was performed in PAUP* (Swofford 2002). Ambiguously aligned sites were excluded from all analyses. Unweighted parsimony (UP) analysis were performed. Gaps were treated as missing data. Maximum parsimony analysis was performed for all data sets using the heuristic search option with 1000 random taxa additions and tree bisection and reconstruction as the branch-swapping algorithm. Branches of zero length was collapsed and all multiple, equally parsimonious trees were saved. The robustness of the trees obtained was evaluated by 1,000 bootstrap replications. Other measures calculated include tree length, consistency index, retention index, and rescaled consistency index (TL, CI, RI, and RC, respectively). The resulting phylogenetic tree was printed with TreeView version 1.6.6 (Page 1996). Restriction digestion of PCR products was conducted directly without further purification with restriction endonucleases to obtain RFLPs; each sample was digested M. eumusae M. fijiensis M. musicola et al et al DNA extraction and sequencing Sequence alignment and phylogenetic analysis Restriction Fragment Length Polymorphism (RFLP) analysis reaction mixture contained 5μL DNA suspension; 2.5μL ; 2.5μl 600μMdNTPs; 0.25μL of each primer at 60μM; 0.2μL U/μL); 0.25μl internal control, and was filled up with MilliQ to a final volume of 25μL μ 2 PCR assay based on the ITS regions for identification of species on Banana - Iman HidayatMycosphaerella 21 BIOTROPIA Vol. 19 No. 1, 2012 with I, III, I, or I in single enzyme digests. Per each 20mL restriction digest, 10 mL of unpurified, amplified PCR reaction was mixed with the appropriate restriction reaction buffer and 10 U of the appropriate enzyme and then incubated for 6h at 37°C for the I, III, or I digests or at 65°C for the I digests. Restriction fragments were separated by electrophoresis in 2% (wt/vol) and 2.5% (wt/vol) Sepharide Gel Matrix in 1× TAE (40mM Tris acetate, 1mM sodium EDTA) with EtBr at 100 ng/mL in the gel and running buffer. DNA bands were visualized by fluorescence under UV light and photographed. Sequences obtained from ITS region were aligned with Clustal X (Thomson . 1997) dan Bioedit (Hall 1999). A series of species specific primers were designed using Vector NTI software (Invitrogen, Sigma-Aldrich), based on sequence differences among the , , and . The robustness and specificity of various primer combinations were evaluated using DNA from isolates of the , , and . DNA extraction and PCR amplification of these isolates were performed as described above. Alu Hae Taq Rsa Alu Hae Rsa Taq et al M. fijiensis M. musicola M. eumusae M. fijiensis M. musicola M. eumusae Development of specific PCR primers Table 1. List of and obtained in this study.Mycosphaerella Pseudocercospora No. Name Origin Culture Collection Number 1 Mycosphaerella musicola (Mycosphaerella sp.1) Cibinong, West Java, Indonesia LIPIMC 0598 2 Mycosphaerella fijiensis (Mycosphaerella sp.2) Cibalagung, West Java, Indonesia LIPIMC 0599 3 Mycosphaerella musicola (Mycosphaerella sp.3) Wonosobo, Central Java, Indonesia LIPIMC 0600 4 Mycosphaerella eumusae Unknown CBS 114825 5 Mycosphaerella fijiensis Cameroon CBS 120258 6 Mycosphaerella musicola Cuba CBS 116634 7 Pseudocercospora sp. Cibinong, West Java, Indonesia LIPIMC 0601 RESULTS AND DISCUSSIONS Fungal materials and phylogenetic analysis Three isolates of and one isolate of were isolated from specimens collected. The cultures of species collected in this study were compared morphologically to the three species from banana obtained from CBS culture collection (Netherlands). All isolates are listed in Table 1. BLAST result from NCBI GenBank database showed that sequences of sp.1 has 100% similarity to the (AY646445) (Fig. 1), and sp.2 has 99% similarity to the (Gq169763) (Fig. 2). Mycosphaerella Pseudocercospora Mycosphaerella Mycosphaerella Mycosphaerella M. musicola Mycosphaerella M. fijiensis 22 The alignment data matrix of newly ITS sequences of three species and one Speg. species from banana were aligned with sequences of , Fresen., and Fr. retrieved from NCBI GenBank DNA database. The alignment consists of 47 taxa including sequences of (Fresen.) G.A. de Vries and (De Not.) Crous & U. Braun as outgroup. The data matrix yielded 510 total characters included in the analysis of which 320 characters were constant, 27 characters were variable and parsimony-uninformative and 137 characters were parsimony-informative. Twenty- six of the informative characters which were positioned within small insertion/deletions or ambiguous regions were excluded from the analysis. Two maximum parsimonious trees were generated from the analysis. Sum of minimum possible lengths is 232, and sum of maximum possible length was 1091. The best parsimonious tree selected by using KH test was generated in 360 steps (CI = 0. 644, RI = 0. 851, RC = 0.548, HI = 0. 356). The best phylogenetic trees obtained from unweighted maximum parsimony analysis is shown in Figure 3 Mycosphaerella Pseudocercospora Mycosphaerella Cercospora Pseudocercospora Passalora Cladosporium cladosporioides Davidiella tassiana . Figure 1. Pairwise alignment showed 100% similarity between sp.1 and . (Ay646445). Mycosphaerella M musicola Based on this analysis, three major clades were performed. These included clade with anamorph (Clade I) with 61% bootstrap support. Another clade was and anamorph (Clade II, 54% bootstrap support) which is also sister clade to clade with 61% bootstrap support. The last clade is and anamorph (Clade III) with 90% bootstrap support. The sp.1 from this study nested together with species in the clade with 100% bootstrap support. Another species from this study, sp.3 nested together with species within clade with 92% bootstrap support. This information confirms the Mycosphaerella Pseudocercospora Mycosphaerella Cercospora Mycosphaerella Pseudocercospora Mycosphaerella Passalora Mycosphaerella M. musicola Mycosphaerella Mycosphaerella M. fijiensis - 23 PCR assay based on the ITS regions for identification of species on Banana - Iman HidayatMycosphaerella BIOTROPIA Vol. 19 No. 1, 2012 species name of sp.1 as , and sp.3 as . Furthermore, this finding confirms that and exist in Indonesian banana plantation (Java). Another isolate, sp.1 needs more detailed examination as this species does not form monophyletic group with any clades in the phylogenetic tree All species from banana, viz, , , and form a monophyletic clade with 52% bootstrap support. This finding has shown that species from banana is a distinct group of species among the species from various hosts. It has also indicated that the three species of from banana are host specific to the banana trees. Further analysis such as pathogenicity test is necessary to carry out in order to justify the specificity of the three species from banana. Mycosphaerella M. musicola Mycosphaerella M. fijienssis M. fijiensis M. musicola Pseudocercospora Mycosphaerella M. eumusae M. fijiensis M musicola Mycosphaerella Mycosphaerella Mycosphaerella Mycosphaerella . Figure 2. Pairwise alignment showed two nucleotides differences between sp.2 and . (GQ169763) (boxes). Mycosphaerella M fijiensis Restriction Fragment Length Polymorphism ( ) analysisRFLP Polymorphism of fragment size of ITS regions was recognized as reported previously in other fungal group (Gardes & Bruns 1993; Sreenivasadprasad 1996), and it was thought to be variable in the sequences of ITS region because of nucleotide deletions and insertions. In order to identify the species detected by PCR using primers ITS5 and ITS4, the RFLPs of the ITS region were generated using four restriction enzymes, namely, I, III, I, or I. From the analysis, only I was not very useful because it did not cut the amplicon of all species et al. Mycosphaerella Alu Hae Taq Rsa Rsa Mycosphaerella 24 from banana (Fig. 4a). Other restriction enzymes, namely, I, III, and I, generated more fragment per digest, so that the ITS sequences of the , , and could be separated using each RFLPs profile. The III had two recognition sites in the fragments of , and had three recognition sites in the fragments of and (Fig. 4b). One isolate of also be separated from three other isolates of using the III restriction enzyme because it only had two recognition sites in the fragments. A similar result was also found in the restriction fragments of generated by I and (Fig. 4c-d). The majority of RFLPs profiles generated from III were unique for each species. For , the given enzymes ( I and I) probably generated RFLPs profiles which separated isolates at the subspecies level (Fig. 4c-d), but further analysis will be required to justify this result. Alu Hae Taq Mycosphaerella fijiensis M. musicola M. eumusae Hae M. eumusae M. musicola M. fijiensis M. musicola M. musicola Hae M. musicola Alu TaqI Hae Mycosphaerella M. musicola Alu Taq can Figure 3. Single parsimonious tree based on ITS nrDNA sequence data representing placement of spp. and sp.1 found in this study within representatives of the family . The tree is obtained from heuristic search with 1000 random taxon addition of the sequences alignment. Bootstrap values (>50%) from 1000 replicates of Unweighted Maximum Parsimony (UMP) analyses are shown above internodes. Mycosphaerella Pseudocercospora Mycosphaerellaceae 25 PCR assay based on the ITS regions for identification of species on Banana - Iman HidayatMycosphaerella BIOTROPIA Vol. 19 No. 1, 2012 Development of Specific PCR Primers Understanding the banana Sigatoka disease complex is a challenge for plant pathologists (Arzanlou 2007). Therefore, in this study we developed rapid and specific detection method with the feasibility of wide application. Even though a PCR-based detection tool has been developed previously (Johanson & Jeger 1993), those primers could only differentiate from Three species- specific primers were designed, namely, MeuF/MeuR, MfijF/MfijR, and MmusF/MmusR, respectively (Table 2). All primers were designed to operate at relatively high annealing temperatures (54°C-55°C), thereby preventing the co- amplification of non-specific DNA targets. Primer sequences were compared against existing sequences in NCBI GenBank Data Base (http://www.ncbi.nlm.nih.gov/) and DDBJ DNA Data Base of Japan (http://www.ddbj.nig.ac.jp/), and a result of BLAST (Basic Local Alignment Search Tool) showed 100% homology of the primers with sequences of strains belonging to the species of which primers were designed. Single bands of correct size were obtained with species-specific primers from all strains belonging to the three species from banana. et al. M. fijiensis M. musicola. Mycosphaerella Table 2. Primer pairs designed in this study. No. Name Species target Notes 1 MeuF (Forward) (5’-CATCTTTGCGTCGGAGTTCA-3’) Mycosphaerella eumusae Not species-specific (cross reactions with M. fijiensis )MeuR (Reverse) (5’-CCGAAGCGAATTGAAGAATCC-3’) 2 MfijF (Forward) (5’-TCTTTGCGTCGGAGTTTCA-3’) Mycosphaerella fijiensis Species -specific MfijR (Reverse) (5’-TCCGAAGCGAATTGAAAGATC-3’) 3 MmusF (Forward) (5’-TCCTTAACACTGCATCTCTACG-3’) Mycosphaerella musicola Species specific MmusR (Reverse) (5’-TCAGCCGGGAGACTTTGG -3’) M. eumusae M. fijiensis M. musicola M. fijiensis M. musicola Mycosphaerella M. eumusae M. fijiensis M. eumusae M. fijiensis M. fijiensis M. musicola Validation assay on pure cultures of , , and showed that primer pairs of (MfijF/MfijR) (Fig. 5a) and (MmusF/MmusR) (Fig. 5b) are specific to the fungal pathogens as no cross-reactions with others species were observed in the amplification bands. Unfortunately, primer pairs of (MeuF/MeuR) failed to show specificity as cross-reactions were found with sequences of (Fig. 5c). It is probably due to small nucleotide differences between DNA sequences and from ITS region. The specificity of primer pairs of MfijF/MfijR to and MmusF/MmusR to is good indication for the development of molecular diagnosis technique and understanding of the Sigatoka diseases complex of banana in Indonesia. The molecular technique developed in this study may also significantly contribute to plant quarantine because of its reliability, specificity and simplicity. This assay could be done within 1-2 days laboratory works and analysis. 26 Figure 4. Restriction patterns of internal transcribed spacer (ITS) regions of ribosomal DNA amplified from three species from banana. a. I b. III c. I d. I (1-3: ; 4-7: ; 8-9: ). Mycosphaerella Rsa Hae Alu Taq M. eumusae M. musicola M. fijiensis 27 PCR assay based on the ITS regions for identification of species on Banana - Iman HidayatMycosphaerella Figure 5. Species-specific amplification of species from banana using specific primers. a. Primer pairs MfijF/MfijR to the b. primer pairs MmusF/MmusR to the c. primer pairs MeuF/MeuR to the and (2-4: ; 5-8: ; 9-10: ). Mycosphaerella M. fijiensis M. musicola M. eumusae M. fijiensis M. eumusae M. musicola M. fijiensis BIOTROPIA Vol. 19 No. 1, 2012 28 CONCLUSIONS Diagnosis of the banana Sigatoka disease complex is a challenge for plant pathologists. RFLP analysis using III is capable in discriminating the and The rapid and specific PCR-based detection method using species-specific primers of MfijF/MfijR and MmusF/MmusR has been successfully developed to detect and , respectively, from pure cultures. Further examination/validation directly on samples from infected banana leaves with diseases symptom are necessary to test the sensitiveness of this method This research was funded by SEAMEO BIOTROP awarded to Dr. Iman Hidayat. Laboratory of Plant Molecular Systematics, Botanical Division, Research Center for Biology (LIPI) is acknowledged for providing the authors with molecular facilities. The author also thanks Sulistiani and Aerma Hastuty for laboratory assistance. Hae restriction enzyme M. eumusae, M. fijiensis, M. musicola. M. fijiensis M. musicola Mycosphaerella . ACKNOWLEDGMENTS REFERENCES Arzanlou M, Abeln ECA, Kema GHJ, Waalwijk C, Carlier J, de Vries I, Guzmán M, Crous PW. 2007. Molecular diagnostics for the Sigatoka disease complex of banana. Phytopathology 97: 1112-8. Bonants P, Hagenaar-deWeerdt M, van Gent-Pelzer M, Lacourt I, Cooke D, Duncan J. 1997. Detection and identification of Hickman by the polymerase chain reaction. Eur J Plant Pathol 103: 345-55. 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