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Morphological Characterization of Kurnool Strains of Chickpea Collar Rot Casual Agent Sclerotium rolfsii Sacc

Chickpea (Cicer arietinum L.; 2n=14) is a major legume of the world. It is an annual crop and had the capability to fix atmospheric nitrogen in the soil. It is a crop of subsistence farming and completes its life cycle within 90 days and provides economic benefits to the farming community. The desi and kabuli types are highly cultivated in India and the yields of these genotypes are highly fluctuating over the years. The reason for yield fluctuations is mainly due to the various biotic and abiotic stresses. Among the biotic stresses, diseases caused by the pathogens affecting root like wilts and root rots are of special significance as they affect the crop stand in the initial stages of crop growth and reduce the yield. Sclerotium rolfsii is one of the major pathogen of soil borne nature and International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 9 (2020) Journal homepage: http://www.ijcmas.com


Introduction
Chickpea (Cicer arietinum L.; 2n=14) is a major legume of the world. It is an annual crop and had the capability to fix atmospheric nitrogen in the soil. It is a crop of subsistence farming and completes its life cycle within 90 days and provides economic benefits to the farming community. The desi and kabuli types are highly cultivated in India and the yields of these genotypes are highly fluctuating over the years. The reason for yield fluctuations is mainly due to the various biotic and abiotic stresses. Among the biotic stresses, diseases caused by the pathogens affecting root like wilts and root rots are of special significance as they affect the crop stand in the initial stages of crop growth and reduce the yield. Sclerotium rolfsii is one of the major pathogen of soil borne nature and International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 9 (2020) Journal homepage: http://www.ijcmas.com Sclerotium rolfsii causing collar rot is a major disease affecting crop stand and yield in chickpea. A comprehensive study was performed to report the variability in Sclerotium rolfsii isolates present in a major chick pea growing area, Kurnool District of Andhra Pradesh. A roving survey was conducted to collect 50 samples and was used for isolation of disease causing pathogen. 18 isolates of Sclerotium rolfsii were isolated to study the variability in morphological and cultural characteristics in the present study. The variation noted for mycelial traits and sclerotial characters were very huge. The trait, total colony diameter, values after 72 hours of incubation ranged from 42 mm to 72 mm and was exploited to classify the isolates as slow, medium and fast. The colour of the mycelia colony was pure white to dull white while, colony morphology or topography varied from compact to fluffy in some isolates. Further, the trait, sclerotia initiation, varied significantly from 5 to 10 days indicating the aggressiveness of the isolates in causing the disease. Sclerotia colour was light brown to dark brown and black in KCSR 16 and the shape was round, spherical to oval and irregular in few. The test weight (100 sclerotia) was ranged from 40 to 320mg and the number varied from 25 to 124 indicating presence of sufficient variability among the isolates for characterization. This characterization of S.rolfsii isolates is necessary to devise effective management strategies to control the disease. causes collar rot in chickpea. This disease is getting lot of importance at present because of its effect on the seedlings mortality in early stages of plant growth i.e., 54.7-95% in conducive environment (Aycock et al., 1966 andKotasthane et al., 1976) and yield loss of up to 10-30% in India (Maurya et al., 2008). In Andhra Pradesh, the incidence of this disease is reported to be increasing year after year i.e., 6.31% -12.21% (Nagamani et al., 2015).
The management strategies for this disease are with limited success as the pathogen has wide host range of 500 species, presence of variations among the isolates, growth habit in favourable environments, saprophytic nature and production of dormant structures like sclerotia during unfavourable environmental conditions. The existence of geographical variability among S. rolfsii populations was clearly presented by the authors like Harlton et al., (1995), Nalim et al., (1995), Okabe et al., (1998), Punja and Sun (2001), Sarma et al., (2002) and Shukla and Pandey (2007) but the presence of variation within the geographical regions is not well studied. This forms a crucial step in designing strategies to manage the disease as this helps in documentation of changes happen in the population (Sarma et al., 2002) and also to understand the population structure in designing efficient management strategies to reduce disease outbreak (Epsita Swain et al., 2018). Development of efficient breeding strategies to incorporate the resistance to these isolates in the cultivated types will help to overcome the problem and effective for a longer period of time. But isolates variability documentation in the cultivated regions of chickpea is very crucial for making proper management strategies. We are making concrete efforts to document the variability in chickpea growing areas of Kurnool district by collecting the samples. In the present study, efforts were made to document morphological variability among the Sclerotium rolfsii isolates of Kurnool district of Andhra Pradesh.

Materials and Methods
A roving survey was conducted to record occurrence and distribution of collar rot disease incidence in the fields of chickpea in Kurnool district of Andhra Pradesh during rabi 2019-2020.

Isolation of pathogen
The selected plant samples in the roving survey were used for the isolation of the pathogen. The pathogen was isolated using tissue segment method as suggested by Rangaswami and Mahadevan (1999). The infected collar region of plant was cut into small pieces of about 0.5 cm through sterilized scalpel blade and were surface sterilized with 1% sodium hypochlorite for 30sec followed by washing with sterile distilled water for three times. They were kept on blotting paper to avoid excess water and were later transferred onto petriplates containing PDA medium and incubated at 23±1°C. These cultures were kept under observation from time to time to note the growth of fungus. Axenic cultures of the fungus was obtained by single hyphal tip method and maintained on PDA. The fungus was identified based on mycelial and sclerotial characters (Barnett and Hunter, 1972).

Cultural and morphological variability
Eighteen isolates of S. rolfsii (KCSR 1-18) collected from different Mandals of Kurnool District, Andhra Pradesh were studied for their cultural and morphological characters on PDA. The mycelial disc of 4mm diameter of each isolate was placed in the centre of the plate and replicated twice. The inoculated plates were incubated at 23±1°C for 20 days. They were studied for eight morphological characters based on mycelial (mycelia growth, colony colour, mycelial dispersion and appearance) and sclerotial (sclerotial colour, weight and shape, number of sclerotia and their arrangement on surface of media) parameters recorded from 3 rd to 20 th day of incubation, respectively, for each isolate (Bansal et al., 1990) along with total growth and growth rate. Radial growth of each colony in two directions at right angles was measured. Visual observations on sclerotial formation were recorded.

Assessment of aggressiveness of isolates by pot culture method
The pathogen was further purified and multiplied on jowar grains to carry out aggression test. Jowar grains were boiled in fresh water for 30 minutes and drained excess water allowed to cool down by adding 5 per cent sucrose. 200g of jowar seeds are transferred into 500ml flasks and autoclaved. The flasks were allowed to cool at room temperature and inoculated with 5 mm culture discs of 5 days old S. rolfsii grown on PDA. Four discs per flask were added and the flasks were incubated for one week at 28 ± 1°C.
A pot culture experiment in the glass house was conducted to know the variation in aggressiveness of the eighteen isolates of S. rolfsii and to the pathogenic potential in terms of time taken for disease expression of each isolate of S. rolfsii and per cent of disease incidence on chickpea susceptible variety, L 550.
Soil inoculation method was adopted to test the aggressiveness among S. rolfsii isolates (Haware and Nene, 1980). The plastic pots of size 45x30 cm each containing 3kgs of autoclaved soil were used for raising the host plants. The two week old mass cultured S. rolfsii isolates was mixed with the soil in pots @ 50 g Kg -1 soil. The seeds of chickpea (var. L 550) were sown in the pathogen inoculated soil @ 10 seeds per pot. The observations were recorded up to 20 DAI by maintaining two replications in each treatment. Per cent disease incidence was calculated by using the following formula.

Results and Discussion
The present study with 18 isolates of Sclerotium rolfsii collected from the chickpea growing villages of Kurnool district of Andhra Pradesh indicated the existence of considerable variation in terms of morphological and cultural traits of the mycelia and sclerotia.

Morphological variability in mycelial parameters
The data pertaining to the mycelia growth and growth rate of different isolates is presented in the Table 1 and Plate 1. The isolates showed great variability for total growth from 40 mm (KSCR 16) to 72 mm (KCSR 1) after 72 hrs/ 3DAI. The average total growth of the isolates was 56.22 mm. Ten isolates showed more than average total growth while eight isolates recorded lower than the average value of total growth. Each isolate growth rate varied from 15 mm/day (KCSR 16) to 25 mm/day (KCSR 11)  , showed cottony growth on the media. The isolates, KCSR 6 and KCSR 7, appearance was dense mat like and the isolates, KCSR 4 and KCSR 9, showed wavy like pattern on the growth media. Among the isolates, KCSR 16 showed upright growth and KCSR 17 appearance was sparse on the media indicating these isolates different in its appearance and growth of the mycelia per day. Some isolates were similar in their morphological traits and growth pattern which is due to the genetic differences only as they were grown in controlled conditions on PDA (Okereke and Wokocha, 2007).

Morphological variability in sclerotial parameters
The sclerotial characteristics like time for their formation, colour, site of production, number and weight of 100 sclerotia varied in the studied isolates of S. rolfsii and the results are presented in the Table 3 and Plate 2.
The number of sclerotia ranged from 25 (KCSR 18) to 124 (KCSR 13) among isolates after 20 days of incubation. The weight of 100 sclerotia varied from 40mg (KCSR 1) to 320mg (KCSR 16). But the number of sclerotia is inversely proportional to the weight of 100 sclerotia as it was clearly seen in the isolates, KCSR 2, KCSR 3, KCSR 16 and KCSR 18 where the number is less but the weight is more while some of the isolates showed high sclerotia number with minimum weight as seen in KCSR 1. Some produced very less number of sclerotia with with minimal weight. The variation in sclerotial forming capacity could be a useful parameter in characterizing the isolates in the present study as the number of sclerotia showed great variability in the study.

Assessment of aggressiveness of isolates by pot culture method
The pot culture experiment was conducted to test the aggressiveness i.e., quantitative variation of pathogenicity on susceptible host, of each isolate on chickpea susceptible variety, L 550 through Percentage of Disease Incidence (PDI). The pathogen was inoculated in jowar grains for mass multiplication and the multiplied isolates were mixed with the soil in pots. The seedling mortality was recorded at 20 days after pathogen inoculation and calculated the per cent of disease incidence (PDI).
The aggressiveness of isolates was studied on chickpea variety L 550 along with a control and the results are presented in the  The presence of huge variations among the isolates of S. rolfsii can be attributed to the differences in physiological and metabolic rates exist in the isolates arising from different geographical and ecological systems and also their biochemical variability to adapt to the local conditions. The existence of variability among S. rolfsii populations was reported by earlier workers (Harlton et al., 1995 andOkabe et al., 1998). The studies on variability within the population of a geographical region are crucial as these studies documented the changes occurring in the population over time periods and facilitate us to design a effective strategy to manage the disease.
Thus, the study reported great variability in the pathogen in a limited area. The variation in cultural, mycelial and sclerotial morphological traits and pathogenicity amongst the isolates revealed that S. rolfsii can be characterized by a combination of these traits. The differences in morpho and cultural traits can be taken as potential parameters for differentiating isolates. Further, morphological characterization is a basic key for identification and for devising effective strategies to manage the disease including resistance breeding programmes.