Identification of Colletotrichum isolates from Capsicum chinense in Amazon

Abstract

Chili pepper (Capsicum chinense) is a great economic important culture on the State of Amazonas, and it represents, approximately, a production of 1.9 thousand tons per year. It is one of the hosts of Colletotrichum genus in the North region of Brazil. The aim of the study was to differentiate and to identify isolates of Colletotrichum collected from C. chinense in Amazon. Molecular characterization, using RFLP-PCR, ERIC-PCR and ISSR, was carried out initially for screening of morphologically similar isolates. Furthermore, phylogenetic analyses were performed using combined regions: Actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for the three isolates, INPA 2066, INPA 2286 and INPA 1858, plus superoxide dismutase (SOD2) for INPA 2066. We showed that the molecular markers were able to distinguish the isolates of Colletotrichum studied and these results were confirmed with the phylogenetic analyses, three different occurrences of Colletotrichum species (C. siamense, C. scovillei and C. brevisporum) causing anthracnose in C. chinense in the State of Amazonas. This study represents the first report of the species C. siamense and C. scovillei in this host in Brazil.

Introduction

Colletotrichum (teleomorph Glomerella), comprises a big range of cosmopolitan fungi species and are usually described as being the anthracnose disease agent. It is known ~600 species of the genus Colletotrichum, which are pathogens of over 3000 species of plants including cereals, legumes, vegetables, perenial crops, and tree fruits (O��?Connell et al., 2012). There are some Colletotrichum species complexes already described in the literature, such as C. gloeosporioides, C. acutatum, C. boninense and C. orbiculare (Weir et al., 2012; Damm et al., 2012a,b, 2013). In Brazil, there are many reports of plant infection caused by Colletotrichum species, especially in economically important plants and fruits, such as coffee berries, sugarcane, strawberry, maize, sorghum, banana, avocado and many others. The tropical and subtropical climate favors the spread of Colletotrichum species on these plants.

It is notable the great economic interest of chili pepper (Capsicum chinense Jacq.) culture in many parts of the world. It happens because of its great potential of growth in almost every country, since it is a tropical regions native, and its use on food, pharmaceutical, cosmetic, and ornamental products (Dias et al., 2013). C. chinense is one of the most cultivated vegetables in Brazil, mainly in the North region. The anthracnose is a disease that more affects the culture and five species of Colletotrichum were described as the pathogen for this host, C. capsici and C. gloeosporioides in India, Indonesia, Korea, and Thailand, C. acutatum in Australia and Indonesia, C. coccodes in Korea and New Zealand (Ratanacherdchai et al., 2010) and C. brevisporum in Brazil (Almeida et al., 2017).

The identification of Colletotrichum based on morphological characters is problematic due to the few morphological traits that can be used to separate species in this genus (Than et al., 2008). Therefore, it is necessary a precise study and characterization based not only on morphological but also on molecular data utilized for species delimitation and defining of inter- and intraspecific relationships as it has been performed in the past decades. Several molecular techniques have been developed to characterize and to identify different Colletotrichum species. Multilocus phylogenetic analysis using partial sequences of gene such as actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), and manganese superoxide dismutase (SOD2) have been utilized to identify different species of this genus (Weir et al., 2012; Damm et al., 2012a).

Additionally, molecular markers are generally recognized as a reliable method to evaluate genetic diversity and differentiation of Colletotrichum spp isolates, such as restriction fragments length polymorphism (RFLP-PCR), inter-simple sequence repeats or microsatellites (ISSR) and enterobacterial repetitive intergenic consensus (ERIC-PCR). In the present paper, the objective was to differentiate and identify isolates of Colletotrichum collected from C. chinense in Amazon, northern of Brazil, using molecular markers and phylogenetic analysis.

Materials and Methods

Isolation of Colletotrichum from C. chinense

Fruits of chili pepper (C. chinense Jacq.) with typical anthracnose symptoms were observed in Manaus, Amazonas, Brazil.

The isolation was performed by collecting spores directly from the surface of the lesions in C. chinense fruit and then plated on PDA culture medium. A monosporic culture was performed to ensure that this work would be upon a single genetic uniformity.

The isolates of Colletotrichum selected and used for screening analysis (Figures 1, 2 and 3 Table 1).

Table 1. List of primers used for molecular screening and phylogenetic analysis.

Screening Colletotrichum isolates by molecular markers

To confirm if C. chinense, in the State of Amazonas, is host of different species of Colletotrichum, a initial screening on the obtained isolates was carried out only using different molecular profile isolates for phylogenetic analysis. For this screening, we used ERIC-PCR, ISSR and PCR-RFLP of GS (Glutamine sintase) intron techniques. For comparison, we used isolates of reference of C. fructicola, C. gloeosporioides and C. fragariae.

DNA extraction was carried out at the Molecular Biology Laboratory - Embrapa Western Amazon, according to Doyle and Doyle (1987). The primers used for amplification are listed in Table 2.

Table 2: Strains of Colletotrichum spp used for phylogenetic analysis, with collection details and GenBank accessions.

To ERIC-PCR marker, ERIC1 and ERIC2 primers were used at a concentration of 0.2 �?µM; 1X Buffer [100 mM Tris-HCl pH 8.8; 500 mM KCl, 0.8% (v/v)], 25 mM MgCl₂; 0.5 mM dNTPs; 50 ng DNA; 1 U Taq polymerase (DNA Express^�?®), and reaction was set up to a final volume of 25 �?µL. The programming on thermal cycler (Applied Biosystems Veriti^TM96-Well Thermal Cycler) initiated with 94�?°C for 1 min followed by 35 cycles of denaturation (94�?°C for 1 min), annealing (48�?°C for 1 min) and elongation (65�?°C for 5 min) and further extension of 65�?°C for 6 min.

To ISSR marker analysis, the PCR was performed at a 25-�?µL final reaction volume, with 0.2 �?µM of the UBS primer 885; 1X Buffer [100 mM Tris-HCl pH 8.8; 500 mM KCl, 0.8% (v/v)]; 25 mM MgCl₂; 0.5 mM dNTPs; 50 ng DNA; 1.5 U Taq Polymerase (DNA Express^�?®). The pre-cycle was on 94�?°C for 5 min, followed by 40 cycles of denaturation (94�?°C for 1 min), annealing (45�?°C for 1 min) and elongation (72�?°C for 1 min), followed by a final extension at 72�?°C for 7 min. Amplification of intron GS, PCR-RFLP was carried out according to Liu et al. (2012). All PCR and PCR-RFLP products ran at a 1.5% w/v agarose gel electrophoresis.

Phylogenetic analysis

Three loci were amplified by PCR and we used for phylogenetic analysis: ACT, GAPDH and SOD2. For these reactions, 1X Buffer [(100 mM Tris-HCl pH 8.8; 500 mM KCl, 0.8% (v/v)], 25 mM MgCl₂; 10 mM dNTPs; 5 �?µM for each primer; 50 ng/mL DNA; 5 U Taq Polymerase (DNA Express^�?®). The first cycle initiated with 94�?°C for 4 min, followed by 35 cycles of denaturation (94�?°C for 30 s), annealing (60�?°C for 30 s) and elongation (72�?°C for 1 min), followed by a final extension at 72�?°C for 7 min. The primer sequences used for each gene are described in the Table 2. PCR products were purified and sequenced by the Applied Biosystems^�?® 3500 Genetic Analyzers. Sequences from forward and reverse primers were aligned to obtain a consensus sequence (Table 1). The fungal DNA sequences were aligned using MEGA 6 (Tamura et al., 2013) with reference sequences of the Colletotrichum obtained from GenBank. The Bayesian inference analyses employing a Markov Chain Monte Carlo method were performed with all individual and combined sequences. The MrModeltest 2.3 (Posada and Buckley, 2004) was used to determine the best model of nucleotide evolution (HKY+I to ACT; HKY+G to GAPDH; GTR+I+G to SOD2). The phylogenetic analysis was performed on CIPRES web portal (Miller et al., 2010) using MrBayes version 3.2 (Ronquist et al., 2012). Markov chain Monte Carlo method was run for 10,000,000 generations, sampling every 1000 generations and discarding 2500 samples as burn-in. The resulting trees were rooted using outgroup taxon. Trees were visualized in FigTree 1.4.0 (Rambaut, 2012) and exported to graphic programs. The sequences obtained in this study were deposited in GenBank (Table 2).

Results and Discussion

The results obtained reveal that simple molecular settings such as ERIC-PCR, PCR-RFLP and ISSR can be used with efficiency for screening of Colletotrichum isolates aiming to identify different species capable of causing anthracnose in the same host. ERIC-PCR and ISSR unique band profiles can be identified (Figures 1 and 2 Figure 3).

Phylogenetic results revealed that these different profiles correspond to different species of Colletotrichum associated with C. chinense (Figures 4, 5 and 6). This information could be confirmed by Bayesian inference methods from multiple gene sequences.

Sequences from fragments of ACT, GAPDH and SOD2 from isolate INPA 2066, ACT and GAPDH from isolates INPA 2286, and INPA 1858 from C. chinense were compared with sequences from strains of other Colletotrichum species and it showed approximately 99% of similarity with Colletotrichum siamense, 99% of similarity with C. scovillei Damm, P.F. Cannon & Crous, and 96% of identity with C. brevisporum Phoulivong, P. Noireung, L. Cai & K.D. Hyde, respectively.

The combined dataset generated from Bayesian analysis shows similar topology with individual trees. In the phylogenetic trees based on combined dataset of ACT, GAPDH and SOD2 comprised 873 characters including alignment gaps, which showed that the isolate INPA 2066 is closely related to C. siamense H. Prihastuti, L. Cai & K.D. Hyde. This tree was rooted to C. boninense (Figure 4). C. siamense is biologically and geographically diverse, found in many hosts across several tropical and subtropical regions, including Capsicum annuum in Thailand (Weir et al., 2012). Recently, Sharma et al. (2015) using multilocus analysis demonstrated that C. siamense are four distinct species forming the C. siamense species complex.

The analysis of the combined dataset of ACT and GAPDH showed that the isolate INPA 2286 formed a monophyletic clade supported (Bayesian posterior probability = 0.87) with three strains of C. scovillei (Figure 5). And isolate INPA 1858 formed a monophyletic clade with high support (Bayesian posterior probability = 1) with two isolates of C. brevisporum (Figure 6). The trees were rooted to C. orbiculare, and two species from each complex (C. acutatum and C. scovillei), respectively (Figures 5 and 6). Colletotrichum scovillei belongs to C. acutatum species complex and was associated with chilli in Indonesia and Thailand (Than et al. 2008; Weir et al., 2012).

In Brazil, the first report of anthracnose on pepper fruit (C. annuum L.) caused by C. scovillei was by Caires et al. (2014). Colletotrichum brevisporum, which is still not inserted in any Colletotrichum species complex has been reported in Neoregalia sp and Pandanus pigmaeus in Thaliand (Noireung et al., 2012). In Brazil, it has been notified the presence of this pathogen in papaya fruit (Vieira et al., 2013), chaoyte fruits (Bezerra et al., 2016) and chili pepper (Almeida et al., 2017).

In the present study, we showed that the molecular markers were able to distinguish the isolates of Colletotrichum studied through the different band profiles and was possible to differentiate isolates of the C. gloeosporioides and C. acutatum species complex. The phylogenetic analysis results confirmed the occurrence of C. siamense, C. scovillei and C. brevisporum causing anthracnose in C. chinense in the State of Amazonas.

This study represents the first report of the species C. siamense and C. scovillei in this host.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

p>The authors thank Fundação de Amparo à Pesquisa do Estado do Amazonas
(FAPEAM) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for
scholarships.