Chickpea (Cicer arietinum L.) is the world's third most important food legume after dry bean and pea India is the largest producer of chickpea, contributing more than 70 per cent of the total world production. One of the major constraints limiting agriculture production is difficult in managing disease caused by soil borne pathogens. Among the soil borne diseases of chickpea, Collar rot disease caused by Sclerotium rolfsii Sacc. is a serious threat to chickpea that may cause 55-95% mortality of the crop at seedling stage under favourable environment conditions (Gurha and Dubay 1982). Collar rot caused by soil borne fungus Sclerotium rolfsii Sacc is a major threat when weather conditions are conducive. It is an important diseases especially in paddy - chickpea or soybean - chickpea based cropping systems. The disease is favoured by good soil moisture, high soil temperature (25-30oC) and low organic matter in the soil (Mathur and Sinha 1968). Collar rot is a fast spreading and destructive disease of chickpea. The disease is confined at seedling stage (upto 45 days) resulting in reduced plant population. Collar rot can cause 54.7-95% mortality of chickpea seedlings (Kotastthane et al., 1976; Nirmalkar et al., 2017). Diseases caused due to S. rolfsii requires warm climates, occurs more frequently at high moistures and high temperatures (Al-Askar et al., 2013) S. rolfsii initiates the rotting of seeds and seed surfaces are covered with white mycelia mat in soil. Germinated seedlings were killed within 7 days after emergence. Symptoms were evident as yellowing and collapse. The affected roots showed rotting at the collar region and down ward with the whitish mycelium in earlier stages of infection. Rapeseed like sclerotia can be observed attached to mycelium around the collar (Nene et al., 1978).

Isolation of pathogen Sclerotium rolfsii from infected plant samples. Fresh samples of collar rot were brought to the laboratory in paper bags and washed under tap water to remove dust and other inert materials. In Small pieces of specimen were then cut, with each piece containing half infected and half healthy portions. These pieces were disinfected with a sodium hypochlorite (0.1%) solution for 1 minute followed by washing with sterilized distilled water. The pieces were placed on blotting paper and allow to dry. Once properly dried the pieces were transferred into culture slants and subsequently transferred into petri dishes containing PDA media (Neha et al., 2016).

In-vitro, evaluation of fungicides against Sclerotium rolfsiiby poison food technique. The efficacy of 16 fungicides (Metiram 70% WG, Tricyclazole 75% WP, Propiconazole 25% EC, Azoxystrobin 18.2% + Difenoconazole 11.4% SC, Validamycin 3% L, Hexaconazole 5% SC, Iprobenfos 48% EC, Azoxystrobin 11% + Tebuconazole 18.3% SC, Thifluzamide 24% SC, Mancozeb 75% WP, Propineb 70% WP, Tebuconazole 50% + Trifloxystrobin 25% WG, Kasugamycin 3% SL, Tebuconazole 38.39% w/w SC, Hexaconazole 4% + Zineb 68% WP and  Metiram 55% + Pyraclostribin 5% WG) was evaluated in-vitroat different concentrations of 100, 500, 1000 and 2000ppm on growth of Sclerotium rolfsiion Potato dextrose agar (PDA) medium using poisoned food technique (Nene and Thapliyal 1982).

The pathogen Sclerotium rolfsii was grown on Potato Dextrose Agar (PDA) medium for a period of seven days. The PDA medium was prepared and melted. Concentrations of fungicide were added to the cooled melted medium. Each sterilized petri plate was poured with 25-30 ml of the fungicide containing medium. To prevent bacterial contamination, 500 ppm of streptomycin was added to the medium while pouring it into the petri plates. A ycelia disc with a diameter of 5 mm was taken from the periphery of a fresh culture of test plant pathogens i.e. Sclerotium rolfsii and placed in the centre of each petri plate. Three replications were maintained for each treatment. The inoculated plates were then incubated at 28 ± 2°C for 4 days. The percentage of inhibition was calculated using the formula developed by Vincent (1947). 

Where, 

I = per cent inhibition

C = growth in control

T = growth in treatment

In-vivo, Testing the efficacy of fungicides as seed treatment for the control of collar rot of chickpea (Sclerotium rolfsii). Pot experiment was conducted during Rabi season in 2023-2024 to assess the efficacy of different fungicides as seed treatment applied for the control of collar rot of chickpea. Different fungicides were used at 3g/seed as seed treatment to treat the chickpea seeds. The experiment was laid out in completely randomized design (CRD) and each treatment was replicated thrice.

Treatment Details

Preparation of inoculums. Fresh inoculum of collar rot pathogen (Sclerotium rolfsii) was prepared in the laboratory of Plant Pathology on sorghum grains which were boiled with water sealed and packed in polythene bag @ 200 g/bag and kept for sterilization in autoclave (121.6°C for 15 PSI for 15 min) after sterilized bag were allowed with mycelial disc to cool at room temperature followed by inoculation with mycelial disc 8 to 10 disc (5 mm) of 6 days old culture of Sclerotium rolfsii were aseptically inoculated into sterilized seed and incubated for the seven days in BOD incubator at 28 ± 2°C temperature. The cement pots (25 cm diameter) containing sterilized sandy loam soil, were inoculated with two weeks old culture of Sclerotium rolfsii (multiplied on sorghum grain medium) @ 25g/kg soil and allowed to incubate for fifteen days. Pots were regularly observed for the sclerotia formation and top layer of soils along with sclerotia were removed from the pot after Sclerotial formation and mixed with the soil a definite proportion. Number ofsclerotia were determined by the adding 10 gm of sick soil into water. Fresh pot further inoculated with 200g of sick soil which contain and covered and mixed with the soil with the definite proportion @ (920) number of sclerotia. Treated chickpea seeds (variety RVG- 202) were sown in pots (28 × 28 cm) @ 50 seeds / pot. 

Observations recorded:

• Observations on seed germination and pre-emergence were recorded seven days after sowing, while post emergence seedling mortality (PESM) at 30 days after sowing. 

• The percentage seed germination, pre emergence and post emergence seedling mortality was calculated by the formulas. 

Where, C = Per cent rot/ mortality in treatment pots 

 T = Per cent mortality in treated pots

Data presented in Table 1 indicated that the different fungicides tested at different concentrations (100ppm, 500 ppm, 1000 ppm and 2000 ppm) significantly reduced the mycelial growth of Sclerotium rolfsii over untreated control (90.00 mm). However fungicides i.e. Metiram 70%WG, Tricyclazole 75% WP, Propiconazole 25% EC, Azoxystrobin 18.2% + Difenoconazole 11.4% SC, Hexaconazole 5% SC, Iprobenfos 48% EC, Azoxystrobin 11% +Tebuconazole 18.3% SC, Thifluzamide 24% SC, Mancozeb 75% WP, Propineb 70% WP, Tebuconazole 50% + Trifloxystrobin 25% WG, Tebuconazole 38.39% w/w SC, Hexaconazole 4% + Zineb 68% WP and Metiram 55% + Pyraclostribin 5% WG were completely inhibitory at 100ppm concentration showing high potential against Sclerotium rolfsiicompared to other fungicides. Moreover, fungicides i.e. Validamycin 3% L (90.00, 65.83, 62.67 and 49.00 mm) and  Kasugamycin 3% SL (60.67, 0.00, 0.00 and 0.00 mm) respectively at 100ppm, 500ppm, 1000ppm and 2000ppm. Present study also indicated that the new generation fungicides having combination of two molecules and formulated either in wettable powder (WP), emulsifiable concentrate (EC) and suspension concentrate (SC) were found to be significantly more effective compared to other systemic or non-systemic fungicides already recommended against Sclerotium rolfsii. 

The results of present study all in accordance with the findings of Bhat and Shrivastava (2003) who reported the highly effectiveness of triazole fungicides i.e. hexaconazole, propiconazole and penconazole were also found result with higher inhibitory rates ranging from 63.0 per cent to 100 per cent against Sclerotium rolfsii at concentrations of 250, 500, and 1000 ppm. Similarly, Shirsole et al. (2019) also reported the effectiveness of fungicides used under the present study. The higher efficacy of systemic and combo fungicides (combination of two molecules) under the present study might be due to synergistic action of both molecules in inhibiting the mycelial growth of Sclerotium rolfsii. Sangeeta et al. (2022); Singh, et al. (2017) also reported that Hexaconazole (0.05%), Propiconazole (0.15%), and Mancozeb showed 100per cent inhibition.

Testing the efficacy of fungicides as seed treatment for the control of collar rot of chickpea (Sclerotium rolfsii). The pot experiment was carried out to assess the efficacy of fungicides for management of collar rot of chickpea. Fungicides were used as a seed treatment for management of collar rot mortality in chickpea.  

The data recorded in Table 2 on per cent seed germination from various treatments indicated that seeds treated with Hexaconazole 4% + Zineb 68% WP (95.33%) followed by Tebuconazole 50% + Trifloxystrobin 25% WG (93.33%) and Hexaconazole 5% SC (90.33%) were found significantly superior over all other treatments including control (39.33%) for enhancing the per cent seed germination of chickpea. Seed treated with other fungicides i.e. Tebuconazole 38.39% w/w SC (88.33%), Thifluzamide 24% SC (86.33%), Metiram 55% + Pyraclostribin 5% WG (85.66%), Azoxystrobin 11% +Tebuconazole 18.3% SC (85.33%),  Azoxystrobin 18.2% + Difenoconazole 11.4% SC (83.00%),  Propineb 70% WP (81.33%),  Metiram 70% WG (76.66%),  Propiconazole 25% EC (71.33%), Iprobenfos 48% EC (67.33%), Mancozeb 75% WP (66.67%) and Tricyclazole 75% WP (62.00%) had also significant effect on increasing the per cent germination over control(39.33%).

Data represent in Table 2 indicated that the lowest per cent pre emergence mortality was observed in treatment Hexaconazole 4% + Zineb 68% WP (4.67%) which was at par with Tebuconazole 50% + Trifloxystrobin 25% WG (6.67%) and found significantly superior over other treatments including control (60.67%). Other fungicides i.e. Hexaconazole 5% SC (9.67%), Tebuconazole 38.39% w/w SC (11.67%), Thifluzamide 24% SC (13.67%), Metiram 55% + Pyraclostribin 5% WG (14.33%), Azoxystrobin 11% +Tebuconazole 18.3% SC (14.67%),  Azoxystrobin 18.2% + Difenoconazole 11.4% SC (17.00%),  Propineb 70% WP (18.67%),  Metiram 70% WG (23.33%) and  Propiconazole 25% EC (28.67%) were also found significant in controlling pre emergence mortality compare to control (60.67%). Whereas, Iprobenfos 48% EC (32.67%), Mancozeb 75% WP (33.33%) and Tricyclazole 75% WP (38.00%) were found least effective. Moreover these fungicides were found significantly superior over control (60.67%).

Data from Table 2 indicated that the treatment i.e. Hexaconazole 4% + Zineb 68% WP (0.00%), Tebuconazole 50% + Trifloxystrobin 25% WG (0.00%), Hexaconazole 5% SC (0.00%), Tebuconazole 38.39% w/w SC (0.00%),  Thifluzamide 24% SC (0.00%),  Azoxystrobin 11% +Tebuconazole 18.3% SC (0.00%), and Propiconazole 25% EC (0.00%) were found significantly highly effective and completely checking the post emergence mortality of chickpea. Whereas, other fungicides i.e.  Azoxystrobin 18.2% + Difenoconazole 11.4% SC (3.27%),  Propineb 70% WP (28.00%),  Metiram 70% WG (34.83%), Iprobenfos 48% EC (44.33%), Mancozeb 75% WP (28.73%) and Tricyclazole 75% WP (13.33%) were also found significantly effective for suppressing the post emergence mortality over control (75.00%).

The results of present study are in accordance with the findings of Shirsole et al. (2019), who reported that the efficacy of seventeen fungicides (systemic - seven, non-systemic - four and combo - six) as seed treatment under pot experiment (in-vivo). Seed treatment with Hexaconazole 5% EC, Propiconazole 25% EC and Azoxystrobin 35% EC exhibited complete protection from collar rot mortality and 100% reduction in disease incidence over control. Charde et al. (2002) also found that seed treatment with Propiconazole and Hexaconazole were superior in checking stem rot of groundnut caused by S. rolfsii and increasing the shoot and root biomass.

Table 1: In-vitro, evaluation of fungicides at different concentrations (ppm) of fungicides against mycelial growth of Sclerotium rolfsii, the causal agent of collar rot of chickpea (at 96 hour).

*Figures in the parentheses indicate arc sine transformed

Table 2: Effect of different fungicides on percent germination of chickpea seed and controlling pre-emergence and post-emergence collar rot mortality of chickpea caused by Sclerotium rolfsii.

*Figures in the parentheses indicate arc sine transformed

Fig. 1. In-vitro, evaluation of fungicides against Sclerotium rolfsiiat different concentrations (ppm).

Fig. 2. Effect of seed treatment with fungicides on seed germination, pre emergence mortality and post emergence mortality of chickpea against collar rot.

Plate 1. In-vitro, evaluation of fungicide against Sclerotium rolfsiiat different concentrations (ppm).

In-vitro study on the efficacy of fungicides against Sclerotium rolfsii indicated that fungicides  Metiram 70%WG, Propiconazole 25% EC, Azoxystrobin 18.2% + Difenoconazole 11.4% SC, Hexaconazole 5% SC, Azoxystrobin 11% +Tebuconazole 18.3% SC, Mancozeb 75% WP, Propineb 70% WP, Tebuconazole 50% + Trifloxystrobin 25% WG, Hexaconazole 4% + Zineb 68% WP and Metiram 55% + Pyraclostribin 5% WG were found highly effective and can be used as seed treatment for the control of disease caused by Sclerotium rolfsii.

Fungicides found most effective against Sclerotium rolfsii under in-vitro and pot condition may be tested for their efficacy under natural field condition with different combination of novel fungicides and in combination of bioagents i.e. Trichoderma and Pseudomonas as seed and soil treatment and their combination, Integrated management strategy for collar rot of chickpea that is combined the use of microbial agents by fortification techniques. The efficacy of the fungicides may be tested at lower dosages. Compatibility scale should be prepared according to toxic level.

Abhishek Sahu, R.K.S. Tiwari, V.K. Nirmalkar, P.K. Keshary and N.K. Chaure  (2024). In-vitro and in-vivo Evaluation of Fungicides Against Collar Rot of Chickpea (Sclerotium rolfsii). Biological Forum – An International Journal, 16(11): 81-86.