Chickpea (Cicer arietinum L.) is the primary pulse crop cultivated in India. India is about 75 per cent of the world’s chickpea production. In Bundelkhand region of Uttar Pradesh chickpea is the predominant pulse crop. (Kumar et al., 2025).  The crop production in this region limited due to various biotic restrictions. Collar rot is one of the most severe disease of chickpea. It is a well known polyphagous pathogen. The fungus was first reported by Rolfs (1892) as a cause of tomato blight in Florida. Fungi in the genus Sclerotium form sclerotia and sterile mycelia but no spores (Saccardo, 1899) and include more than 40 plant pathogenic species (Farr, 2008). Although there are several geographical variability among S. rolfsii populations was demonstrated by earlier workers (Punja, 1985; Sarma et al., 2002; Remesal et al., 2013; Le et al., 2012) and contemporary studies that there is shift of paradigm in the pathogen’s prevalence due to climatic changes therefore major yield loss (Rajalaxmi, 2020; Sood et al., 2020). Collar rot is a fast-spreading and severe chickpea disease. S. rolfsii have become more important in recent years due to drastic climate change which makes the pathogen more aggressive and increased with adaptability to the environment (Ghatak and Ansar, 2017; Kumar et al., 2017; Savary et al., 2011). Traditional methods on only morphological characters based that is not sufficient for the differentiation of the specimens. Consequently, the current research was conducted to evaluate the diversity of S. rolfsii isolates through morphological and molecular.

Study area
 
The present investigation entitled” Morpho-Pathogenic and molecular Variability of Chickpea collar rot in Buldelkhand region of Uttar Pradesh, India” was carried out in the Department of Plant Pathology’s laboratory and performed in the greenhouse, College of Agriculture, BUAT, Banda (U.P.) during 2020-23.
 
Isolation and purification of pathogen
 
Twenty Nine isolates of S. rolfsii addressing chickpea growing districts of Bundelkhand Region of U.P. was collected, isolated by using PDA in BUAT, Banda, Department of Pathology Laboratory. The fungal cultures were purified by a single hyphal tip from a pathogen culture, which are then transferred on PDA slants or PDA plates. The PDA plates were incubated at 27±1°C three days for growth of test fungus.
 
Morphological variability
 
Twenty-nine isolates of S. rolfsii from different regions of Bundelkhand, Uttar Pradesh, were examined for morphological variation. Five-mm culture discs were positioned centrally on petri dishes with three repetitions for each isolate and incubated at 27±1°C for 15 days. Colony diameter was measured daily for three days, taken at right angles. Mycelial and sclerotial characteristics were noted on the 7^th and 15^th day, respectively. Replicate data were gathered and statistically examined to evaluate morphological differences between isolates.
 
Pathogenic variations in Sclerotium rolfsii
 
Pathogenic variability of 29 isolates of S. rolfsii was evaluated under net house conditions at BUAT, Banda through a pot experiment complete randomized design(CRD). Sterilized soil was combined with sorghum grain-based inoculum @ 25 g/kg soil. Chickpea seeds of three varieties that were surface-sterilized were planted in inoculated pots, while non-inoculated pots served as controls. Every treatment was replicated three times and the pots were arranged in a random manner. Sterilized water ensured sufficient hydration. Data on seedling emergence and incidence per cent were receorded 10-15 days after sowing and final plant stand was counted after 60 days of sowing.                         

       

 
Molecular diversity   
 
Fungal DNA isolation and quantification
 
Isolates of Sclerotium rolfsii were grown in 2% potato dextrose broth for 12 days at 26±1°C. A modified CTAB method was employed to extract DNA from the fungal mycelial mats. Mycelia were crushed using liquid nitrogen and combined with prewarmed 2X CTAB buffer (containing CTAB, NaCl, EDTA, Tris-HCl and β-mercaptoethanol). The extract was cleaned using chloroform-isoamyl alcohol and spun at 10,000 rpm for 5 minutes. DNA was precipitated with cold isopropanol, kept on ice and then centrifuged once more. The pellet underwent a wash with 70% ethanol, was air-dried and then dissolved in TE buffer. DNA quality and quantity were evaluated using gel electrophoresis and a UV transilluminator. The quality and quantity of DNA was checked using gel electrophoresis and UV trasnliminator. In Table 1 show the oligonucleotide primer sequence of RAPD primers.

A roving survey was conducted during Rabi season 2021-22 and 2022-23 for occurrence and distribution of collar rot of chickpea in seven chickpea growing district of Bundelkhand region of U.P. The severe form of incidence is mainly due to monocropping and sowing time when high moisture conditions prevail because of the rainy days. Singh et al. (2019) also reported the incidence of chickpea diseases in all seven districts of Bundelkhand region.
 
Morphological variability
 
Mycelial characters
 
Data in Table 2 show Morphological and cultural characters of 29 isolates significant differences among the isolates for total growth and growth rate on PDA. The average colony diameter varied from 40.00 mm (SRC29) to 90.00 mm (SRC5, SRC25, SRC28), indicating substantial differences among the isolates. Among the isolates, 10 showed rapid growth, 3 demonstrated slow growths and 16 displayed medium growth. Mycelial color ranged from very white to dull white, with very white being frequent among rapidly growing isolates. The mycelial growth distribution also differed: 10 isolates exhibited fluffy growth, 13 displayed dense growth and 6 were sparse. Interestingly, fluffy growth was frequently linked to rapidly growing isolates. The identified morphological variability, with a coefficient of variation (C.V.) of 2.17% and a critical difference (C.D.) of 2.42 at p=0.05, emphasizes the varied phenotypic characteristics of the fungal isolates among regions. Similar, reports were given by Srividya et al. (2018); Manu et al. (2018). Okereke and Wokocha (2007) observed the variation in colony diameter.


 
Sclerotial characters
 
Out of twenty-nine isolates of S. rolfsii obtained from various chickpea cultivation areas showed considerable sclerotial diversity when grown on potato dextrose agar (PDA) medium. Sclerotia yield differed considerably among isolates, spanning from 108.33 (SRC22) to 1005.33 (SRC2) per plate. The weight of sclerotia varied from 66.33 mg (SRC10, SRC20) to 538.33 mg (SRC2) and the diameter of sclerotia ranged from 0.93 mm (SRC5) to 1.59 mm (SRC2). The majority of isolates formed brown to light brown Sclerotia. The distribution was generally uniform, with only a few isolates exhibiting peripheral (SRC1, SRC14) and concentric patterns (SRC15). C.D. values at p=0.05 and low coefficients of variation confirmed notable differences (Table 3). The variability in number of sclerotia produced among the isolates of S. rolfsii collected from different location and hosts was also recorded in earlier reports (Sekhar et al., 2017; Manu et al., 2018). Palaiah and Adiver (2004) also recorded the similar variation in sclerotial test weight of various isolates of S. rolfsii.


 
Pathogenic variability
 
Data in Table 4 show a notable difference in disease occurrence was noted among the 29 S. rolfsii isolates across three chickpea varieties in the 2022 and 2023 growing seasons. Data collected over two years showed considerable differences in the occurrence of S. rolfsii across 29 isolates on chickpea varieties JG14, JG16 and L550. Disease incidence varied between 66.22% (JG14) and 66.93% (L550), with L550 exhibiting marginally greater vulnerability. Mostvirulent isolates such as SRC25, SRC27 and SRC28 resulted in 97.22-100% incidence, whereas those with lower virulence exhibited 33.33-44.72%. Control treatments exhibited low levels of infection (5.55-9.26%). Notable differences (CD at p=0.05) and low CV values (6.68-15.70%) validated the dependability of findings. Similar pathogenic variability has also been reported by Kumari and Ghatak (2018). Sennoi et al., (2010) evaluated pathogenecity test of ten S. rolfsii and found most of isolates were aggressive nature.


 
Molecular variability of S. rolfsii Isolates by RAPD
 
Data in Table 5 show ten random RAPD primers were selected to study the genetic diversity among the 29 isolates of S. rolfsii. RAPD primers employed for detecting genetic diversity produced clear and reproducible banding patterns. These primers generated 357 amplified bands which ranged from 100 to 3,600 bp. The total number of polymorphic bands was 49. The per cent polymorphism ranged from 69.91 (OPY-13) to 35.98 (OPA-11) percent. The minimum size of 300 base pairs was generated from OPA-19 and OPY-01 while maximum size 3600 base pairs were generated with primer OPS-12. The primers OPB-18, OPB-20, OPS-12, OPY-01, OPY-13, OPY-14 and OPS-12 and OPB-19 were found to be most informative based on the level of polymorphism detected by them.


       
Data in Table 6 show Genetic similarities were analyzed through the data obtained on the basis of 10 RAPD primers from the 29 isolates of the S. rolfsii. The genetic similarity between S. rolfsii isolates exhibited different levels, with Jaccard’s similarity coefficients varying from 0.05 to 1.0. The greatest similarity was noted between genotypes SRC-11 and SRC-29, suggesting a close genetic relationship. Conversely, the minimum similarity was noted between SRC-5 and SRC-25, SRC-13 and SRC-7, as well as SRC-18 with SRC-8 and SRC-25. A dendrogram showed (Fig 1) that all isolates, apart from SRC-18, grouped into a larger cluster, while SRC-11 and SRC-29 created a separate pair. 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 study revel wider heterogeneity within a small area in Bundelkhand region of U.P. at the pathogenic, molecular and morphological level in S. rolfsii, which causes chickpea collar rot. The study of aggressiveness and the genetic basis of variability can be based on the identification of isolates based on differences in physical and cultural characteristics. Further, in the isolate, the highest similarity was observed between genotypes SRC-11 and SRC-29. The lowest similarities were reviled between the lines SRC-5 and SRC-25, SRC-13 and SRC7, SRC-18 and SRC8, SRC-25 and SRC-18 registered the minimum similarity value indicating the maximum genetic distance between them.

My advisor and Vice-chancellor of BUAT, Banda UP, India, provided the facilities to perform the this research work.

In relation to publishing this work, the author declare that they have no conflicts of interest.