Volume 6, Issue 1, February Issue - 2018, Pages:124-130
|Authors: Prathibha Veerappa Hanumanthappa*, Nanda Chinnaswamy, Mohan Rao Annabathula, Ramesh SampangiramaReddy, Nagaraja Niduvalli Ramachandrappa|
|Abstract: A total of sixty Colletotrichum isolates were purified from anthracnose disease samples, collected from 15 chilli growing districts of Karnataka. The isolates were evaluated for their morphological and genetic characterization using AFLP marker assay. Based on the morphological characterization, 40 isolates were identified as Colletotrichum capsici/truncatum and 20 as C. gloeosporioides. Considerable morphological variability was observed in C. gloeosporioides isolates compared to C. capsici isolates. AFLP marker assay could clearly differentiate the C. capsici and C. gloeosporioides isolates at 43% genetic similarity, thus complementing species classification based on morphological characterization. However, morphological and AFLP grouping of isolates indicates no clear correlation between clustering in the dendrogram and morphological grouping of C. capsici and C. gloeosporioides isolates this suggested existence of wide variability in both the species.|
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Chilli (Capsicum annuum L.) is an important vegetable cum cash crop of Indian subcontinent. The crop is grown largely for its pungent fruits, which are used both green and ripe to impart pungency to the food. The sustainability in chilli production is threatened by many biotic stresses such as several insect pests and diseases (Isaac,1992). Among these, anthracnose disease caused by Colletotrichum species is a major constraint to chilli production in major chilli-growing regions of the world and often results in high yield losses (Than et al., 2008). This disease caused by a complex of Colletotrichum species, results in both pre and post-harvest fruit decay (Liu et al., 2016).The species of Colletotrichum associated with anthracnose disease includes Colletotrichum capsici, C. gloeosporioides, C. truncatum, C. dematium, C. acutatum, C. siamense, C. fructicola (Hong & Hwang, 1998; Gopinath et al., 2006; Sharma & Shenoy 2014), C. coccodes and C. karstii (Saini et al., 2016).
Colletotrichum infecting diverse hosts including cereals, legumes, vegetables, perennial crops and tree fruits have a high degree of pathogenic variability. Thus the accurate identification of the pathogen and species differentiation is imperative in development of appropriate management practices. Also studies on the variability of the pathogen populations are needed to direct breeding efforts towards long term resistance to anthracnose disease.
Classically, identification and characterization of Colletotrichum spp. was primarily relied on morphological characters such as colony color and growth rate, shape and size of conidia, optimal temperature for growth, presence or absence of setae and existence of the teleomorph (Adaskaveg & Hartin, 1997; Freeman et al., 1998). Although these criteria are valuable but alone are not always adequate for reliable differentiation among Colletotrichum spp. due to variation in morphology and phenotype among species under environmental influences (Cannon et al., 2000).
To overcome the inadequacies of these traditional schemes, molecular techniques combined with morphological studies have proven to be effective for characterization of Colletotrichum species (Sreenivasaprasad & Talhinhas, 2005; Van Hemelrijck et al., 2010). Therefore the present investigation was carried out to assess the variability in Colletotrichum spp. infecting chilli in Karnataka state of India using morphometric and molecular approaches
2 Materials and Methods
2.1 Collection and isolation of Colletotrichum spp.
Chilli fruits showing typical symptoms of anthracnose disease were collected from farmer’s fields and chilli markets of 15 chilli growing districts of Karnataka, India (Table1). The pathogen was isolated by following standard tissue isolation method (Karuna-vishunavat & Kolte, 1998). The pathogen was purified using single spore isolation technique (Karuna-vishunavat & Kolte, 1998). The pathogenicity of monoconidial cultures of all the isolates was conducted by inoculating on hybrid NS1701 by following detached fruit method (AVRDC, 2003).
2.2 Morphological variability
Morphological variability viz., colony characteristics (colony colour, texture, margin and radial growth) and conidial morphology of 60 isolates of Colletotrichum spp. was assessed on Potato Dextrose Agar (PDA) medium. Petri dishes containing 15 ml of PDA medium was inoculated centrally with 5 mm diameter mycelial disc taken from the periphery of 5 days old culture. Three replications were maintained for each isolate and the plates were incubated at 25±1°C. Conidial size, shape and acervuli size of 60 isolates were recorded using Leica bright field microscope. The colony morphology was recorded by following mycological chart (Rayner, 1970).
2.3 Molecular variability
Amplified Fragment Length Polymorphism (AFLP) molecular marker assay was used to study the variability among sixty isolates of Colletotrichum spp. of Karnataka. Two isolates each of C. capsici and C. gloeosporioides sampled from Andhra Pradesh which were earlier characterized for their virulence pattern were also included in the study (Nanda, 2011).
2.4 Isolation of DNA and PCR amplification
The DNA was extracted from five days old mycelia grown on Potato dextrose broth by following procedure of Sharma et al. (2005) with minor modifications. AFLP protocol outlined by Vos et al. (1995) was followed with minor modifications in some steps. A total of nine, three nucleotide selective primer combinations were used for the study of genetic diversity in 60isolates of Colletotrichum spp. The primers were first screened using DNA and the combinations that showed good amplification were selected for final amplifications.
Genomic DNA was digested with 4 units of MseI and 10 units of EcoRI restriction endonucleases at 37° C for 3 hours in a PCR machine. The digested product was loaded on to 1.2 per cent agarose gel to confirm the complete digestion of the genomic DNA. The digested DNA was ligated with MseI and Eco-R I adapters with T4 DNA ligase. Ligation reaction mix was incubated at 37°C for overnight. The digested / ligated product was diluted to 1:10 with T10E1 (pH8) and stored at -20°C.
An initial round of PCR (Pre-amplification) was carried to enrich a subset of the AFLP template; the primers used in pre-amplification have a single base selection. In the Pre-amplification reaction, a 3.0 µl diluted ligation product, 2 µl each of MseI and EcoRI, 2 µl dNTPs, 1µl 10X PCR buffer were mixed in 1.0 unit of Taq polymerase and used in total volume of 10.2 µl. Pre-amplification was performed with an amplification profile of 94°c for 30 s, annealing at 56°c for 1 min, extension at 72°c for 1 min, repeated for 20 cycles and then at 10°c for 30 min. The pre-amplified product was loaded on to 1.2 % agarose gel to check the amplification. Depending on the amplification intensity, the amplified product was diluted to1:3 in TE buffer and used as template for re-amplification using AFLP primers, each containing three-selective nucleotides. Re-amplification PCR was performed in 10.2 µl reactions containing of 3.0 µl template DNA, 2.0 µl each of EcoRI and MseI selective nucleotide primer, 2.0 µl dNTPs, 1µl 10X PCR buffer and 1.0 unit of Taq polymerase. The re-amplification reaction was carried out with cycling parameters of 94°c for 30s, 65°c for 30s reducing by 0.7°C / cycle to 56°C, 72°C for 1 min for 11 cycles, and 94°C for 30 s, 56°C for 30 s and 72°C for 1 min for 24 cycles followed by 10° C for 30 minutes.
PCR were carried out separately for each primer pair and the products were denatured immediately by adding eight microlitres of stop loading dye to each sample. Denaturation was carried out at 94°C for 5 min and then cooled to 10°C for 5 min. Electrophoresis of the samples was carried out on 6% polyacrylamide gels, by loading 3 µl of each final PCR product. Electrophoresis was carried out at 1,200 V for 3.0h until the dark blue dye ran off. The gels were then seperated and developed silver staining technique.
2.5 Analysis of AFLP profiles
The amplicons generated in AFLP, behave as dominant markers. Therefore, the score ‘1’ was assigned for the presence of band and ‘0’ for absence of band at each loci. The variation in band intensity is not taken into consideration to avoid confusion in scoring. The binary data was used to estimate pair wise genetic distance based on Jaccard’s coefficients using NTSYS-pc version 2.0 software. Dendrogram was constructed using Unweighted Paired Group Arithmetic Mean (UPGMA) algorithm based on distance matrix.
3 Results and Discussion
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