Amplification of the cap20 pathogenicity gene and genetic characterization using different markers molecular in Colletotrichum gloeosporioides isolates

AGRICULTURE, AGRIBUSINESS AND BIOTECHNOLOGY

Amplification of the cap20 pathogenicity gene and genetic characterization using different markers molecular in Colletotrichum gloeosporioides isolates

Danielli Barreto Maciel^I; Lílian Vieira de Medeiros^I; Vivian Vieira de Medeiros^I; Mariele Porto Carneiro Leão^I; Luis Eduardo Aranha Camargo^II; Neiva Tinti de Oliveira^I,*

^IDepartamento de Micologia; Universidade Federal de Pernambuco; Campus Universitário; 50670-901; Pernambuco - PE - Brasil

^IIDepartamento de Fitopatologia; Escola Superior de Agricultura Luiz de Queiroz; Universidade de São Paulo; Campus Universitário; 13418-900; São Paulo - SP- Brasil

ABSTRACT

Studies were performed to analyze the genetic characterization using RFLP-ITS and Intron (primer EI1) markers and the amplification of the cap20 pathogenicity gene by PCR in Colletotrichum gloeosporioides isolates of different hosts plant. The genetic variability was accessed using RFLP-ITS and Intron markers and grouping by UPGMA method. Primers to cap20 gene were constructed using selected sequences of the GenBank (National Center of Biotechnology Information, http://www.ncbi.nlm.nih.gov) with the Primer 3 program. The dendrograms analysis showed that the RFLP-ITS marker was more informative to separate the Colletotrichum sp, and that primer EI1 demonstrated greater genetic diversity. The amplification of the DNA of the Colletotrichum isolates to the cap20 gene with primers P1 and P2 indicated that this gene could present variations into C. gloeosporioides related with the host, and also that it was present in other Colletotrichum sp.

Key words:cap20 gene, Colletotrichum gloeosporioides, pathogenicity.

RESUMO

Estudos foram realizados para analisar a caracterização genética usando marcadores de RFLP-ITS e ISSP e a amplicação do gene de patogenicidade cap20 por PCR em isolados de Colletotrichum gloeosporioides de diferentes hospedeiros. Primers para o gene cap20 foram construídos a partir de seqüências selecionadas do GenBank (National Center of Biotechnology Information, http://www.ncbi.nlm.nih.gov) com o programa Primer 3. A análise dos dendrogramas revelou que o marcador RFLP-ITS foi mais informativo em separar as espécies de Colletotrichum, e que o primer EI1 evidenciou maior diversidade genética. A amplificação do DNA dos isolados de Colletotrichum para o gene cap20 com os primers P1 e P2 indicou que este gene pode apresentar variações dentro de C. gloeosporioides relacionada ao hospedeiro, e que também está presente em outras espécies de Colletotrichum.

INTRODUCTION

Anthracnose caused by Colletotrichum species is the main disease in post-harvest fruit and is considered disease of high economic importance in the Northeast of Brazil (Serra and Silva, 2004). Colletotrichum is a filamentous fungus that affects fruit of many varieties of botanical species in pre and post-harvest, causing quiescent infections particularly in tropical and subtropical regions, resulting in losses of billions of dollars annually (Korsten and Jeffries, 2000; Kramer-Haimovich et al., 2006).

Several factors are responsible for the post-harvest losses, including post-harvest fungal diseases, which cause considerable losses on the products collected, fruits and vegetables. The losses range around 20 to 50%, which depending on the year and quality of storage, can be high in developing countries (Smith et al., 2003; Agrios, 2005).

The Colletotrichum genus displays two modes of nutrition, biotrophic and necrotrofic and develops a series of specialized infection structures, such as: germ tube, appressorium, peg penetration, vesicles infectives, primary and secondary hyphaes (O'Connell et al., 2000). The stages of formation of these structures are marked by the production of specific metabolites, which may act as pathogenicity factors. The penetration of the tissue is favored by the action of pectinolytic enzymes, excreted on the host tissue and by the mechanical force exerted by appressorium on the peg of penetration (Agrios, 2005).

Recently several genes of the phytopathogenics fungi have been identified, that after deletions, resulted in reduction or total loss of symptoms of the disease. Some of these genes are responsible for the degradation on the cell wall of the plant; others induce the formation of the specialized structures such as appressorium that penetrates into the epidermis and others produce toxins to overcome the defenses of the plants (Idnurm and Howlett, 2001). Oliver and Osboum (1995) sugested that the pathogenicity genes were those required for the development of the disease and have been identified, in recent years, to increase the knowledge of process and control of the disease (Idnurm and Howlett, 2001).

Differential screening of a cDNA library produced by a subtractive hybridization approach yielded the genes designated cap expressed uniquely during appressorium formation of C. gloeosporioides induced by the wax present on the surface of the fruit of avocado. The cap20 gene encodes a protein of 20 kDa which may have a role in the apressorium function. The CAP20 peptide showed homology with the proteins of the cell wall and could be part of the wall of the apressorium. Mutants with disruption of this gene was unable to express a protein of 20 kDa and showed drastic reduction in virulence in the fruits of avocado and tomato (Hwang et al., 1995).

Several methods can be used for inter and intraspecific characterization of Colletotrichum sp.. Currently, a promising tool for the study of population dynamic is the use of molecular markers that can infer the genetic variability (Katan, 2000). The existence of species-specific primers based on nucleotides sequences of the ITS1 rDNA region have made the Polymerase Chain Reaction (PCR) a powerful tool for the identification of Colletotrichum species (Peres et al., 2002; Afanador-Kafuri et al., 2003; Bueno, 2005; Tozze Junior et al, 2004; Andrade et al., 2007).

The aim of this study was to amplify the pathogenicity gene cap20 by PCR and intraspecific genetic characterization using molecular markers RFLP-ITS and ISSP in C. gloeosporioides isolates obtained from different hosts plant.

MATERIAL AND METHODS

Strains Colletotrichum

Twenty eight isolates of Colletotrichum sp of different hosts were obtained from the Department of Mycology, Federal University of Pernambuco (URM-UFPE) (Table 1). The isolates were maintained on potato dextrose agar (PDA) medium for seven days. Out of these, 17 (1-17) were used for genetic characterization with molecular markers CgInt-ITS4, RFLP-ITS and ISSP and three isolates of other species (20-21 and 25) were used as outgroup for comparative analysis.

For the amplification of the cap20 gene were used them all the 28 isolates, of wich nine isolates of other species (20-28) were used as outgroup for comparative analysis.

DNA extraction

Flasks containing 100 ml liquid minimum medium were inoculated with 3 ml of the conidial suspension of Colletotrichum sp. (10⁶ conidia/ml), maintained under agitation (27 rpm) and incubated at 27ºC for 120 h. The mycelium were harvested by filtration, washed with sterile-distilled water and stored at -20ºC until use. Total genomic DNA was extracted as described by Kuramae and Izioka (1997). The mycelium was ground into the fine powder under liquid nitrogen and suspended in 700 µL extraction buffer (1M Tris-HCl pH 8.0; 5M NaCl; 0,5mM EDTA pH 8.0; 10% SDS). Upon homogenization, the tubes were incubated for 30 minutes at 65ºC.

DNA samples were purified with equal volumes of chloroform: isoamyl alcohol (24:1) mixture (1X), and precipitaded with isopropanol. The tubes were centrifuged at 15400g (Eppendorf® centrifuge) for 10 minutes and DNA pellets were rinsed with 70% ethanol, air dried, suspended in TE buffer (pH 8.0) and stored at 4°C until use.

DNA amplification with primers CgInt/ITS4

For specific confirmation of the C. gloeosporioides isolates, species-specific primers CgInt-ITS4 was used. Amplification reactions were prepared to final volume of 25 µL containing 1x Taq buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 25 ng template DNA, 1.5 mM MgCl₂, 0.2 mM dNTP, 0.5 µM of each primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and CgInt (5'-GGCCTCCCGCCTCCGGGCGG-3') and 0.04U Taq DNA polymerase (Invitrogen), as described by Freeman et al (2000). Thermal cycling consisted of initial denaturation of 5 minutes at 95ºC, followed by 40 cycles of 30 seconds at 95ºC, 30 seconds at 45ºC and 1 minute and 30 seconds at 72ºC. The amplicons were visualized in 1.0 % (w/v) agarose gel at 3 V/cm in TBE buffer (pH 8.0) after ethidium bromide staining using 100-pb ladder marker (Invitrogen).

RFLP-ITS

Amplification reactions were prepared to final volume of 25 µL containing 1x Taq buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 25 ng template DNA, 1.5 mM MgCl₂, 0.2 mM dNTP, 0.2 µM of each ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAA-3') and 0.04U Taq DNA polymerase (Invitrogen), as described by White et al (1990). Thermal cycling consisted of initial denaturation of 5 minutes at 95ºC, followed by 40 cycles of 30 seconds at 95ºC, 30 seconds at 50ºC and 1 minute and 30 seconds at 72ºC. Amplification products were visualized in 1.4 % (w/v) agarose gel at 3 V/cm in Tris-Borato-EDTA (TBE) buffer (pH 8.0) after ethidium bromide staining. Aliquots of 4 µL of the amplicons were subjected to enzymatic digestion with DraI, MspI or HaeIII, according to manufacturer instructions. Fragments were separated in 1.5% (w/v) agarose gel and their molecular weights were determined using to 100-pb ladder marker (Invitrogen).

ISSP

Fingerprinting analysis were performed with EI1 type I Intron Splice Site Primer (5'-CTGGCTTGGTGTATGT-3') as described by De Barros Lopes et al (1996). The amplification reactions contained 1x Taq buffer (20 mM Tris-HCl pH 8.4, 50mM KCl), 25 ng template DNA, 1.5 mM MgCl₂, 0.25mM dNTP, 0.5 µM of EI1 (5'-CTGGCTTGGTGTATGT-3') primer and 0.04U Taq DNA polymerase (Invitrogen) into final volume of 25 µL. Thermal cycling consisted of initial denaturation of 5 minutes at 95ºC, followed by 40 cycles of 30 seconds at 95ºC, 30 seconds at 45ºC, 1 minute and 30 seconds at 72ºC, with final extension of 5 minutes at 72ºC. The amplicons were visualized in 1.0 % (w/v) agarose gel at 3 V/cm in TBE buffer (pH 8.0) after ethidium bromide staining.

Genetic analysis

The variable binary similarity matrix was prepared using Jaccard coefficient by the NTSYS program (Numerical Taxonomy System of Multivariate program) version PC 2.1 (Rohlf, 1988). Dendrogram were prepared by UPGMA (Underweight Pair Group Method with Arithmetical Average) analysis.

Amplification of the cap20 gene by PCR

Primers for the PCR reactions were constructed from the sequences of C. gloeosporioides selected from GenBank (National Center of Biotechnology Information, http://www.ncbi.nlm.nih.gov). The amplification reactions were prepared to final volume of 25 µL with following conditions: 1x Taq buffer (Tris-HCl 20mM pH 8.4; 50mM KCl), 1.5 mM MgCl₂, 0.3 mM dNTP, 0.2 µM of each primer P1 (5'-GCAACATCTCGTCCGCTCT-3') and P2 (5'-TGAAGTGGGGAGAAGGGAA-3'), 0.04U Taq DNA polimerase (Invitrogen Life Tecnologies) and 25 ng of DNA. The PCR reaction involved an initial step of denaturation of 5 minutes at 95C, followed by 10 cycles involving denaturation of 30 seconds at 95ºC; anelament of 30 seconds at 50°C and extension of 30 seconds at 72ºC, and a second step with 30 cycles involving initial denaturation of 30 seconds at 95ºC, anelament of 30 seconds at 47°C and extension of 30 seconds at 72ºC. These reactions were repeated four times (4x) to get the generated confirmation results. The products amplified were visualized in 1.0 % (w/v) agarose gel at 3 V/cm in TBE buffer (pH 8.0) after ethidium bromide staining and their molecular weights were determined using 100-pb ladder marker (Invitrogen).

Enzimatic digestion of the amplified cap20 gene products

Aliquots of 6 µl of the amplicons were subjected to enzymatic digestion with MspI (Invitrogen), according to manufacturer instructions. The restriction fragments were separated on 1.5% (w/v) agarose gel, with 0.5x TBE buffer. The gel was stained with ethidium bromide (0.5mg/mL), and the DNA fragments visualized under UV light, using marker of molecular weight 1-kb plus (Invitrogen).

RESULTS AND DISCUSSION

Specific characterization with primers CgInt-ITS4

The amplification using the primers CgInt-ITS4 (Fig. 1) was positive for all of the C. gloeosporioides isolates, generating fragments of approximately 450-bp and negative for of the C. acutatum and C. sublineolum isolates.The use of these primers confirmed the identity of the isolates of the C. gloeosporioides identified by traditional methods. Mills et al. (1992) and Freeman et al. (2000) used these markers for the identification of the C. gloeosporioides isolates, which showed fragments of 450-bp. The results were similar to those found here.

Polymorphism of fragments digestion of the ITS rDNA regions of C. gloeosporioides

The amplification of the products ITS rDNA regions using primers ITS4 and ITS5 revealed fragments around the 600-pb for Colletotrichum sp. isolates. The ITS rDNA region was not informative to differentiate the Colletotrichum species (Fig. 2A).

Several authors working with primers ITS1 and ITS2 observed that the majority of isolates identified as C. gloeosporioides, showed a single fragment of approximately 590-600 bp (Abang, 2002; Martínez-Culebraz et al., 2000; Saha, 2002).

The digestion of the fragments of the ITS with DraI produced a monomorphic fragment of approximately 400-bp, for all Colletotrichum sp. isolates that made impossible to differentiate the species (Fig. 2B). MspI generated three distinct fragments of 300, 150 and 100-bp for all of the C. gloeosporioides isolates, except for the URM4900 isolate which presented fragments of 350, 150 and 100-bp; fragments of 300, 170 and 100-bp for URMPI15 and URMAcu of C. acutatum isolates and fragments of 300, 130 and 100-bp for URMSub of the C. sublineolum isolate (Fig. 2C). The restriction of the products amplified with the HaeIII enzyme generated three fragments distinct in sizes of 280, 180 and 150-bp for all the C. gloeosporioides and C. acutatum isolates, and fragments of 300, 180 and 170-bp for URMSub of the C. sublineolum isolate, distinguishing only this last specie (Fig. 2D) (Table 3).

In this study, the MspI and HaeIII enzymes showed polymorphism in the number and length of the resulting fragments, supporting the idea of the existence of intraspecific genetic diversity among different C. gloeosporioides isolates.

The MspI restriction enzyme better separated the three of the Colletotrichum species, in comparison with the other enzymes tested; however was not efficient in separating the C. gloeosporioides isolates from the host and geographic region, except for URM4900 isolate. The URM4900 isolate from cashew leaf of the C. gloeosporioides showed different amplification profiles of the other isolates of the same host.

This could be due to the great morphological plasticity, adaptability and specificity that existed among the isolates of the C. gloeosporioides specie or by pathogenic specialization caused by the mutations, requiring further studies. Results obtained with the HaeIII restriction enzyme suggested that C. gloeosporioides and C. acutatum species presenting the same profile of digestion, could be related and that these species probably differed recently.

The analysis of the grouping profiles generated from three enzymes allowed the construction of the dendrogram where two distinct groups could be observed in the level of similarity of size of fragments of 85%. The first group contained all of the C. gloeosporioides isolates with 100% of similarity between themselves (except the URM4900 isolate), and the second group with the two isolates of C. acutatum also with 100% of similarity between themselves. The URM4900 isolate showed similarity of size of fragments with the others two groups in amount around 73%. The URM4900 of the C. gloeosporioides isolate and URMSub of C. sublineolum isolate were different (Fig. 3).

Martínez-Culebras et al (2000), studying the genetic variability of Colletotrichum sp. isolates from the strawberry separated the isolates of C. gloeosporioides from the other species examined for the restriction profiles of ITS rDNA region with nine restriction enzymes whose specific profiles of C. gloeosporioides was observed with the MvnI enzyme. Martínez-Culebras (2003) differentiated 80 species of the Colletotrichum genus by ITS1 and ITS2 of the region 5.8S rDNA, which showed that the C. acutatum isolates of the same geographical region belonged to the same group. Vinnere (2002), using profiles of restriction of the fragments of ITS region distinguished the C. acutatum, C. gloeosporioides and C. dematium species between the isolates which were identified as a classical form of C. gloeosporioides. The ITS-RFLP technique was useful for the identification of the Colletotrichum species, whose specific profile was observed with the MspI enzyme and efficient for the analysis of intraspecific genetic diversity between C. gloeosporioides isolates using the MspI and DraI enzymes.

Analysis of the intron site of splicing

The amplification profiles of the region of intron using the primer EI1 in Colletotrichum sp. isolates and dendrogram generated are illustrated in the Figs. 4 and 5, respectively. The dendrogram generated from the amplification profiles revealed the formation of three distinct groups with 85% similarity in the size of fragments, indicating high intraspecific genetic diversity among the C. gloeosporioides isolates. The first group was represented by four isolates of C. gloeosporioides from the onion (URM4626, URM4627, URM4628 and URM4629); the second group was formed by two isolates of C. gloeosporioides from the mango (URM4852 and URM4858), and the third group was represented by three isolates from the mango (URM4854, URM4857 and URM4859) of C. gloeosporioides. The groups formed presented 100% similarity in the size of fragments for the isolates between themselves.

In this study, all the C. gloeosporioides isolates from the onion showed the same profile of bands; however, the cashew isolates of the species (URM4894, URM4896, URM4900, URM4905, URM4908) were more heterogeneous in the number, and the length of the bands generated showed high genetic diversity. It was observed that the URM4856 of the C. gloeosporioides isolate from the pink mango did not form group with other isolates of the same host, and the URMCi and URMPI13 isolates were different.

The ISSP marker showed high intraspecific genetic diversity among the C. gloeosporioides isolates, presenting certain degree of grouping for the host, but the grouping for the species was not evidenced (Fig. 5). This is the first report on the use of primer EI1 for genetic analysis of the Colletotrichum species.

Brasileiro et al (2004) had used marker EI1 to analyze the intraspecific polymorphism in Fusarium solani isolates. They reported that some F. solani isolates understood the I-EI1 group that represented a clonal strain. Moreover, other isolates of the same species showed high genetic divergence to the primer EI1, detected by the analysis of ITS region.

Medeiros (2008), studying the genetic characterization by ISSR molecular markers of 20 isolates of Colletotrichum sp. from the various hosts, found that this technique was able to group the isolates of different species according to the origin host. Similar, in this study the homology observed between the isolates from the onion and the grouping between some isolates of C. gloeosporioides from the mango and cashew.

In this work, the ITS-RFLP and ISSP techniques were effective to demonstrate the intraspecific genetic characterization between the C. gloeosporioides isolates; however, the ISSP molecular marker was more informative to separate the intraspecific isolates, generating a higher polymorphism and consequently, greater genetic diversity in relation to ITS-RFLP technique, which was a very important method to differentiate the species of fungi. Moreover, these two techniques are appropriate tools to discriminate all the species at the interspecific level.

Amplification of the cap20 gene by PCR

The amplification of cap20 gene fragments, using the specific primers P1 and P2 for the Colletotrichum sp., is illustrated in Fig. 6. Between the C. gloeosporioides isolates, the tests with primers constructed indicated a variation in the structure of cap20 gene, therefore 12 of the C. gloeosporioides isolates amplified one fragment of 950-bp, and of the URM4905 isolate proceeding from the cashew presented consistently two bands in all the repetitions of the reaction, one common to the isolates of the C. gloeosporioides (950-bp) and other of size lesser (700-bp) similar to the band generated in C. boninense, C. capsici and C. dematium. Moreover, all mango isolates (except URM4856) and C. gloeosporioides URM1633 isolate from the chilli, did not present amplification. Also there was variation in the structure of the gene between the species, therefore, C. boninense, C. capsici and C. dematium presented the bands of different sizes of the C. gloeosporioides from majority of the isolates. Hwang et al (1995) found that the cap20 gene in C. gloeosporioides was a apressorium functional gene whose expression was clearly induced by the wax present in the fruits of avocado and tomato, and its delection caused drastic reduction in the pathogenicity to these fruits. However this gene presented only one copy in the genome.

This work showed the amplification of the cap20 gene in C. gloeosporioides isolates from the onion, cashew, mango, chilli and ciclamen related to the host of origin and in C. boninense, C. capsici, C. dematium and C. gossypii var. cephalosporioides species and absence in C. sublineolum, C. graminicola, C. acutatum and C. coccodes. The majority of C. gloeosporioides isolates derived from the mango did not present amplification with primers tested. This suggested that primers constructed had a structural difference in the isolates of this host or that primers had not been adjusted for the amplification of all the isolates.

This is the first report on the presence of cap20 gene in C. gloeosporioides isolates of other hosts, except the avocado, and other species of Colletotrichum.

Analysis of the digestion fragment of amplification cap20 gene with the MspI restriction enzyme

The amplification products of cap20 gene of the C. gloeosporioides isolates amplified with primers P1 and P2 were submitted to the digestion with the MspI enzyme. The digestion generated two fragments of approximately 500-bp and 250-bp for all the C. gloeosporioides isolates (Fig. 7), not showed differences in the number and length of this gene among the isolates tested.

ACKNOWLEDGEMENTS

This work was carried out with the support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and CNPq/RENEBRA.

Received: September 10, 2008; Revised: July 30, 2009; Accepted: July 23, 2010

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Publication Dates

• Publication in this collection
  18 Jan 2011
• Date of issue
  Dec 2010

History

• Received
  10 Sept 2008
• Accepted
  23 July 2010
• Reviewed
  30 July 2009