Cross-host infectivity of Sclerotium rolfsii associated with finger millet foot rot

Introduction

Finger millet (Eleusine coracana L. Gaertn.), commonly known as ragi, bird's foot millet, mandua, nagli and nachni, is one of the most important small millets cultivated in tropical and subtropical regions of Asia and Africa. The crop originated in the Ethiopian highlands and was subsequently introduced into India, where it has become an important staple food crop due to its adaptability to diverse agro-climatic conditions and resilience under marginal environments (Phillips, R.W., 1966) [2] . Finger millet is nutritionally superior to many cereals because of its high calcium, dietary fibre, essential amino acids, vitamins and mineral contents, making it an important food source for nutritional security (Malleshi and Hadimani, 1993) [7] . Owing to its low glycaemic index and gluten-free nature, finger millet is increasingly recognized as a functional food beneficial for diabetic patients and individuals with celiac disease (Geetha et al., 2020) [8] . India is the leading producer of finger millet, accounting for a major share of global production with Karnataka being the principal cultivating state. Despite its inherent tolerance to drought and environmental stresses, finger millet productivity is constrained by several diseases caused by fungi, bacteria, viruses and nematodes (Govindu and Shivanandappa, 1967) [9] . Among fungal diseases, foot rot caused by Sclerotium rolfsii Sacc., has emerged as an important soil-borne disease, particularly under irrigated and high rainfall conditions, causing severe yield losses and threatening sustainable crop production.

Sclerotium rolfsii is a ubiquitous necrotrophic fungus belonging to the family Atheliaceae with the teleomorphic stage Atheliarolfsii (Tu and Kimbrough, 1978) [10] . The pathogen survives in soil through resilient sclerotia and infects plants primarily at the collar region, resulting in tissue rotting, wilting and eventual plant death (Kwon et al., 2017) [6] . The fungus possesses an exceptionally broad host range, infecting more than 500 plant species including cereals, pulses, oilseeds, vegetables, ornamentals and medicinal plants (Aycock, 1966) [1] . Important hosts include groundnut, soybean, chickpea, chilli, onion, potato, tomato, wheat, barley and finger millet (Desai and Schlosser, 1999) [5] . In finger millet, foot rot disease can cause substantial reductions in grain and fodder yield, with losses reported up to 27.40 and 23.31 per cent, respectively (Pawar et al., 2013) [3] . The wide host range of S. rolfsii contributes significantly to its persistence and epidemiology in agricultural ecosystems. Alternate and collateral hosts facilitate long-term survival of the pathogen in soil and serve as reservoirs for inoculum, increasing disease incidence in subsequent crops. Therefore, understanding host susceptibility is essential for developing suitable crop rotation strategies and integrated disease management practices. Host range studies help identify susceptible and resistant plant species, elucidate crossinfectivity patterns and improve understanding of pathogen ecology and survival mechanisms (Punja, 1985) [4] . In addition to broad host adaptability, S. rolfsii populations exhibit considerable variability in morphological, cultural, pathogenic and genetic characteristics. Variations among isolates have been documented with respect to colony morphology, sclerotial production, growth rate, virulence and aggressiveness across hosts and geographical regions (Cooper, 1961; Kim, 1974; Punja, 1985) [11,12,4] . Such pathogenic variability influences disease severity and host specificity, thereby posing challenges for breeding resistant cultivars and designing effective management strategies. Understanding variability among pathogen isolates is therefore important for identifying highly virulent strains and predicting disease outbreaks. Characterization of pathogenic variability coupled with host range assessment provides crucial insights into the epidemiology and evolution of S. rolfsii populations. Information on differential virulence and host adaptation can support resistance breeding programmes and facilitate the development of targeted disease management strategies. Hence, the present investigation was undertaken to study the host range and pathogenic variability of Sclerotium rolfsii causing foot rot in finger millet with the objective of understanding its infectivity across different crop species and variability in pathogenic behaviour among isolates.

Materials and Methods

Isolation of the Pathogen

Finger millet plants exhibiting typical foot rot symptoms at the collar and stem base regions were collected. Small pieces (5-10 mm) from the advancing margins of infected tissues were excised and surface sterilized in 1% sodium hypochlorite solution for 1 min, followed by three rinses in sterile distilled water. The sterilized tissue pieces were aseptically transferred onto potato dextrose agar (PDA) plates and incubated at 27±1 °C for 3 days. Emerging fungal colonies were transferred to fresh PDA plates and purified by the hyphal tip culture technique. Pure cultures obtained from different samples were maintained on PDA slants and designated as individual isolates.

Hyphal Tip Culture

To obtain genetically pure cultures, hyphal tip isolation was carried out on 2% water agar medium. A dilute mycelial suspension (10 ⁻⁶ dilution) was prepared in sterile distilled water and 1 mL was spread uniformly on water agar plates. Under a microscope, actively growing hyphal tips were identified and marked. Small mycelial fragments from the hyphal tips were aseptically transferred to PDA plates and incubated at 27±1 °C until sufficient growth was obtained. Pure cultures were maintained for further studies.

Identification of the Pathogen

The fungal isolate was identified based on its cultural and morphological characteristics. On PDA, the pathogen produced abundant cottony white mycelial growth, which later became dull white. Spherical to irregular mustard seedlike sclerotia developed after 6-7 days of incubation and gradually changed from white to brown upon maturity. Based on these characteristics, the pathogen was identified as Sclerotium rolfsiiSacc.

Maintenance of Cultures

Pure culture of S. rolfsii was maintained on PDA slants and incubated at 27±1 °C for 10 days. The cultures were subsequently stored at 4 °C and sub-cultured at monthly intervals to maintain viability.

Mass Multiplication of Inoculum

Sorghum grains were used for mass multiplication of the pathogen. The grains were soaked in water for 24 h and 250 g of grains were filled into autoclavable polypropylene bags. The grains were sterilized at 121 °C (1.1 kg cm ² pressure) for 20 min. After cooling, two to three 5-mm ⁻ mycelial discs from actively growing cultures of S. rolfsii were inoculated into each bag under aseptic conditions. The inoculated bags were incubated at 28 °C for 30 days and shaken periodically to ensure uniform colonization. Fully colonized sorghum grains were used as inoculum for preparing sick soil.

Soil Sterilization and Preparation of Sick Soil

A potting mixture consisting of red sandy loam soil, sand, and compost in the ratio of 1:1:0.5 was sterilized by drenching with 4% formaldehyde solution until saturation. The treated soil was covered with a polyethylene sheet for five days and subsequently exposed to open air for seven days to allow complete dissipation of formaldehyde residues.For preparation of sick soil, 20 dayold S. rolfsii inoculum grown on sorghum grains was thoroughly mixed with the sterilized potting mixture. The inoculated soil was filled into pots and maintained under suitable moisture conditions to facilitate pathogen establishment.

Host Range Studies

Seeds of different millet species were surface sterilized with 0.1% sodium hypochlorite solution, washed thoroughly with sterile distilled water and sown in pathogeninfested soil. Pots without pathogen inoculum served as controls. Alternate wetting and drying conditions were maintained throughout the experiment to promote disease development. The following formula is used to calculate the percent disease incidence and incubation period,

Results and Discussion

The host range study conducted under greenhouse conditions revealed differential susceptibility of small millet species to the highly virulent isolate of Sclerotium rolfsii (SrVcM), the causal agent of foot rot in finger millet. The absence of infection in several millet species suggests their potential suitability for crop rotation strategies aimed at reducing pathogen populations in infested fields.

The findings of the present investigation corroborate earlier reports describing the broad host range of S. rolfsii. Aycock (1966) [1] reported that the pathogen infects more than 500 plant species belonging to diverse botanical families. Similarly, Punja (1985) [4] highlighted the remarkable ecological adaptability and host diversity of S. rolfsii, which contributes to its persistence in agricultural ecosystems. Host range studies conducted by Epps et al. (1951) [13] demonstrated variability in pathogenicity among different isolates of S. rolfsii on various hosts. Furthermore, Shwetha (2012) [14] reported that S. rolfsii causing wilt of Stevia rebaudiana infected several medicinal and aromatic plants, confirming the pathogen's capacity for cross-host infectivity.

The present study establishes that finger millet is the most preferred host of the highly virulent isolate SrVcM, while proso millet and little millet can serve as alternate hosts under favorable conditions. The resistant reaction observed in brown top millet, kodo millet, foxtail millet, barnyard millet, pearl millet, and sorghum indicates their potential utility in designing sustainable crop rotation systems for effective management of foot rot disease caused by Sclerotium rolfsii. The pathogenic response of different millet species to the highly virulent isolate of Sclerotium rolfsii was assessed through disease severity, seedling mortality, plant survival, and pathogen re-isolation studies (Table 2). Significant differences in host susceptibility were observed among the millet species tested. Among all the hosts evaluated, inoculated finger millet (Eleusine coracana) exhibited the highest disease severity score (4.6 on a 0-5 scale), accompanied by maximum seedling mortality (68.7%) and the lowest plant survival (31.3%). The pathogen was successfully reisolated from all symptomatic plants, resulting in 100 per cent pathogen recovery. The severe disease reaction observed in finger millet confirms its high susceptibility to S. rolfsii and demonstrates the aggressive nature of the isolate used in the study. Moderate disease development was recorded in proso millet (Panicum miliaceum) and little millet (Panicum sumatrense). Proso millet exhibited a disease severity score of 2.3, with 28.5 per cent seedling mortality and 71.5 per cent plant survival. Similarly, little millet recorded a disease severity score of 1.8, seedling mortality of 20.0 per cent, and plant survival of 80.0 per cent. Pathogen re-isolation frequencies of 82.0 and 75.0 per cent were obtained from proso millet and little millet, respectively, confirming successful infection and colonization by S. rolfsii. These findings indicate that both crops can serve as alternate hosts and may contribute to pathogen survival in millet-based cropping systems. In contrast, brown top millet (Urochloaramosa), kodo millet (Paspalum scrobiculatum), foxtail millet (Setariaitalica), barnyard millet (Echinochloa esculenta), pearl millet (Pennisetum glaucum), and sorghum (Sorghum bicolor) showed complete resistance under greenhouse conditions. No disease symptoms were observed on these hosts, resulting in zero disease severity, no seedling mortality, 100 per cent plant survival, and absence of pathogen re-isolation. Likewise, the uninoculated finger millet control remained symptomless throughout the experimental period. These results suggest that these millet species are either non-hosts or possess effective resistance mechanisms that restrict pathogen infection and establishment. Statistical analysis revealed significant differences among the millet hosts for disease severity, seedling mortality, plant survival, and pathogen re-isolation. The calculated SEm (±) values were 0.18, 2.36, 2.36, and 3.12, respectively, while the corresponding CD values at 5 per cent probability were 0.54, 7.08, 7.08, and 9.36, indicating that the observed differences among susceptible and resistant hosts were statistically significant. The present findings support earlier reports describing the broad host range and pathogenic variability of S. rolfsii. Aycock (1966) [1] reported that the pathogen infects a wide range of cultivated crops and weeds, enabling its long-term persistence in agricultural soils. Punja (1985) [4] further emphasized that alternate hosts play a crucial role in pathogen survival and disease epidemiology. Similar host range studies conducted on other crop-pathogen systems have demonstrated varying levels of susceptibility among related plant species, reflecting differences in host defense mechanisms and pathogen adaptation. The successful infection of proso millet and little millet indicates their potential role as collateral hosts capable of maintaining inoculum between cropping seasons. Conversely, the resistant reaction observed in brown top millet, kodo millet, foxtail millet, barnyard millet, pearl millet, and sorghum highlights their potential suitability in crop rotation programmes aimed at reducing soil inoculum levels. Therefore, inclusion of these resistant millet species in cropping systems may contribute to sustainable management of foot rot disease caused by Sclerotium rolfsii in finger millet-growing regions. [4] emphasized that severe infection by S. rolfsii often results in extensive root and collar rot, leading to reduced plant growth, premature senescence, and significant yield losses. The present study clearly demonstrates that S. rolfsii infection adversely affects growth and biomass production in susceptible millet hosts, particularly finger millet. The absence of growth reduction in brown top millet, kodo millet, foxtail millet, barnyard millet, pearl millet, and sorghum further supports their resistant or non-host status and highlights their potential suitability in crop rotation programmes for managing foot rot disease in finger milletbased production systems.

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