A Species-Specific PCR Based Assay for Rapid Detection of Mango Anthracnose Pathogen Colletotrichum gloeosporioides Penz. and Sacc.

Mango (Mangifera indica L.) is considered as one of the most popular fruits grown throughout the tropics and subtropics worldwide [1]. India is the world`s largest producers, shares around 56% of total global production [2]. Production of mango is affected by a large number of fungal pathogens. The genus Colletotrichum contains many morphologically similar taxa comprising endophytic, saprobic and plant pathogenic fungi [3]. Of which, C. gloeosporioides an incitant of mango anthracnose is the most important biological constraint which restricts mango production in Southeast Asia [4]. C. gloeosporioides affects mango production both in the pre and post harvest stages particularly when attempting to extend storage life resulting in huge economic losses about 5-20% in the form of damage on the stems, leaves, fruit decay and damage [1,5]. Anthracnose disease is clearly identified by morphological symptoms but sometimes the symptoms are masked as this disease survived in the form of latent infection in absence of congenial environment. Symptom based identification and characterization is not accurate and reliable due to incipient infection. The diagnostic techniques, thus will assist in the monitoring the spread and distribution of pathogens. PCR technology can provide very accurate quantitative data required for control and quarantine decisions. The ability to design PCR primers to target specific regions of DNA has led to rapid, accurate, and sensitive detection which is a greater understanding for managing Colletotrichum diseases.


Introduction
Mango (Mangifera indica L.) is considered as one of the most popular fruits grown throughout the tropics and subtropics worldwide [1]. India is the world`s largest producers, shares around 56% of total global production [2]. Production of mango is affected by a large number of fungal pathogens. The genus Colletotrichum contains many morphologically similar taxa comprising endophytic, saprobic and plant pathogenic fungi [3]. Of which, C. gloeosporioides an incitant of mango anthracnose is the most important biological constraint which restricts mango production in Southeast Asia [4]. C. gloeosporioides affects mango production both in the pre and post harvest stages particularly when attempting to extend storage life resulting in huge economic losses about 5-20% in the form of damage on the stems, leaves, fruit decay and damage [1,5]. Anthracnose disease is clearly identified by morphological symptoms but sometimes the symptoms are masked as this disease survived in the form of latent infection in absence of congenial environment. Symptom based identification and characterization is not accurate and reliable due to incipient infection. The diagnostic techniques, thus will assist in the monitoring the spread and distribution of pathogens. PCR technology can provide very accurate quantitative data required for control and quarantine decisions. The ability to design PCR primers to target specific regions of DNA has led to rapid, accurate, and sensitive detection which is a greater understanding for managing Colletotrichum diseases.
The development of species-specific primers has provided a powerful tool for the detection of plant pathogens. The identification of fungal pathogens based on polymerase chain reaction (PCR) using species-specific primers is now widely used, especially for economically important plant pathogens such as quarantine listed fungi or those that are difficult to isolate or cause symptomless [6,7]. The internal transcribed spacer regions (ITS1 and ITS4) within the nuclear ribosomal gene clusters are particularly attractive loci for the design of PCR-based detection assays since they are readily accessible using universal primers [8] typically present in high copy number increasing PCR sensitivity and often exhibiting sufficient inter-specific sequence divergence for the designing of species-specific primers [9]. In recent years, molecular tools have been employed to infer the evolutionary relationships of Colletotrichum species. Based on nu-rDNA ITS sequence data and morphological characteristics, some species have been segregated from the Colletotrichum gloeosporioides complex. Although ITS sequence data may help in Colletotrichum species identification, it cannot alone be used to adequately address species delimitation for closely related species [10]. Researchers have recently tried to examine multiple genes sequence data to distinguish species in Colletotrichum [10,[11][12][13][14][15][16].
These regions can be further exploited for diagnostic purpose and the primers can be designed against species specific regions from rDNA. Therefore, a more contemporary approach aimed to develop a molecular diagnostic assay for rapid and accurate diagnosis against mango anthracnose pathogen for species-specific identification.

Materials and Methods
Thirty isolates of Colletotrichum gloeosporioides were collected from different agro-climatic mango growing regions of the India and reference pure culture were maintained and collected from leaf tissue and fruits affected with anthracnose. For further PCR assay standardization the 11 most pathogenic isolates of C. gloeosporioides collected from mango were procured. The details of colletotrichum gloeosporioides isolates collected and place of origin is given in Table 1.

Fungal DNA isolation
DNA extraction: The fungal mycelium and conidia from pure cultures, grown on one potato dextrose agar (PDA) (Himedia) petridish for 2 weeks at 25°C in the dark, were scraped and mechanically disrupted by grinding to a fine powder using a mortar and pestle. Total DNA was extracted with the Fast DNA Extraction Kit (MP Bio) following the manufacturer's instructions. Genomic DNA isolated was visualized under UV light on 0.8% (wt/v) agarose gel stained with ethidium bromide and stored at -20°C.

Species-specific primers designing
Species-specific primers were designed using advanced bioinformatics tools and the sequence retrieved from the NCBI database (http//:www.ncbi.nlm.niv.gov.in) from accession no. HM102505 of Colletotrichum gloeosporioides [17] and the conserved sequences were then picked up through Genmark software and the multiple sequences were aligned through Clustal W. The primer designed through the software Primer 3 and confirmed through the repeated NCBI-Primer BLAST and synthesized by Metabion International Pvt. Inc. A sequence based species-specific primer were designed according to Kamel et al. [18] (Figure 1) for the identification of Colletotrichum gloeosporioides and validated with other species of Colletotrichum (Table 2).

ITS-PCR
The ITS rDNA region including ITS1, 5.8S and ITS4 were amplified in 11 isolates of C. gloeosporioides selected from different mango growing regions of India ( Figure 2). The isolates were PCR amplified using the universal primers ITS1/ITS4 [8]. Each PCR reaction contained 1×PCR buffer, 2.5 mM MgCl 2 , 200 μM of each dNTP, 0.2 μM of each primer, 0.7 U of Taq DNA polymerase (Biochem), and 1 μl of template DNA. The PCR reaction mix was adjusted to a final volume of 25 μl with nuclease free water. PCR amplifications were performed on a Thermal Cycler (Eppendorf, India Limited). The program consisted of an initial step of 1 min at 94ºC for 1 min, followed by 30 cycles of 60 s at 94°C, 2 min at 58°C, and 60 s at 72°C; and a final extension step of 5 min at 72°C. PCR products were analyzed in 1.5% agarose gel, stained with ethidium bromide and visualized under UV light. 100-bp DNA ladder plus was used as a molecular weight marker (Biochem).

Results and Discussion
Nuclear rDNA, including the small and large subunits, 5.8 S, and the Internal Transcribed Spacer (ITS) region, proved an ideal target for specific PCR primers, as each sequence is variable at the family, genus, or species level [8]. Internal transcribed spacer (ITS) regions have been used successfully to generate specific primers capable of differentiating closely related fungal species [19]. Amplification of target DNA through PCR with taxon-specific primers is a potentially more sensitive and accurate approach than conventional microscopic techniques [20][21][22]. Unlike identification based on culture techniques, PCR does not

Sequencing and data analysis
The sequence information obtained for the twenty C. gloeosporioides isolates were analyzed by local alignment tool using BLASTn. Taxonomic correlation of the isolates upon NCBI web proved that the isolates were more related to C. gloeosporioides. All annotations were based on BLAST searches with a score threshold of e" 200 for BLASTn and e-values 10 -5 with a minimum of 99-100% identity over at least 80% of the length of the nucleotide sequence which are the commonly used thresholds for reliable sequence annotation. Sequences were confirmed for identification using repeated blast with already available sequences for the identification and taxonomy of the C. gloeosporioides using the NCBI-BLAST search for sequence identification of BLAST 2.0 (http:// www.ncbi.nlm.nih.gov/BLAST) Figure 3.

PCR amplification of species-specific designed primer
DNA fragment of approximately 380 bp were amplified for C. gloeosporioides using primer-pair MKCgF and MKCgR (Figure 4). No cross-amplification in other Colletotrichum sp., viz., C. falcatum, C. capsici ( Figure 5). Thus, the diagnostic PCR approach yielded highly consistent and reproducible results. The first amplification with fungus specific primers preferentially increased the population of fungal ITS molecules, thereby increasing the ratio of fungus by host ITS molecules. Then, the PCR amplification with species-specific designed primer amplifies a PCR product of 380 bp as specific for Colletotrichum gloeosporioides. Similarly, the existence of species-specific primers require the presence of viable organisms and can work even when there is a limited amount of sample [23]. In the present investigation, a sensitive PCR-based diagnostic assay were developed with the aim to detect Colletotrichum gloeosporioides mango anthracnose pathogen in infected plant and fruit tissue using species-specific designed primerpair. The specificity of this sensitive PCR-based assay was also verified by the absence of reactivity with DNA from uninfected (healthy) tissues and other Colletotrichum species. The 10 pg to 10 mg of genomic DNA of Colletotrichum spp. was found to be sufficient for a detectable PCR amplification. This is for the first time a PCR based diagnostic assay for early detection of mango anthracnose pathogen C. gloeosporioides was carried out using species-specific designed primer which amplifies a uniform band of 380 bp among all the isolates ( Figure 3) and clearly discriminated other species having no amplification with the designed primer as specific.

Validation of species-specific primer with other
Colletotrichum species PCR amplification of species-specific designed primer MKCgF and MKCgR gives amplicon size of 380 bp uniformly amplifies in all the isolates of Colletotrichum gloeosporioides but, no amplification in other with other Colletotrichum species viz. C. accutatum, C. falcatum and C. capsici. In context to our findings, the designed primer-pair does not amplified in other Colletotrichum species which; clearly discriminates Colletotrichum gloeosporioides the pathogen of mango anthracnose ( Figure 5) on the basis of species-specificity.
Identification of the causal agent and prevalence of a disease is very essential for adequate and timely management of disease, which in turns depends on accurate diagnosis and early detection of the pathogen. Often, it may be desirable to examine the planting material for prevalence of any potential pathogen even before the crop is sown. So, in the present investigation the species-specific designed primer-pair MKCgF and MKCgR for Colletotrichum gloeosporioides specifically for mango was designed and the primer has been validated with other Colletotrichum species. Thus, the designed primer proved to be an efficient marker for species-specific discrimination which would be useful in developing a rapid and sensitive diagnostic PCR based assay for early detection and timely management of Colletotrichum gloeosporioides causing mango anthracnose disease.