In-vitro efficacy of systemic, non-systemic and combi-fungicides against Alternaria porri causing purple blotch of onion

Introduction

Onion (Allium cepa L.) is one of the most widely cultivated and consumed vegetable crops globally, occupying an important position in Indian agriculture for its culinary, nutritional, and economic value. India is the second-largest onion producer in the world, with an annual output of approximately 26.74 million metric tonnes from 1.42 million hectares during 2022-23 (NHB, 2023) [12] . Maharashtra is the leading producer, contributing about 33-35% of the national yield, with major cultivation in Nashik, Pune, Ahmednagar, and Jalgaon districts. Despite its significance, onion production is seriously hampered by fungal diseases, among which purple blotch, caused by Alternaria porri (Ellis) Cif., is one of the most destructive foliar pathogens. Depending on environmental conditions, cultivar susceptibility, and crop stage, yield losses may range from 20% to 70% (Mishra et al., 2014; Pandey et al., 2019) [8,13] . In Maharashtra, outbreaks have become more frequent during the post-monsoon and Rabi seasons, when high humidity and moderate temperatures (25-30 °C) favour disease development.

The disease manifests initially as small, water-soaked, whitish sunken lesions on older leaves, which enlarge into purplish-brown concentric rings with yellow halos. These lesions lead to early leaf senescence, reduced bulb size, and lower marketable yield. Windborne spores contribute to rapid secondary spread, especially in dense canopies and under poor crop rotation practices. Fungicide application remains the primary control strategy, but indiscriminate use has resulted in resistance development, phytotoxicity, and environmental concerns (Prajapati et al., 2021) [14] . To mitigate these issues, recent research has focused on the rational use of systemic and protective fungicides, along with combi-formulations that can minimise resistance risks and expand control spectra. Triazole-based fungicides such as tebuconazole, hexaconazole, and propiconazole have been shown to effectively suppress A. porri growth under both in vitro and field conditions (Mohan et al., 2004; Yadav et al., 2017) [9,18] . Protective fungicides like mancozeb and copper hydroxide remain integral in integrated disease management programmes, while combi-fungicides are increasingly valued for their dual mode of action and high performance under severe disease pressure (Ravichandran, 2012) [16] . Considering this background, the present study was undertaken to assess the in vitro efficacy of selected systemic, non-systemic, and combi-fungicides against the most virulent isolate of A. porri (ChAp1) collected from infected onion fields in Maharashtra, with the aim of identifying fungicides that can be integrated into sustainable disease management programmes.

Materials and Methods

The present study on purple blotch of onion, caused by Alternaria porri (Ellis) Cif., was conducted in vitro at the Department of Plant Pathology, VNMKV, Parbhani, Maharashtra.

Isolation and Identification of the Pathogen

The most virulent isolate of A. porri (designated ChAp1) was obtained from naturally infected onion leaves showing typical purple blotch symptoms. Infected leaf tissues were surface sterilised with 0.1% sodium hypochlorite for 30 seconds, rinsed thoroughly in sterile distilled water, and placed on potato dextrose agar (PDA) medium. Plates were incubated at 25±2 °C for seven days. Fungal colonies were sub-cultured to obtain pure cultures. Identification was confirmed based on morphological characteristics such as conidial shape, beak length, and septation, following the descriptions of Ellis (1971) [3] .

Preparation of Culture Medium

PDA was prepared by boiling 200 g of peeled potatoes in 1 L of water for 30 minutes, filtering the extract, and adding 20 g dextrose and 15 g agar. The medium was autoclaved at 121 °C for 20 minutes and poured into sterile Petri plates under laminar airflow.

In Vitro Evaluation using the Poisoned Food Technique

The poisoned food technique described by Nene and Thapliyal (1993) [11] was used to evaluate fungicide efficacy. The required quantity of each fungicide was incorporated into molten PDA (cooled to approximately 45 °C) to achieve the desired concentration before pouring into sterile Petri plates. Once solidified, a 5 mm diameter mycelial disc from the actively growing margin of a seven-day-old A. porri culture was placed in the centre of each plate. Each treatment was replicated three times, and an untreated control was maintained for comparison. Plates were incubated at 25±2 °C for seven days, after which radial mycelial growth was recorded in millimetres. The percentage inhibition of mycelial growth was calculated using the formula given by Vincent (1927) [21] :

Where C is the mycelial growth in the control (mm) and T is the mycelial growth in the treatment (mm).

Evaluation of Systemic Fungicides

The efficacy of systemic fungicides against A. porri (ChAp1) was assessed at concentrations of 500 and 1000 ppm using PDA as the basal medium. The experiment was laid out in a Completely Randomised Design (CRD) with three replications and eight treatments:

Evaluation of Non-Systemic Fungicides

Non-systemic/contact fungicides were evaluated at 2000 and 2500 ppm in vitro using the same poisoned food technique and PDA medium. The experiment followed a CRD with three replications and eight treatments:

Evaluation of Combi-Fungicides

The efficacy of combi-fungicides was tested at 2000 and 2500 ppm in vitro using PDA as the basal medium and the poisoned food technique. The experiment was conducted in CRD with three replications and eight treatments: Observations on radial mycelial growth and sporulation were recorded at 24-hour intervals until the growth in the control plates completely covered the surface.

Results and Discussion

(Table 1) revealed that among the systemic fungicides tested (each @ 500, 1000 ppm) were found effective against the A.porri pathogen. The mycelial growth recorded with the systemic fungicides ranged from 00.00 mm to 33.83 mm @ 500 ppm and from 00.00 mm to 26.83 mm @ 1000 ppm. Whereas, mycelial growth inhibition with the systemic fungicides ranged from 62.40 to 100 percent and 70.18 to 100 percent, respectively @ 500 and 1000 ppm. At 500 ppm concentration of systemic fungicides, there was no growth of Alternaria porri was observed in the treatments of propiconazole, Hexaconazole, Difeneconazole and Tebuconazole (00.00 mm). The next best treatments followed by these were pyraclostrobin (8.96 mm), Thiophanate methyl (24.83 mm). Amongst all fungicides tested, Azoxystrobin was found comparatively less effective with having maximum growth of A.porri i.e. (33.83 mm).

Among the fungicides tested at 500 ppm, cent percent inhibition of the test pathogen was observed in the treatment of propiconazole, Hexaconazole, Difeneconazole and Tebuconazole, are significantly superior over other treatments including control and these are statistically at par with each other, which was followed by Pyraclostrobin (90.03%) and Thiophanat methyl (72.40%). Among the, Azoxystrobin was found comparatively less effective with minimum mycelial inhibition of pathogen i.e. 62.40 percent. At 1000 ppm concentration of systemic fungicides, there was total inhibition of Alternaria porri was observed in the treatments of propiconazole, Hexaconazole, Difeneconazole and Tebuconazole (00.00 mm) followed by this were pyraclostrobin (8.16 mm), Thiophanate methyl (14.83 mm). Among the all fungicides tested, Azoxystrobin was found comparatively less effective with having maximum growth of A.porri i.e. (26.83 mm.) Among the fungicides tested at 1000 ppm, cent percent inhibition of the test pathogen was observed in the treatment of propiconazole, Hexaconazole, Difeneconazole and Tebuconazole, are significantly superior over other treatments including control and these are statistically at par with each other, which were followed by Pyraclostrobin (90.92%) and Thiophanat methyl (83.51%). Among all systemic fungicides, Azoxystrobin was found comparatively less effective with minimum mycelial inhibition of pathogen i.e. 70.18 percent. The observationss of the present studies are found similar with the reports of earlier researchers where Mohan et al., (2004) [9] studied the efficacy in-vitro of nine fungicides against stemphylium blight (S.botryosum) of onion. The fungicides Tebuconazole (Folicur 250 EW) @ 0.05%, Difenconazole (Score 25 EC) @ 0.05%, Hexaconazole (Contaf 5 EC) @ 0.05%, Propineb (Anthracol 70 WP) @ 0.25%, Iprobenphos (Kitazin 48EC) @ 0.10%, Chlorothalonil (Kavach 75WP) @ 0.30%, Tricyclazole (Sivic 75WP) @ 0.10%, Carbendazim (Bavistin 50 WP) @ 0.10% and Mancozeb (Indofil M-45 WP) @ 0.30%.were evaluated highest growth inhibition of the test fungus was caused with the fungicides, Tebuconazole followed by Difenconazole, Hexaconazole, Carbendazim, Iprobenphos. In vitro evaluation of Non-systemic fungicides against Alternaria porri: Results (Table 2 Plate 2 and Fig 2 ) revealed that among the seven nonsystemic fungicides tested (each @ 2000 and 2500 ppm) were found effective against the A.porri pathogen. The mycelial growth with the non-syestemic fungicides ranged from 6.16 mm to 27.16 mm @ 2000 ppm and 5.33 mm to 20.16 mm @ 2500 ppm. Whereas, mycelial growth inhibition with the non-syestemic fungicides ranged from 69.81 to 100 percent and 77.59 to 100 percent @ 2000 and 2500 ppm respectively. At 2000 ppm concentration of non-systemic fungicides, the least growth of Alternaria porri was observed in the treatment of Copper hydroxide (6.16 mm). The next best treatment followed by this was Mancozeb (8.16 mm) which was followed by Dinocap (9.83 mm), Propineb (13.00 mm), Copper oxychloride (14.83 mm), Chlorothalonil (22.16 mm).Amongst all the treatments Thiram was found comparatively less effective with maximum mycelial growth of Alternaria porri (i.e. 27.16 mm). Among all the fungicides tested at 2000 ppm concentration Copper hydroxide, Mancozeb and Dinocap were significantly superior over rest of treatments including control with highest mycelial growth inhibition of 93.14%, 90.92% and 89.07% respectively. These were followed by Propineb (85.55%), Copper oxychloride (83.51%), Chlorothalonil (75.37%). Among these Thiram was found less effective with 69.81 percent inhibition of A.porri pathogen. At 2500 ppm concentration of non-systemic fungicides, the least growth of Alternaria porri was observed in the treatment of Copper hydroxide (5.33 mm) and Mancozeb (6.16mm) which were found significantly superior over all the treatments followed by Dinocap (8.33 mm), Propineb (10.66 mm), Copper oxychloride (11.66 mm), Chlorothalonil (18.16 mm). Among the treatments Thiram was found comparatively less effective with maximum mycelial growth of Alternaria porri (i.e. 20.16 mm) .Among all non-syetemic fungicides tested at 2500 ppm concentration Copper hydroxide, Mancozeb and Dinocap were significantly superior over rest of treatments including control with highest mycelial growth inhibition of 94.06%, 93.14% and 90.23% respectively. These were followed by Propineb (87.03%), Copper oxychloride (88.14%), Chlorothalonil (79.81%). Among these Thiram was found less effective with 77. In vitro evaluation of combi-fungicides against Alternaria porri: Results (Table 3 Plate 3 and Fig 3) revealed that among the combifungicides tested (each @ 2000 and 2500 ppm), were found effective against the test pathogen. The mycelial growth with the combi-fungicides ranged from 0.00 mm to 12.16 mm @ 2000 ppm and from 0.00 mm to 10.33 mm @ 2500 ppm. Whereas, mycelial growth inhibition with the combi-fungicides ranged from 86.48 to 100 percent and 88.51 to 100 percent @ 2000 and 2500 ppm respectively. At 2000 ppm concentration combi-fungicides, the least growth of Alternaria porri was observed in the treatment of Captan 70% + Hexaconazole 5% (0.00 mm) is significantly superior over rest of the treatments including control. The next best treatment followed by this was Trifloxystrobin 25% + Tebuconazole 50% (6.66 mm) which was followed by Hexaconazole 4% + Zineb 68% (7.83 mm), Thiophanatemethyl 45% + Pyraclostrobin 5% (9.16 mm), Azoxystrobin 20% + Difenoconazole 12.5% (9.33 mm), Metalaxyl 8% + Mancozeb 64% (10.00 mm). Among the treatments Metiram 50% + Pyraclostrobin 5% was found comparatively less effective with maximum mycelial growth of Alternaria porri i.e. 12.16 mm. Among all the fungicides tested at 2000 ppm concentration Captan 70% + Hexaconazole 5%was significantly superior over rest of treatments including control with highest mycelial growth inhibition of 100%. [16] studied the invitro efficacy of twelve fungicides against A. porri Among the tested fungicides, Mancozeb, (Mancozeb 12% + Marbendazim 63% WP) and (Zineb 68% + Hexaconazole 4% WP) at 0.1, 0.15 and 0.20 percent recorded 100 percent mycelial inhibition.

While, Hexaconazole at 0.05 and 0.10 percent and Propiconazole, Tebuconazole at 0.10 percent recorded 100 percent inhibition in the mycelial growth. Yadav et al., (2017) [18] studied efficacy of six systemic fungicides and two non-systemic fungicides against purple blotch of onion. The study revealed that, Tebuconazole at 0.025, 0.50, 0.10 and 0.20 percent followed by Difenconazole and (Tebuconazole 50% + Trifloxystrobin 25% WP) at (0.10 and 0.20%) and Propiconazole at (0.20%) recorded 100 percent inhibition in the mycelial growth of the test pathogen.

Conclusion

The present study clearly demonstrates the effectiveness of various systemic, non-systemic, and combi-fungicides against the most virulent isolate of Alternaria porri (ChAp1), the causal organism of purple blotch in onion. Among the systemic fungicides, Propiconazole, Hexaconazole, Difenoconazole, and Tebuconazole provided complete inhibition of fungal growth at both 500 and 1000 ppm concentrations, indicating their high efficacy. In the category of non-systemic fungicides, Copper hydroxide and Mancozeb were found significantly effective, achieving over 93% inhibition at ppm. Among the combiformulations, Captan + Hexaconazole achieved 100% inhibition at both concentrations and was found superior over other treatments. These findings suggest that certain triazole-based systemic fungicides and copper-based non-systemic and combifungicides are highly effective in suppressing A. porri under in vitro conditions. The use of these fungicides, either alone or in rotation, may serve as a key component of integrated disease management (IDM) strategies for controlling purple blotch in onions. However, further in vivo validation under field conditions is recommended to confirm their efficacy and economic feasibility before recommending them for large-scale use.

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