Journal of Plant Pathology (2013), 95 (3), 519-524 Edizioni ETS Pisa, 2013 Tao and Cai 519 MOLECULAR DIAGNOSIS OF COLLETOTRICHUM KAHAWAE BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) G. Tao1, 2, 3 and L. Cai1 1State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, West Bei Cheng Road, Beijing 100101, P.R. China 2Guizhou Institute of Plant Protection, Guiyang 550006, P.R. China 3Guizhou Key Laboratory of Agricultural Biotechnology, Guiyang 550006, P.R. China SUMMARY 2009; Phoulivong et al., 2010). The epitypification of C. gloeosporioides is an important milestone for taxonomic Colletotrichum kahawae, a very aggressive pathogen studies of the C. gloeosporioides complex (Cannon et al., causing coffee berry disease (CBD), is specific to Arabica 2008). The ‘gloeosporioides’ complex currently comprises coffee (Coffea arabica) in Africa. This species is a signifi- C. asianum, C. fructicola, C. horii, C. kahawae, C. gloeo- cant quarantine pathogen in many countries which are still sporioides and C. siamense (Waller et al., 1993; Johnston free from CBD. C. kahawae is morphologically similar to et al., 2008; Cai et al., 2009; Prihastuti et al., 2009; Yang C. gloeosporioides but is phylogenetically and phenotypi- et al., 2009), plus several species that were described or cally distinct. Loop-mediated isothermal amplification epitypified more recently, e.g. C. ignotum, C. musae and (LAMP) is a simple, cost-effective, and rapid method for C. tropicale (Rojas et al., 2010; Su et al., 2011). Morphologi- specific DNA-based detection. In this paper, the Apn2/ cal characters of species in the ‘gloeosporioides’ complex MAT locus was selected as candidate marker, on whose often overlap and are ambiguous (Cai et al., 2009; Hyde et basis species-specific primers were designed for loop- al., 2009), thus differentiating species in this complex re- mediated isothermal amplification (LAMP) diagnosis of mains a challenge for taxonomists and plant pathologists. C. kahawae. The accuracy of this assay was tested using Colletotrichum kahawae Bridge and Waller was formerly the type strains of other closely related species in the C. referred to as C. coffeanum (Nutman and Roberts, 1960), gloeosporioides species complex. The sensitivity of LAMP and later recognized as C. gloeosporioides, C. acutatum is high, with 8×10-2 pg µl-1 genomic DNA as the lowest (Hindorf, 1970) and C. kahawae (Waller et al., 1993). Thus detectable concentration. It offers a new and effective way for a long time, C. kahawae, the cause of Coffee berry dis- for the rapid, specific and cost-effective diagnosis of severe ease (CBD), was regarded as a synonym of C. gloeosporioi- fungal pathogens, thus representing a favourable option des. Waller and Bridge (1993) described the causal agent to conventional PCR and real-time PCR assays. LAMP of CBD as C. kahawae, a distinct Colletotrichum species diagnosis of C. kahawae is likely to provide a useful tech- based on morphological, cultural and biochemical charac- nological option for quarantine and disease management ters and more recently this was confirmed by multi-locus and control to prevent further spreading of this pathogen. phylogenetic analyses (Prihastuti et al., 2009). C. kahawae emerges as a specialized pathogen causing CBD on Ara- Key words: Apn2/MAT locus, Coffea Arabica, coffee bica coffee (Coffea arabica) throughout the African con- berry disease, Colletotrichum gloeosporioides, molecular tinent (Waller et al., 1993). It is the only Colletotrichum diagnosis, plant disease. species listed as a quarantine pathogen in China (Ministry of Agriculture of the People’s Republic of China, 2007), as well as in many other countries of Asia and Latin America INTRODUCTION (Flood and Waller, 2001). Some molecular detection methods for important Col- Colletotrichum gloeosporioides, known as one of the letotrichum pathogens such as C. acutatum and C. gloeo- world’s most important pathogens, is a species complex sporioides have been developed (Sreenivasprasad et al., comprising morphologically indistinguishable but geneti- 1996; Natalia et al., 2002). However, they were established cally and biologically separated cryptic species (Johnston before the availability of type-derived sequences and et al., 2008; Cai et al., 2009). These cryptic species have failed to detect species among the “gloeosporioides” com- recently been described based on multi-locus phylogeny in plex. Up to now, there are very few simple, cost-effective, combination with phenotypic characters (Prihastuti et al., and rapid molecular tools available to detect important pathogens in Colletotrichum, except for a species-specific Corresponding author: L. Cai real-time PCR protocol for the diagnosis of C. kahawae Fax: +86.010.62560523 developed by Tao et al. (2013). E-mail: [email protected] 4. JPP1457RP - Tao.indd 519 06/11/13 15:54 520 Molecular diagnosis of C. kahawae Journal of Plant Pathology (2013), 95 (3), 519-524 Table 1. List of Colletotrichum species and sequences of Apn2/ in less than 1 h under isothermal conditions, and opti- MAT locus used in this study mized for rapidness, specificity, and sensitivity (Notomi et al., 2000). Compared to conventional PCR and real-time Fungal species Strain No. GenBank accession No. PCR, LAMP has the advantages of reaction simplicity (with a constant temperature) and higher amplification C. kahawae IMI319418 JQ899281 efficiency (within 1 h) (Parida et al., 2008). LAMP has C. kahawae IMI363578 JQ899282 potential applications for diagnosis of pathogenic fungi C. asianum MFLU 090233 JQ899285 as well as surveillance of plant diseases without requiring C. fragariae ICMP17927 JQ899279 sophisticated equipment and special expertise (Notomi et C. fructicola MFLU 090228 JQ899290 al., 2000; Parida et al., 2008; Mori and Notomi, 2009). C. gloeosporioides CBS953.97 JQ899278 The objectives of this study were to design a C. ka- C. horii ICMP12942 JQ899280 hawae-specific primer set for LAMP using the Apn2/ C. jasmini-sambac LLTA-01 JQ899273 MAT locus as candidate marker, and to develop a LAMP C. musae CBS116870 JQ899271 protocol to diagnose C. kahawae specifically, rapidly C. siamense MFLU 090230 JQ899289 and efficiently from its closely related species in the C. simmondsii BRIP28519 - ‘gloeosporioides’ complex. ITS region has been routinely employed to explore the MATERIALS AND METHODS phylogenetic relationships, and has also been proposed as the universal DNA barcode for the Kingdom of Fungi Fungal strains. The fungal isolates of C. kahawae were (Schoch et al., 2012). However, it has also been widely ac- the ex-type cultures deposited in the culture collection of knowledged that ITS does not give sufficient resolution to CABI, UK. Other reference taxa employed in this study differentiate Colletotrichum species (Crouch et al., 2009a). include ex-type strains of C. asianum, C. fragariae, C. fructi- ITS sequence data has been applied to resolve species of cola, C. gloeosporioides, C. horii, C. jasmini-sambac, C. mu- the ‘gloeosporioides’ complex (Sreenivasaprasad et al., sae, C. siamense and C. simmondsii from the culture collec- 1993, 1996; Johnston and Jones, 1997), but the resolution is tion centers worldwide (Table 1). Sequences of the Apn2/ not satisfactory (Crouch et al., 2009a). On the other hand, MAT locus from the 10 species above were retrieved from as shown by Crouch et al. (2009b) and Silva et al. (2012), the GenBank (Table 1). the sequence data from the Apn2/MAT locus provides a much better phylogenetic resolution for the graminicolous Genomic DNA extraction, C. kahawae-specific primer species and those of the ‘gloeosporioides’ complex. set design. Fungal strains were grown on potato dextrose Loop-mediated isothermal amplification (LAMP) is a agar (PDA) at 25°C for 7 days. Genomic DNA was ex- simple, cost-effective and rapid diagnostic protocol for the tracted using a Biospin Fungus Genomic DNA Extrac- early detection of microorganisms (Notomi et al., 2000; tion Kit (Fluxion, USA) according to the manufacturer’s Parida et al., 2008). The amplification can be completed instructions. Quality and quantity of DNA were estimated Fig. 1. The positioning and orientation of the species-specific primers for C. kahawae LAMP assay are delimited in boxes within the nucleotide sequence of the Apn2/MAT locus region. GenBank accession Nos. are JQ899281 (C. kahawae IMI319418) and JQ899282 (C. kahawae IMI363578). The forward internal primer (FIP) is composed of the complementary sequence to F1 (F1C) and the sequence F2. The backward internal primer (BIP) is composed of the complementary sequence to B1 (B1C) and the se- quence B2, modified with a TT (FIP) and T (BIP) linker sequence between the sense and antisense sequences to ensure loop for- mation. Two outer primers (F3 and B3) are designed for the strand displacement during the non-cyclic step reaction of LAMP. 4. JPP1457RP - Tao.indd 520 06/11/13 15:54 Journal of Plant Pathology (2013), 95 (3), 519-524 Tao and Cai 521 visually by staining with GelRed on 1% agarose gel elec- (Eiken Chemical Co., Japan). Four microliters of LAMP trophoresis. reaction products were run on a 2.0% (wt/vol) agarose Design of a highly sensitive and specific primer set is gel, and visualized by staining with GelRed under UV crucial for performing LAMP reactions. In the case of C. illumination. kahawae, the Apn2/MAT locus sequences from 10 Colle- totrichum species (listed above) were retrieved from Gen- Specificity and sensitivity of C. kahawae LAMP assay. Bank, aligned with ClustalX 1.81 (Thompson et al., 1994) For specificity tests, nine species in the C. gloeosporioides and manually edited by BioEdit 7.0 (Hall, 1999). The complex and C. simmondsii as reference were selected alignment was analyzed for divergences among the se- (Table 1). The genomic DNA of these Colletotrichum quences and divergent regions were used to develop prim- strains was diluted to a final concentration of 20-30 ers (F3, F2, F1C, B1C, B2, B3) (Fig. 1) that would specifi- ng µl-1 measured by Biospec-NANO (UV-VIS Spectro- cally amplify C. kahawae, using the LAMP program of photometers, Japan), and used as templates for LAMP Primer Explore (available at http://primerexplorer.jp/e/, amplification.