Studies on Diversity of Sclerotium rolfsii Causing Collar Rot in Chickpea using Morphological and Molecular Markers

Chickpea (Cicer arietinum L.), a rich source of protein (20 to 25%), is one of the major grain legumes grown worldwide and ranks second in the global farming and forms an important component of subsistence farming in the Indian sub-continent. The ability to derive more than 70 per cent of its nitrogen from symbiotic nitrogen fixation makes chickpea a promising crop for the sustainable agriculture.
        
The chickpea production globally is limited by various biotic and abiotic stresses. Among the biotic constraints, diseases, insect pests, nematode and parasitic weeds, are responsible for instability in yield. Insects and diseases cause yield loss of 5 to 10% in temperate and 50 to 100% in tropical regions in chickpea (Van et al., 1988). In chickpea, root diseases particularly Fusarium wilt, Verticillium wilt, dry root rot, black root rot, collar rot etc. are very common and affect the plant from sowing to harvest. Collar rot caused by Sclerotium rolfsii Sacc. is one of the devastating soil-borne diseases of fungal origin and cause sufficient yield losses to become the disease of prime importance for designing better management practices in chickpea. The disease generally appears within two weeks after sowing. The younger seedlings exhibit clear rotting at the collar region and collapse but older seedlings turn yellow and may dry without collapsing. Drying plants with foliage turned slightly yellow before death are scattered throughout the field and is an indication of collar rot infection. It causes 55-95% seedling mortality (Gurha and Dubey, 1982). This disease is favoured by good soil moisture, high soil temperature (25-30°C) and low organic matter in the soil (Mathur and Sinha, 1968). S. rolfsii has wide host range with prolific growth and ability to produce persistent sclerotia to inflict large economic losses (Ramesh et al., 2014).
        
The Central and Southern India form an important region of chickpea production. The hot and rainfed climatic conditions, presence of susceptible hosts and aggressive isolates of pathogen had made the emergence of collar root rot as the major constraint for chickpea production in these regions.  Development and cultivation of resistant varieties is the ideal and feasible management practice for the efficient management of disease but identification of the sources for resistance/ tolerance has become main hurdle. Identification of resistant sources against this disease has been reported (Sugha et al., 1991) but stable resistance could not be achieved due to the prevalence of aggressive isolates of S. rolfsii (Sharma and Jodha, 1984). Geographical variability among S. rolfsii populations was demonstrated indicating the importance of studies on variability within the population in a geographical region. The variability in S. rolfsii isolates is largely due to the mycelial compatibility and nuclear exchange through anastomosis in hyphae (Nalim et al., 1995).
        
Molecular markers play a major role in analyzing genetic basis of genotypic variation among fungal population. Welsh and McClelland (1990) described randomly amplified polymorphic DNA (RAPD) marker technique to detect genetic polymorphisms in fungi. RAPD analysis is used as a powerful tool for the investigation of genetic relatedness and diversity among closely related strains and was found to be a valuable method for differentiating the genetic variability of S. rolfsii isolates (Saude et al., 2004).
        
Thus, the present study was undertaken to assess the diversity among the S. rolfsii isolates collected from different chickpea growing regions using morphological and molecular markers like RAPD markers.

A roving survey was done in major chickpea growing areas of Andhra Pradesh i.e., Kurnool and Anantapur districts for documenting the occurrence and distribution of collar rot in chickpea during rabi 2017. Plants were assessed based on visual typical symptoms of collar rot on plant for the confirmation of disease and five severely affected 25-30 days old chickpea plants were collected from each village for variability studies. The pathogen was isolated on potato dextrose agar medium (PDA) (Rangaswami and Mahadevan, 1999) and the pathogen was identified as S. rolfsii based on its mycelial and sclerotial characteristics through standard mycological keys (Barnett and Hunter, 1972).
 
Cultural and morphological variability
 
The mycelial disc of 0.5 cm diameter of each isolate was inoculated in the Petriplates with PDA media and the inoculated plates were incubated at 26±1°C for 20 days. S. rolfsii isolates were studied for their cultural and morphological characters (mycelia growth- moderate/ fast; colony colour- white/orange and appearance- dense mat/ other) and sclerotial parameters (sclerotial colour- brown/ orange; shape- spherical/irregular and their arrangement on surface of the media- scattered/peripheral) at 7 and 20 days of incubation, respectively, for each isolate.
 
Fungal cultures for DNA extraction and quantification
 
The total genomic DNA was extracted using the procedure suggested by Murray and Thompson (1980) with slight modifications. The quality and quantity of DNA was analyzed by running 2 μl of each sample mixed with 3 μl of 6X loading dye on 1% agarose gel. The DNA was quantified by comparing with the 1 kb size marker and by spectrophotometer.
 
RAPD
 
Twenty nine random primers (Operon Technologies Inc.) were used for screening to generate polymorphism among the isolates. The Oligonucleotide primer sequences of RAPD primers are given in Table 1. PCR amplifications were carried out in 0.2 ml eppendorf tubes with 25 μl reaction mixture having 2.5 μl of 10X Taq buffer, 2.0 μl of 25 mM MgCl₂, 2.0 μl of primer (1 picomolar/μl), 1.0 μl of 10 mM dNTP mix, 0.4 μl of Taq polymerase enzyme (conc. 5 U μL^-1) and 15.6 μl of sterile PCR grade water and 1 μl (200 ng) of DNA sample. Amplification was carried out by 5 minutes of initial denaturation at 94°C followed by 45 cycles of denaturation at 94°C for 1 min; annealing at 37°C for 1 min; extension at 72°C for 2 min with final elongation at 72°C for 8 min.
 

        
Amplified PCR products were subjected to 1.5 per cent agarose gel electrophoresis and visualized under UV transilluminator with ethidium bromide staining (10 mg/ml). The banding profiles of RAPD products were documented in gel documentation system (Vilber Lourmat, France). Each amplified band was considered as RAPD marker and recorded for all samples. The RAPD pattern of each isolate was evaluated assigning 1 to bands that were reproducible and detected in the gel and 0 for the absence of band. The data matrix was used to calculate Jaccards’ similarity coefficient (Jaccard, 1908). The cluster analysis and dendrogram preparation were done using the Statistical Package for Social Science (SPSS) package.

The roving survey in Kurnool and Anantapur districts of Andhra Pradesh during rabi 2017 indicated that chickpea collar rot disease incidence range was 4.66 to 18.00 per cent during 10 to 20 days after sowing. The severe form of incidence is mainly due to monocropping and time of sowing i.e., last week of October to second week of November when high moisture conditions prevail because of the rainy days. Nagamani et al., (2015) and Divya et al., (2016) also reported the presence of disease and loss of crop stand at seedling stage in Andhra Pradesh.
 
Morphological variability
 
The isolated pathogen was identified as S. rolfsii based on mycelial characters of silky white mycelia with radial spreading (Plates1-3). Morphological and cultural charactersof 20 isolates were studied based on mycelia and sclerotial parameters and the results indicated significant differences among the isolates for total growth and growth rate on PDA (Table 2). The maximum radial growth was recorded for the isolates, CSR 1, 2, 5, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19 and 20 (90 mm). Among these isolates, CSR 14, 18 and 20, recorded 90 mm growth within 3 days after inoculation (DAI) and considered as very fast growing isolates. The isolates, CSR 1, 2, 5, 7, 8, 9, 10, 11, 12, 13, 16, 17 and 19, recorded 90mm diameter growth at 4 DAI and considered as fast growing. The isolates, CSR 4, CSR 3 and CSR 6, recorded growth of 81 mm, 75 mm and 70 mm, respectively, at 6DAI and categorized as moderate growing isolates. The isolate, CSR 15, recorded the least growth (50 mm) and was considered as the slowest growing isolate.
 

 

 

 

        
The results on cultural characteristics of isolates indicated that seven isolates (CSR 3, 4, 5 12, 16, 17 and 19) had fluffy growth; four isolates (CSR 1, 2, 10 and 15) showed cottony growth and the remaining eight isolates recorded dense mat growth. The isolate, CSR 20, formed thick dark brown mycelial growth. Flower pattern appearance of mycelium was recorded for the isolates, CSR 7 and CSR 8, while wavy pattern was seen in CSR 11, 12 and 16 isolates.
        
Sclerotial characters like, time taken for production, colour and site of production varied among the isolates. The isolates, CSR 4, 5, 6, 13 and 15, took the maximum time of 22 days for the sclerotial production; CSR 2 and CSR 3 took 18 days; CSR 7 took 15 days; CSR 1, 8, 10, 16, 19 and 17 took 11 days and CSR 9, CSR 11 and CSR 12 took 6 days. The isolates, CSR 14, CSR 18 and CSR 20, took only4 days for sclerotial production indicating the fastness of the pathogen growth and formation of sclerotial bodies. The colour of sclerotia varied from brown (CSR 2, CSR 3, CSR 4 and CSR 5), light brown (CS1, CSR 6, CSR 8 and CSR13), dark brown (CSR 7, CSR 9, CSR 11, CSR 12, CSR 15, CSR 16 and CSR17), dark blackish brown (CSR 18, CSR 19 and CSR 20) to light orange (CSR 14). The site of sclerotial production varied among the isolates as most of the isolates produced sclerotial bodies on periphery and spread over the medium whereas the isolates, CSR1 and CSR 14, produced the sclerotial bodies in the centre. Variability in cultural and morphological traits among S. rolfsii isolates have been reported by Thilaghavathi et al., (2013) and Reddi et al., (2014) on various hosts and are exploited for the identification of isolates.
 
Genetic diversity studies in S. rolfsii isolates by RAPD
 
The genetic diversity studies of S. rolfsii isolates with 29 RAPD markers yielded maximum number of amplification products with high intensity, minimal smearing, good resolution, reproducible and scorable clear bands in all the isolates. Number of amplification products obtained were specific to each primer and ranged from 4 to 15. A total of 254 alleles were produced with 100% polymorphism (Table 3). The size of amplification products ranged from 200 bp to 2000 bp. The highest number of scorable bands were obtained with the primer OPA 19, while the lowest was observed with the primer OPA 4. Maximum isolates were amplified by the primers, OPA 10, OPA 17, OPB 1 and OPB 9 and minimum by the primer OPA 8. Sources of polymorphism in RAPD assay might be due to deletion, addition or substitution of bases within the priming site sequence. Gel electrophoresis patterns of the primers are presented in the plates 4 to 12 (OPA 1, 9, 11, 14, 17, 20 and OPB 1, 9, 10).
 

 

 

        
Unique bands were observed for the primers OPA 1 (200bp; CSR 8), OPA 11 (1000-1500bp; CSR 14 and 20), OPA 17 (2000 bp; CSR 14), OPA 18 (250-500 bp; CSR 20), OPA 20 (300 bp; CSR 8), OPB 1 (1500-2000 bp; CSR 14) and OPB 4 (1000-1500 bp; CSR 20). These may be considered during DNA fingerprinting and characterization of these isolates. Similarly, the findings of Perez-Moreno et al., (2002) demonstrated the genetic polymorphism among isolates of Sclerotium cepivorum from Mexico based on RAPD analysis. Prasad et al., (2010) observed the polymorphic and distinguishable DNA banding pattern with five random primers among the eight isolates of S. rolfsii.
        
The binary data from the polymorphism was used for computing the similarity indices. A similarity matrix was prepared to estimate genetic diversity and relatedness among the 20 S. rolfsii isolates (Table 4). The similarity coefficients values ranged from 0.15 to 0.72. The highest similarity matrix index (0.723) was observed between CSR 15 and CSR 16 isolates while, the lowest similarity matrix index (0.15) was noticed between the isolates, CSR 12 and CSR 18. The dissimilarity index in rest of the isolates was found to be in between 0.735 to 0.837. The isolate, CSR 18, was found to be distinct with the isolates, CSR 3, 4, 5, 7, 10, 11, 12, 16, 17 with dissimilarity index range of 76.1 to 85.0%, while the isolate, CSR 20, was found to be distinct with CSR 1, 2, 6, 7, 9, 13, 15 and 19 with dissimilarity range of 73.6 to 85.0%.
        


Subsequently, UPGMA dendrogram grouped the isolates into two major clusters (Fig 1). The detailed analysis of the dendrogram revealed that maximum number of isolates (18) were grouped in cluster I and it was further partitioned into three sub-clusters, sub-cluster IA with ten isolates (CSR 1, CSR 2, CSR 3, CSR 6, CSR 7, CSR 12, CSR 13,CSR 15, CSR 16, CSR 17); sub-cluster IB withseven isolates (CSR 4,CSR 5, CSR 8, CSR 9, CSR 10, CSR 11, CSR 19) and sub-cluster IC with single isolate (CSR 14). The isolate, CSR-14, was distantly present in the cluster I. Cluster II had 2 isolates (CSR 18 and 20). The clustering pattern of the isolates revealed that the isolates are genetically diverse. These results are in agreement with the reports mentioned by Molina et al., (2005) that the isolates obtained from the same habitat have different RAPD patterns indicating that many populations of this fungus are made up of more than one strain and that few are derived clonally. Punja and Sun (2001) and Prasad et al., (2010) also reported similar findings in their studies involving S. rolfsii. Paramasivan et al., (2009) reported that a wide diversity among fungal groups can occur within a limited area, within a host or in geographically isolated regions. Hence, studying the morphological and genomic background of isolates promotes clear understanding of the ecology and pathogenicity aspects of S. rolfsii.
        
The isolate, CSR 14, recorded maximum disease incidence on eighth day, this isolate is present separately in the cluster I. This strain also showed the unique banding pattern with the primers OPA 11 (1000-1500 bp), OPA 17 (2000 bp) and OPB 1 (1500-2000 bp) indicating usefulness of these primers in easy identification of this aggressive isolate among the isolates of S. rolfsii. The markers utilized in present investigation can be effectively used for identification and characterization.

This is the first report on existence of broader variability in limited area at morphological and molecular level in S. rolfsii causing collar rot in chickpea. The differences among the isolates for cultural and morphological traits can be used for the identification of isolates and this form the basis for the study of aggressiveness and genetic basis of variability. Further, the isolate, CSR 14, showed unique DNA banding pattern and morphological traits indicating the usefulness of both these traits for generating the clear picture of this isolate for identification and characterization.

First author is highly grateful to Acharya NG Ranga Agricultural University for providing stipend during the course of study. This study is a part of M.Sc (Ag.) research programme.