In vitro evaluation of fungicides and bioagents against Sclerotium rolfsii, causing collar rot of chickpea

Introduction

Pulses are the source of vegetarian protein in Indian food. Chickpea (Cicer arietinum L.) is significant pulse crop contributing about 27% of global production (ICAR -Indian Institute of Pulses Research) which provides 23% protein, 64% carbohydrates and essential minerals (Miao et al., 2009) [7] . In India chickpea is grown in 9.12 Mha with total production of 11.11 MT and productivity 1218 kg/ha (Anonymous, 2024-25) [1] . In Madhya Pradesh total area under chickpea production is 1.35Mha, (14.84% of total area of the country), total production is 2.11MT (18.98%) and productivity is 1558 Kg/ha. (Anonymous, 2024-25) [1] . Madhya Pradesh is the third leading state in chickpea area, second in total production and first in productivity (Anonymous, 2024-25) [1] . As compare to cropping year 2023-24, area and production under chickpea in MP (2.11Mha and 2.91MT respectively) has been decreased because the crop has been increasingly threatening by various fungal diseases for last many years like-Ascochyta blight (Ascochyta rabiei), Botrytis gray mold (Botrytis cinerea), Alternaria blight (Alternaria alternata), Colletotrichum blight (Colletotrichum dematium), Phoma blight (Phoma medicaginis), Rust (Uromyces ciceris-arietini), Powdery mildew (Leveillula Taurica), Sclerotinia stem rot (Sclerotinia sclerotiorum), Fusarium wilt (Fusarium oxysporum f. sp. Ciceri, Verticillium wilt (Verticillium albo-atrum), Collar rot (Sclerotium rolfsii), Dry root rot (Sclerotium rolfsii), Phytophthora root rot (Phytophthora medicaginis), Foot rot (Operculella padwickii) (ICRISAT 2012 or Nene et.al., 2012) [9] . Since, Collar rot caused by Sclerotium rolfsii was a minor disease but from last few years it is becoming major disease causing quantitative and qualitative losses. In Vindhyan plateau of Madhya Pradesh 12.00-18.20% disease incidence was recorded during 2018 and 2019 cropping seasons (Singh et.al., 2022) [17] . Rolfs (1892) [12] first time reported Sclerotium rolfsii as a causal organism of tomato blight in Florida. The causal organism of tomato blight was first time given by Saccardo (1911) [14] . The pathogen survives as macrosclerotia in soil and infects roots when plants are under abiotic stress. In vitro effect of temperature and pH shows that fungus survives best at 30 0 C and at pH 5.5-7.0 (Premlata et.al. 2025) [11] .

These environmental and soil conditions not found only in Madhya Pradesh but also found in many other States like U.P., Bihar, Haryana, Punjab, Maharashtra, therefore, this disease may prevalent in these states also. The aim of the present study was to know the effect of different fungicides and bio-agents control/management collar rot of chickpea disease caused by Sclerotium rolfsii.

Materials and Methods

Survey of disease

A survey was conducted to record the occurrence and distribution of collar rot of chickpea in Sohawal block, Satna in central India -Madhya Pradesh, during the Rabi season of 2024-25. Chickpea plants displaying typical collar rot symptoms were collected from the surveyed areas, packed in labelled polythene bags and were brought to the laboratory for pathogen isolation.

Isolation and Purification of Fungus from Infected Roots:

The fungus was isolated and purified from infected roots by using Potato Dextrose Agar (PDA) medium having pH 6 (Premlata et.al., 2025) [11] in sterilized Petri dishes. The inoculated Petri dishes were incubated at 27 ± 2°C in a BOD incubator to promote fungal growth for further microscopic examination. Based on morphological characteristics of the mycelium and sclerotia the isolates were identified as Sclerotium rolfsii. The pure cultures were stored on PDA slants in a refrigerator at 4 ± 1°C for further studies.

In vitro Evaluation of Different Fungicides Against

Sclerotium rolfsii: An in vitro study was carried out to evaluate the efficacy of seven chemical fungicides and three biocontrol agents Trichoderma viride, neem oil, and Pseudomonas fluorescens (Table 1) against Sclerotium rolfsii, the causal agent of collar rot in chickpea. The poisoned food technique was employed using Potato Dextrose Agar (PDA) having pH 6 as the basal medium. Fresh (24hours to 48 hours old) and pure culture of Trichoderma viride was inoculated at four points around the disc of Sclerotium rolfsii, on the same day (T 1 ), after one day (T 2 ) and after 2days (T 3 ). The radial growth of tested fungus, Sclerotium rolfsii was measured upto 5 days after inoculation. Based on the active ingredient concentration the required quantity of each fungicide was accurately weighed and mixed thoroughly into autoclaved and cooled PDA medium (approx. 40°C) in conical flasks to achieve the desired concentrations. Twenty millilitres of each fungicide-amended medium was poured aseptically into 90 mm sterile glass Petri dishes and allowed to solidify at room temperature. Each plate was then inoculated at the centre with a 5 mm mycelial disc cut from the actively growing margin of a 7-day-old Sclerotium rolfsii culture and placed in an inverted position in BOD incubation. Control plates (without fungicide or biocontrol agents) were also inoculated with the same culture to serve as untreated controls. Three replications were maintained for each treatment. Plates were incubated at 30 0 C temperature (Premlata et.al., 2025) [11] . After incubation the two directional mycelium growth (in cm) of the fungus (Sclerotium rolfsii) was measured and mean of two direction growth was calculated. The percent inhibition of mycelial growth was calculated using the following formula:

Where, C= growth of the test fungus in control plates (cm) T= growth of the test fungus in treated plates (cm) Where,

Survey and Symptoms of Disease

The survey was conducted from mid Dec.-mid Jan. 2025. The disease caused by Sclerotium rolfsii, was observed in many fields with varying degrees of severity. The infected seedlings turned slightly yellow before death and found scattered throughout the field was an indication of collar rot infection. The disease was seen mostly at the seedling stage particularly in high soils moisture. Young seedlings were collapsed due to sophisticated stage but older seedlings were completely dry without collapsing and having leaves attached to the plant. Infected plants were easily uprooted due to rotting of secondary roots from collar region to downwards of the root. The rotten portion of the root was found covered with whitish mycelial strands.

Isolation and Identification of the Fungus (Sclerotium rolfsii):

Collar rot causing fungus (Sclerotium rolfsii) isolated from infected plants on PDA. The isolated fungus grown on PDA medium exhibited higher radial growth which filled 90 mm diameter petri plates within 5 days. The colony colour of Sclerotium rolfsii appeared cottony white with dense radiating mycelial growth (Fig. 1A) and numerous brown to dark brown macro-sclerotia were developed throughout the mycelial mat (Fig. 1B). The hyphal cells of Sclerotium rolfsii were septate and hyaline. The results presented in Table 2 and figure 2 revealed the impact of varying concentrations of copper oxychloride 50% WP on the radial growth of Sclerotium rolfsii at 3, 5 and 7 days after inoculation (DAI). The maximum fungal growth inhibition (38.66, 21.85 and 18.83%) at 3, 5 and 7 respectively was found with 0.25% (T 4 ) concentration (Table 2) followed by (30.20, 17.20 and 16.14%) at 0.20% (T 3 ) concentration at 3, 5 and 7 DAI with 0.20% concentration of copper oxychloride (Table 2). A similar trend was also observed with lower concentration (0.15%).

Although growth inhibition was very low but Copperoxychloride 50% WP significantly inhibited the growth of Sclerotium rolfsii (Fig. 2).

Effect of Carbendazim 50% on the Growth of Sclerotium rolfsii

Carbendazim 50% WP exerted a less inhibitory effect on the mycelial growth of the pathogen (Sclerotium rolfsii) at 3, 5 and 7 DAI (Table 3).

The highest inhibition (20.58%) was observed with 0.04% (T 4 ) concentration followed by (12.32%) with 0.03% (T 3 ) at 3 DAI but there was not significant inhibition at 5DAI with all concentrations. However, at 7 DAI, three treatments (T 1 , T 2 and T 3 ) showed very similar radial growth inhibition (5.56%, 5.56% and 5.00% respectively) and T 4 showed negligible inhibition (1.11%) which was not significant (Fig. 3). 4). Statistical analysis confirmed that all treatments (0.1/T 1 , 0.15/T 2 , 0.2/T 3 and 0.25% /T 4 ) were significantly inhibited the growth of Sclerotium rolfsii when compared to the control (Fig. 4) across all three observation intervals (3, 5, and 7 DAIs) but 0.15 (T 2 ) and 0.20% (T 3 ) have given best results (Fig. 4).

Effect of different concentrations of Metalaxyl 4% + Mancozeb 64% on radial growth of Sclerotium rolfsii

The present study revealed that all tested concentrations of Metalaxyl 4% + Mancozeb 64% (0.1%, 0.15%, 0.2%, and 0.25%) were completely inhibited the radial growth of Sclerotium rolfsii at 3, 5, and 7 DAI. No fungal growth inhibition was recorded in untreated (control) petri plates (Fig. 5) at any intervals. The consistency of complete inhibition across all concentrations and time points indicated that the fungicidal combination of Metalaxyl 4% + Mancozeb 64% possesses strong and sustained antifungal activity against Sclerotium rolfsii.

Effect of Carbendazim 12% + Mancozeb 63% on the Growth of Sclerotium rolfsii:

At all-time intervals (3, 5 & 7 DAI) and all tested concentrations (0.1%, 0.15%, 0.2%, and 0.25%) of Carbendazim12% + Mancozeb 63% (SAAF) completely inhibited the mycelial growth of Sclerotium rolfsii (Fig. 6). In contrast, the untreated control recorded normal radial growth (zero percent growth inhibition). These findings indicated that Carbendazim 12% + Mancozeb 63% is highly effective in arresting the initial growth of the pathogen even at the lowest concentration tested. The impact of Carboxin 37.5% + Thiram 37.5% at different concentrations (0.1%, 0.15%, 0.2%, and 0.25%) on the radial growth of Sclerotium rolfsii was assessed at 3, 5 and 7 DAI which exhibited complete inhibitory effect (Fig. 7) on the mycelial growth of the pathogen (Sclerotium rolfsii).

Effect of Pseudomonas fluorescens on the Growth of Sclerotium rolfsii

It was shown that maximum fungal growth inhibition was found with only 1% concentration of Pseudomonas fluorescence at 3 DAI (Table 5 and Fig. 9) but not at any other concentration (1.5 and 2.0%). At 5 and 7 DAI there was no inhibition with any concentration (1.0%/T 1 , 1.5%/T 2 and 2.0%/T 3 ) of Pseudomonas fluorescence (Fig. 9). It is confirmed that Pseudomonas fluorescence 1% can inhibit Sclerotium rolfsii at only early stages. This indicated that the inhibitory effect observed at 3 DAI diminished over time and Pseudomonas fluorescens had a limited or transient impact on the pathogen (Sclerotium rolfsii) under the given experimental conditions.

Effect of Neem oil on the growth of Sclerotium rolfsii:

The antifungal efficacy of Neem oil with all tested concentrations/treatments (0.5%/T 1 , 1.0%/T 2 and 1.5%/T 3 ) was evaluated against Sclerotium rolfsii through poison bait technique shown the results in Table (6) and Figure (10). At 3 DAI, all concentrations/treatments (0.5%/T 1 , 1.0%/T 2 and 1.5%/T 3 ) showed increasing inhibition of fungal growth (80.47, 82.48 and 84.46% respectively). The same trend of inhibition (77.94, 80.14 and 81.47%) was found at 5 DAI with all tested concentrations (Table 6) but at 7 DAI growth inhibition of the tested fungus was not found same as at 3 and 5 DAI. However, neem oil indicated statistically significant inhibition of Sclerotium rolfsii at all time period intervals (3, 5 and 7 DAI). Overall, Neem oil exhibited a strong, dose-dependent antifungal activity with 1.5% concentration being the most effective in reducing the mycelial growth of Sclerotium rolfsii across all observations. Effect of Trichoderma viride on the Growth of Sclerotium rolfsii: When Trichoderma viride and Sclerotium rolfsii were inoculated same day (T 1 ) the inhibition of was found 68.93% at 5 DAI (Table7) but when Trichoderma viride was inoculated one and two days after inoculation of Sclerotium rolfsii, the inhibition was found 30.40% and 20% respectively (Table 7) at 5 DAI. The results presented in Table 7 along with Figure10 indicated that Trichoderma viride had a marginal inhibitory effect on the mycelial growth of the pathogen. However, Trichoderma viride checked further growth of Sclerotium rolfsii in T 2 and T 3 treatments (Fig. 11).

Present study showed that Trichoderma viride has antagonistic effect against Sclerotium rolfsii. Statistical analysis was not possible due to size of the experiment (Only 3 treatments and 3 replications) but mean inhibition percent was calculated.

Discussion

Among all fungicides tested, Metalaxyl 4% + Mancozeb 64%, Carbendazim 12% + Mancozeb 63% (SAAF), Carboxin 37.5% + Thiram 37.5%, and Propiconazole 13.9% + Difenoconazole 13.9% were highly effective, exhibiting complete inhibition of fungal growth (Fig. 3.5, 3.6 and 3.7) at all tested concentrations and time intervals (3, 5, and 7 DAI). These results indicated a strong and broad-spectrum fungicidal activity of these systemic and contact fungicides against Sclerotium rolfsii. Similarly, Divyashree et.al. (2024) [3] observed that Carboxin 37.5% + Thiram 37.5% and Carbendazim 12% + Mancozeb 63% were highly effective in preventing sclerotial germination and mycelial spread of S. rolfsii. Carbendazim 12% + Mancozeb 63% was also found most effective in inhibiting complete mycelial growth of S. rolfsii (Kumari et.al. 2023) [5] . Shirsole et.al. (2019) [16] also found 100% inhibition of mycelial growth of Sclerotium rolfsii treated with Propiconazole 13.9% + Difenconazole13.9% EC at the rate of 50ppm, 100ppm and 150ppm. Carbendazim 12% + Mancozeb 63% (0.15%) and Carboxin 37.5% + Thiram 37.5% also showed cent per cent inhibition (Sangeeta et.al., 2022 and Shirsole et.al., 2019) [15,16] as in present study. Thiram 75% WP exhibited moderate antifungal activity. The maximum growth inhibition (72.55% to 98.02%) was observed at all tested concentrations of Thiram 75% (0.1, 0.15, 0.20 and 0.25%) at 3 to 7 DAI. The present findings are supported by Kumar et.al. (2021) [4] that Thiram exhibited moderate antifungal activity (33.33%) against Sclerotium rolfsii in chickpea with 150ppm concentration. Carbendazim 50%, a commonly used systemic fungicide exhibited limited and inconsistent inhibition of S. rolfsii especially at higher concentrations (0.03% and 0.04%) where no significant difference was found as compare to control. Rothe et.al. (2019) [13] found 56.13% inhibition of S. rolfsii growth by Carbendazim 50% WP. Pseudomonas fluorescens (1% concentration) exhibited a significant inhibitory effect (14.5%) on the radial growth of Sclerotium rolfsii at only 3 days after inoculation (DAI) but other concentrations (1.5 and 2.0%) did not show significant inhibition (Fig. 3.8). Kumar et.al. (2021) [4] found 76.3%, Meena et.al. (2023) [6] found 68.89% and Bagul et.al, (2024) [2] found 74.04% mycelial growth inhibition of S. rolfsii with Pseudomonas fluorescence. Neem oil exhibited strong and sustained antifungal activity against S. rolfsii at all tested concentrations (0.5, 1.0 and 1.5%) consistently up to 76.32 -84.46% (Table 6). Singh et.al. (2023) [18] also recorded up to 47.22 and 59.44% mycelial growth inhibition of S. rolfsii at 10 and 15% concentrations of NSKE. Trichoderma viride also exhibited a progressive (61.23-68.93%) and consistent antagonistic effect against Sclerotium rolfsii from 2 to 5 DAI (Table 7). These findings are in close agreement with those of Prajapati et.al. (2015) [10] who reported complete inhibition of S. rolfsii radial growth by T. viride. Kumar et.al. (2021) [4] observed 72.22% inhibition of S. rolfsii while Meena et.al.

(2023) [6] found 78.61% growth inhibition of S. rolfsii by T. viride. These are closely related to the present findings. In another experiment 63.33% inhibition of S. rolfsii fungus causing collar rot of chickpea was recorded by Nagamma and Nagaraja (2015) [8] . Collar rot of chickpea caused by Sclerotium rolfsii has become a serious problem for chickpea producing farmers due to similarity with wilt caused by Fusarium oxysporum f. sp. Ciceri and dry root rot caused by Rhizoctonia bataticola. The farmers have been using few numbers of fungicides, bioagents and botanicals only for management of wilt disease for many years but not for collar rot disease. The present study can give the multiple options for management of collar rot disease of chickpea.

Acknowledgement

AcknowledgementsWe acknowledge the AKS University, Satna, M.P., India for undertaking the different activities of research presented in this manuscript.

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