Survey and characterisation of Fusarium oxysporum f.sp. cepae causing basal rot of onion (Allium cepa L.) in Perambalur district of Tamil Nadu

Introduction

Onion (Allium cepa L.) is one of the most important bulb crops cultivated worldwide and is often referred to as the "Queen of the kitchen" due to its indispensable culinary and nutritional value. Belonging to the family Alliaceae, it is rich in essential nutrients such as vitamin C, vitamin B6, folic acid and dietary fibre and possesses several pharmacological properties including antioxidant, antimicrobial and anticancer activities [26,14] . India ranks second globally in onion cultivation, with Tamil Nadu contributing significantly through districts such as Dindigul, Tiruppur, Perambalur and Namakkal [16] . Despite its economic importance, onion productivity is severely constrained by soil-borne diseases, among which basal rot, caused by Fusarium oxysporum f.sp. cepae, is highly destructive. The pathogen infects the basal plate, leading to yellowing, wilting and eventual plant death, causing yield losses of up to 50% [4] . The survival of the fungus as chlamydospores for extended periods (8-10 years) in the soil further complicates its management [17] . Chemical control and resistant cultivars have shown limited success due to environmental concerns, cost factors and the emergence of virulent pathogen strains [9] . Although several studies have reported the occurrence of Fusarium wilt in onions, detailed investigations on the incidence, isolation and pathogenic variability of F. oxysporum f.sp. cepae under Tamil Nadu conditions remain scarce. Moreover, limited information exists on the occurrence of native antagonists with potential for biocontrol applications in the region. Understanding the pathogenic and cultural variability among isolates and identifying effective native biocontrol agents are crucial for developing sustainable disease management strategies. Therefore, the present investigation was undertaken with the following objectives: 1. To conduct a survey to assess the incidence of basal rot of onion in major onion-growing areas of Tamil Nadu and to isolate the pathogen Fusarium oxysporum f.sp. cepae and native antagonists.

2. To assess the cultural, morphological and pathogenic characterisation among the isolates of Fusarium oxysporum f.sp. cepae.

Materials and Methods

Survey to Assess the Incidence of Basal Rot of Onion

A field survey was conducted across ten major onion-growing regions of Perambalur district, Tamil Nadu, to evaluate the incidence of basal rot. In each field, 100 plants were randomly selected and the number of infected plants was recorded. The mean disease incidence was calculated as a percentage using the formula of McKinney [11] . Onion plants exhibiting characteristic symptoms of Fusarium basal rot were collected along with their rhizosphere soil for pathogen isolation. Additionally, data on locality, crop variety and soil type were recorded [10] .

Isolation and Identification of Fusarium oxysporum f. sp. cepae Onion samples collected from various growing regions of Perambalur district, Tamil Nadu, were used for pathogen isolation. The pathogen was isolated from infected onion bulbs using the tissue segment method as described by Rangaswami [19] . The infected bulb tissues were cut into small pieces with a sterile scalpel and surface sterilised with 1.0% sodium hypochlorite for 30 seconds. The tissues were then rinsed three times with sterile distilled water and transferred aseptically onto Petri plates containing sterilised Potato Dextrose Agar (PDA).

Plates were incubated at room temperature (28 ± 2 °C) for 5-7 days and observed for fungal growth. The hyphal tips of emerging colonies were subcultured onto PDA slants for maintaining pure cultures. The isolated fungi were identified based on their cultural and morphological characteristics.

Morphological and Cultural Characteristics of Fusarium oxysporum f. sp. cepae Ten isolates of Fusarium spp. obtained from infected onion bulbs were examined for variations in their morphological and cultural characteristics on both solid and liquid media. Ten-dayold cultures of each isolate were inoculated separately and incubated at 28 ± 2 °C for seven days. After incubation, parameters such as radial mycelial growth, microconidia and macroconidia population, colony characteristics, sporulation intensity and size of microconidia, macroconidia and chlamydospores were recorded. The conidial and chlamydospore counts and measurements were carried out following standard mycological procedures.

Mass Multiplication of the Pathogen Sand-Maize Medium

Mass multiplication of F. oxysporum f. sp. cepae isolates was carried out using the sand-maize medium following the method of Riker and Riker (20). Sterilised sand and ground maize seeds were mixed in a 19:1 ratio and adjusted to approximately 50% moisture content. The mixture was filled into glucose bottles and autoclaved at 20 psi for 2 hours. Seven-day-old actively growing mycelial discs (9 mm diameter) were inoculated into each bottle under aseptic conditions. The bottles were incubated at room temperature (28 ± 2 °C) for 15 days. The colonised substrate was then used as inoculum for subsequent experiments.

Sorghum and Cumbu Grain Medium [6] Sorghum and cumbu grains were filled up to three-fourths of the glucose bottles and plugged with cotton wool. The bottles were sterilised at 15 lbs pressure for 2 hours on two consecutive days. Six mycelial discs (9 mm) from 10-day-old F. oxysporum f. sp. cepae cultures grown on PDA were inoculated into each bottle. The cultures were incubated at room temperature (28 ± 2 °C) for 15 days and the resulting inoculum was used for soil inoculation.

Inoculation

Five kilograms of sterilised garden soil were filled into earthen pots (30 cm diameter). The soil was sterilised in an autoclave at 15 lbs pressure (1.04 kg cm⁻²) for two successive days and inoculated by thoroughly mixing the freshly prepared Fusarium inoculum (multiplied on sand-maize medium) at a rate of 50 g kg⁻¹ soil [13] . Two onion bulbs were planted in each pot, with three replications per treatment. The pots were maintained in a greenhouse under uniform and regular watering conditions and observed periodically for symptom development. The percentage disease incidence for each isolate was recorded 60 days after inoculation.

Statistical Analysis

All data collected were statistically analysed using the WASP Version 2.0 software developed by the Indian Council of Agricultural Research (ICAR), Goa, following the procedure of Gomez and Gomez [7] . Prior to analysis of variance (ANOVA), percentage data of the disease index were arcsine-transformed.

ANOVA was performed at a significance level of P<0.05 and mean comparisons were made using Duncan's Multiple Range Test (DMRT). Pot culture, laboratory and field experiments were designed following the Randomised Block Design (RBD). Percentage values were subjected to arcsine or square root transformation as appropriate.

Results and Discussions Survey and Incidence of Basal Rot of Onion in Perambalur District of Tamil Nadu

An extensive field survey was conducted in major oniongrowing areas of Perambalur district, Tamil Nadu, to assess the incidence of basal rot caused by Fusarium oxysporum f. sp. cepae. The disease incidence varied between 11.25% and 60.15% across the surveyed locations (Table .1, Plate 1). Among the villages, Settikulam recorded the highest basal rot incidence, which could be attributed to the cultivation of a susceptible variety and favourable environmental conditions for pathogen development. In contrast, Nakkasalem village showed the lowest disease incidence.

Findings from the present study are supported by earlier reports [26] , which documented basal rot incidence in major oniongrowing regions of Tamil Nadu, with the highest (18.07%) observed in Pollachi, Coimbatore district. Similarly, in Karnataka, the incidence of Fusarium wilt ranged from 1.71% to 42.77%, with the highest incidence (42.77%) at Annigeri village in Dharwad district [2] . Further surveys in the districts of Tirunelveli, Thoothukudi and Tenkasi also reported basal rot incidence in all locations, with the highest (78.50%) in Vallanadu village, Thoothukudi district [22] . Soil texture was also found to influence the incidence of basal rot. In the present survey, higher disease incidence was observed in red soils compared to black or clay loam soils (Table 1). Similar findings were reported earlier [5] , indicating higher Fusarium wilt populations in sandy soils, followed by loamy sand and loam textures.

Isolation and Cultural Characteristics of Various Isolates of

Fusarium oxysporum f. sp. Cepae

Isolation and Colony Characters

The pathogen Fusarium oxysporum f. sp. cepae was isolated from diseased onion bulb samples on PDA medium. The isolates exhibited considerable variation in colony colour and texture, ranging from normal white to violet, dirty white and pink. Most isolates produced fluffy to moderately fluffy aerial mycelium, except the isolate Foc10, which showed thin, flat mycelium distinct from others (Table 3).

Earlier reports [18] indicated that the pathogen exhibited diverse colony colours such as light pink, dark pink, creamy white and pale white with pink, along with growth patterns like adherent, smooth and fluffy. Similar groupings among isolates of Fusarium oxysporum f. sp. cepae were also observed [22] . Colony colours described as purplish white, whitish orange, creamy white and cottony white were reported in previous studies (2017). Comparable diversity was also noted among F. oxysporum f. sp. cubense [21] and F. oxysporum f. sp. lycopersici [23, 24] . These earlier findings are in agreement with the present observations.

Mycelial Growth

All ten isolates of Fusarium oxysporum f. sp. cepae exhibited moderate, fluffy mycelial growth with dirty white to violet pigmentation on PDA medium. Among the isolates, Foc3 recorded the maximum mycelial growth (90 mm), followed by Foc9, Foc1, Foc5, Foc6, Foc2, Foc7 and Foc8, while Foc10 showed the minimum (78.78 mm) (Table 3). Comparable variations in mycelial growth among F. oxysporum f. sp. cepae isolates have also been reported earlier [22] .

Size of Micro-and Macroconidia

The isolates produced both micro-and macroconidia, ranging from 0.6 to 2.8 × 10⁶ conidia ml⁻¹. Among them (Plate 4 & 5), Foc3 recorded the largest conidial size, with microconidia measuring 10.07 × 4.58 µm and macroconidia 29.25 × 4.78 µm, whereas the smallest conidia were observed in Foc10, with microconidia measuring 5.43 × 3.25 µm and macroconidia 21.63 × 3.14 µm. All isolates produced aseptate microconidia and 1-4 septate macroconidia (Table 3). Earlier studies [2] reported that F. oxysporum f. sp. cepae produces white, cottony mycelium with abundant hyaline, ovoid to ovate microconidia (3.5-8.0 × 2.5-3.5 µm) and sparse 3-5 septate macroconidia (19.5-29.5 × 3-5 µm). Comparable observations on conidial and chlamydospore variability among Fusarium spp. were also documented [27,8,21,23] . Further, microconidia ranging from 4.3-12.5 × 2.4-3.1 µm and macroconidia 15.5-31.5 × 3.0-4.0 µm were recorded in F. chlamydosporum [12] .

Mass Multiplication of Fusarium oxysporum f. sp. cepae Mass multiplication of Fusarium oxysporum f. sp. cepae was carried out using different substrates sand-maize medium, sorghum grains and cumbu grains (Table 4). Among these, maximum fungal growth was recorded in the sand-maize medium, followed by sorghum grains, while the least growth was observed in cumbu grains (Plate 3). Earlier studies [13] reported the use of sand-maize and sorghum grains for the mass multiplication of F. oxysporum f. sp. cepae. Similarly, sandmaize and sorghum media were used for the multiplication of F. oxysporum f. sp. lycopersici [24] . Sorghum grains were also identified as a suitable substrate for inoculum multiplication of F. oxysporum f. sp. ciceris [1] . In addition, pathogen isolates multiplied on wheat and sorghum grains and incorporated into the collar region at 20 g kg⁻¹ soil were found to cause maximum collar rot incidence in tomato [3] .

Conclusion

The present study revealed wide variability in the incidence and morphology of Fusarium oxysporum f.sp. cepae isolates from Perambalur district of Tamil Nadu. Sand-maize medium proved most effective for pathogen multiplication under laboratory conditions. Settikulam recorded the highest disease incidence, indicating the possible influence of environmental and varietal factors on pathogen development. The observed cultural and conidial diversity among the isolates suggests the presence of multiple virulent strains in the region. These findings provide a basis for future screening of resistant onion genotypes and the formulation of integrated management strategies tailored for Perambalur district and similar onion-growing regions.

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