Bio-efficacy of phytoextracts, bioagents and fungicides against Alternaria porri inciting purple blotch disease in white onion

Introduction

Onion (Allium cepa L.) indeed holds significant importance globally, not just as a culinary staple but also for its numerous health benefits. As a member of the Alliaceae family, it has been cultivated for centuries and is widely recognized for its medicinal properties. One of the key components responsible for the characteristic pungency of onion bulbs is Allyl-propyldi-sulphide which is not only responsible for the onion's flavour but also contributes for its medicinal properties. Allyl-propyl-di-sulphide possesses a different beneficial effect including anti-cancerous antibacterial, antifungal, anti-helminthic, anti-inflammatory, antiseptic and antispasmodic properties (Augusti, 1996) [4] . The onion contains a variety of bioactive compounds, such as organosulfur compounds, flavanols, ascorbic acids and carbohydrate prebiotics. Additionally, its by-products contain a higher concentration of flavonoids compared to the bulb. The major organosulfur compounds found in onions are diallyl monosulfide, diallyl disulfide, diallyl trisulfide and diallyl tetrasulfide, while quercetin, kaempferol, anthocyanin and luteolin are considered as the main flavonoids. Ascorbic acid and fructo-oligosaccharides are also recognized as bioactive compounds. These bioactive compounds in onions have strong antioxidant potential for neutralizing the oxidative stress of cells. Onions offer not just versatility in cooking but also important health-promoting compounds, adding to their nutritional and medicinal value (Kabrah et al., 2016) [13] . Table 1.1. lists the nutritional value of white onions according to the Food Data Central database, USDA (United States Department of Agriculture). [2] .

Materials and Methods

The present study was conducted at the Department of Plant Pathology, College of Agriculture, Dapoli during year 2023-24 and 2024-2025. [17] and by using PDA as basal medium. For obtaining 10% plant extract, 10 ml of standard plant extract was mixed with 90 ml of PDA in 250 ml conical flask. Later, 20 ml of the PDA medium containing plant extracts was poured into sterilized Petri plates under aseptic conditions. Mycelial discs of 5 mm size from seven days old actively grown pure culture were used to inoculate the poured Petri plates. Control was maintained on PDA without adding any plant extract in a medium. Three replications were taken for each treatment. These plates were incubated at 27±2 0 C for seven days and radial colony growth was measured. Observations on radial mycelial growth was recorded in all of the replicated treatments after control (untreated) plates were fully covered with mycelium growth of the test pathogen. The cumulative data was averaged and Per cent inhibition of the growth of the test pathogen, over untreated control was calculated by applying formula given by Vincent, 1947 [23] .

Experimental details

Where, I = Per cent Inhibition C = Growth (mm) of test fungus in control plate. T = Growth (mm) of test fungus in treated plates.

In vitro efficacy of bioagents

Eight highly promising bioagents (fungal and bacterial) were assessed for their efficacy against Alternaria porri using the 'Dual Culture Technique' (Dennis and Webster, 1971) [9] and PDA as the base culture medium. The test bioagents and the pathogen were grown on their respective culture media for seven days before being used in the study. A 5 mm culture disc of both test pathogen and the fungal bioagent (cut out with a sterilized cork borer) were inoculated with positioned at equal distances and directly opposite each other on autoclaved and solidified PDA medium in Petri plates. For bacterial bioagents, a 5 mm culture disc of the test pathogen was placed along the periphery of the PDA plate and opposite to it, a pure culture suspension of the test bacterial bioagent was streaked using a sterilized inoculation needle. Three PDA plates were inoculated for each test bioagent and all treatments were replicated three times. Untreated control was maintained with PDA plates only inoculated with the pure culture disc of the test pathogen. The experimental details were as follows. Observations on mycelial growth of the test fungus and bioagents was recorded after 7 days. Per cent inhibition of test fungus over untreated control was calculated by using formula given by Vincent (1947) [23] .

Experimental details

Where, I = Per cent Inhibition C = Growth (mm) of test fungus in control plate. T = Growth (mm) of test fungus in intersecting plate.

In vitro efficacy of fungicides

Efficacy of seven systemic and eight contact and combi fungicides were evaluated for their efficacy in vitro against the Alternaria porri, using PDA as basal culture medium and applying Poisoned food technique (Nene and Thapliyal, 1993) [17] .

The required quantity of each test fungicide was calculated based on the active ingredient. After calculation, the fungicides were dispensed and thoroughly mixed with sterilized and cooled PDA medium in separate glass conical flasks with a capacity of 250 ml. This was done in order to achieve the desired concentrations of the fungicide-amended PDA medium. The PDA medium amended with each test fungicide was then aseptically poured into sterilized Petri plates @ 20 ml per plate and allowed to solidify at room temperature. Three PDA plates per treatment were maintained and each treatment was replicated thrice for each test fungicide and its concentration. On solidification of poisoned medium each plate was inoculated with 5 mm culture disc of test pathogen centrally. The culture disc was obtained from a 7-day-old pure culture of the test pathogen isolate. PDA plates without fungicide, inoculated with the pure culture disc of the test pathogen, were maintained as untreated control. Both treated and untreated control plates were then incubated in an inverted position in a BOD incubator at 27 ± 2 o C. The study was conducted for systemic and contact plus combi fungicides, separately. The experimental details were as provided below.

Experimental details

• Design: Completely Randomized Design (CRD)

• Replications: Three Treatments details Observations on radial mycelial growth was recorded in all of the replicated treatments after control (untreated) plates were fully covered with mycelium growth of the test pathogen. The cumulative data was averaged and per cent inhibition of the growth of the test pathogen, over untreated control was calculated by applying formula given by Vincent, 1947 [23] .

Where, I = Per cent Inhibition C = Growth (mm) of test fungus in control plate. T = Growth (mm) of test fungus in treated plates.

Results and Discussions 3.1. In vitro efficacy of phytoextracts against Alternaria porri

Using the poisoned food technique and PDA as a basic culture medium, the present investigation assessed the in vitro efficacy of aqueous extracts of eight plant species (each at a 10% concentration) against Alternaria porri, which causes purple blotch disease in onion. Table 3.1, Plate I and Fig. 1 present the outcomes of the study, which are described below.

The results (Table 3.1, Plate I and Fig. 1) indicated that all the plant extracts evaluated each at 10% concentration exhibited antifungal activity against Alternaria porri. The mycelial growth of test fungus was varied from 21.83 to 39.50 mm. Whereas, inhibition of mycelial growth of test fungus was ranged from 56.10 to 75.73 per cent. Among the eight plant extracts evaluated, soapnut rind extract (10 %) outperformed all treatments, with the least colony growth of 21.83 mm and the highest suppression of mycelial growth (75.73%) of A. porri. The second most effective treatment was ginger rhizome extract (10%) which showed 24.00 mm mycelial growth and 73.33 per cent mycelial growth inhibition. Tulsi leaf extract @ 10% was likewise found effective, with a mycelial growth of 28.83 mm and the inhibition of mycelial growth of 67.95 %. (2012) [7] studied in vitro efficacy of various phytoextracts against purple blotch disease of onion and found that garlic extract was significantly superior over neem oil, pongamia oil, cinnamon, turmeric and clerodendron in inhibiting the mycelial growth. Garlic extract caused significantly maximum inhibition (100%) at 20 per cent concentration followed by 88.50 per cent, 52.50 per cent and 42.33 per cent inhibition at 15 per cent, 10 per cent and 5 per cent concentrations, respectively. Maharana et al. (2016) [15] studied in vitro efficacy of eleven phytoextracts against A. porri causing purple blotch disease of onion and found that at 10 % concentration, maximum inhibition was found in neem (82.60%) followed by tulsi (78.40%) and at a concentration of 20%, the extracts of neem, tulsi, eucalyptus, garlic and ginger were the most effective in inhibiting the growth of mycelium, with mean inhibition rates of 90.40%, 86.80%, 79.50%, 78.00% and 75.20%, respectively. Similarly, Jhala et al. (2017a) studied in vitro efficacy of four phytoextracts against A. porri causing purple blotch disease in onion at 2 and 4 per cent concentrations and found that neem leaf extract caused maximum inhibition of linear growth (62.09% and 68.61%) of A. porri followed by garlic extract (53.13 and 57.42%). Bhandekar et al. (2019) [6] reported that tulsi (Ocimum tenuiflorum) leaf extract and neem leaf extract (10%) was most effective in arresting maximum mycelial growth of A. porri to the tune of 60.83 and 58.62%, respectively. Also, Arunakumara et al. (2023) [3] studied in vitro efficacy of various phytoextracts at 15% concentration against A. porri causing purple blotch disease of garlic and found that garlic (Allium sativum) cloves extract was most effective in inhibiting the mycelial growth (64.64%) followed by Clerodendron inerme (57.10%), Aloe vera (55.14%), Eucalyptus globes (48.30%) and least inhibition was recorded in Gliricidia maculata (26.04%). Dibisa et al.

(2023) [10] also reported highest per cent inhibition of mycelium growth of A. porri due to garlic clove extract (76.34%) followed by Aloe vera leaf extract (72.67%) and Ginger (66.48%).

In vitro efficacy of bio-agents against Alternaria porri

The efficacy of eight distinct bioagents against Alternaria porri were assessed in vitro using the standard "Dual culture technique." Table 3.2, Plate II and Fig. 2 display the findings on A. porri mycelial development and inhibition. The results illustrated in Table 3.2, Plate II and Fig. 2 stated that all the bio-agents evaluated were significantly effective in inhibiting the mycelial growth of A. porri. Pseudomonas fluorescens was shown to have the greatest (78.88%) mycelial growth inhibition potential of A. porri among the eight bioagents evaluated. However, Trichoderma longibrachiatum was also found equally effective with least colony growth of test pathogen (20.16 mm) with 77.58 per cent inhibition of mycelial growth and was at par with P. fluorescens. It was followed by T. viride (22.16 (2012) [7] reported that among all the antagonists evaluated Trichoderma harzianum was found most effective against A. porri inciting purple blotch disease of onion which recorded 79.35 per cent inhibition of mycelial growth. Similarly, Mishra and Gupta (2012) [16] reported that Trichoderma viride (55.95%) was most effective in inhibiting the mycelial growth of the test pathogen over control followed by T. harzianum (53.17%) and Trichoderma koningii (46.65%). Similarly, Rahman et al. (2015) [19] found maximum inhibition of colony growth of A. porri incitant of purple blotch disease of onion due to T. viride which was statistically significant over all the other bioagents tested. Maharana et al. (2016) [15] reported that among the bio control agents evaluated against A. porri, the higher percentage of growth inhibition was recorded in T. harzianum (88%) followed by T. viride (83%). Kapgate et al. (2019) [14] resulted that T. viride showed maximum mycelial suppression (76.55 per cent) of A. porri followed by T. harzianum (70.74 per cent), Pseudomonas fluorescence (56.27 per cent) and Bacillus subtilis (58.53 per cent). Bhandekar et al. (2019) [6] also resulted that T. viride recorded maximum mycelial suppression (85.45%) of A. porri inciting purple blotch disease of onion followed by B. subtilis (60.34%) and P. fluorescence (57.75%), respectively. Hariprasad et al. (2021) [11] recorded that maximum mycelial inhibition of (94.50%) of A. porri inciting purple blotch disease of onion in T. viride followed by T. harzianum with mycelial inhibition per cent (90.58). The results of present study also in close conformity with results of Vijaykumar et al. (2022b) [22] who found that maximum mycelial growth inhibition (83.89%) of A. porri causing purple blotch disease of garlic was due to with T. harzianum followed by P. fluorescens (63.40 %) and T. viride (61.20 %). The least mycelial growth inhibition of 38.97 per cent was recorded with B. subtilis.

Plate II: In vitro efficacy of bio-agents against Alternaria porri

In vitro efficacy of fungicides against Alternaria porri 3.3.1. In vitro efficacy of systemic fungicides against

Alternaria porri

Using the poisoned food technique and PDA as a basic culture medium, the present investigation assessed the in vitro efficacy of different systemic fungicides against Alternaria porri, incitant of purple blotch disease in white onion. 3 illustrated that all the systemic fungicides evaluated were significantly effective in reducing mycelial growth of A. porri. Among the systemic fungicides, Tebuconazole 25.9% EC, Hexaconazole 5% EC and Propiconazole 25% EC (each @ 0.1% concentration) were found most significantly effective in inhibiting the mycelial growth of A. porri with 100 per cent inhibition over control and was closely followed by Difenoconazole 25% EC (0.1%) which resulted in minimum mycelial growth of 7.66 mm with inhibition of 91.47 %. Carbendazim 50% WP (0.1%) (17. In vitro efficacy of contact and combi fungicides against Alternaria porri A total of eight contact and combi fungicides at different concentrations were studied in vitro for their efficacy against Alternaria porri by using poisoned food technique and PDA as a basic culture medium. Table 3.4 presents the outcomes of the study, which are described below. 4 illustrated that all the contact and combi fungicides tested were significantly effective in reducing mycelial growth of A. porri. Among the contact and combi fungicides, Tebuconazole 50% + Trifloxystrobin 25% WG (0.05%) and Copper oxychloride 50% WP (0.25%) were found most significantly effective in inhibiting the mycelial growth of Alternaria porri with 100 per cent inhibition over control. Among rest of the fungicides, Cymoxanil 8% + Mancozeb 64% WP (0.2%) resulted with minimum mycelial growth of 10.50 mm with inhibition of 88.32 % followed by Captan 50% WP (0.2%) (12.50 The current investigation results are in accordance with those of several previous researchers. According to Rohan et al. (2018) [20] who studied in vitro efficacy of different fungicides at 0.05%, 0.1% and 0.15% concentration against A. porri and found that Tebuconazole 50% + Trifloxystrobin 25% WG at 0.15% revealed 100 per cent inhibition of the mycelial growth of A. porri. Propiconazole 25% EC @ 0.15% (94.44 %) was next best fungicide which was at par with Tebuconazole 25.9% EC @ 0.15% (92.59%) followed by Hexaconazole 5% EC @ 0.15% (91.85%). Aujila et al. (2013) [5] reported that complete mycelial growth inhibition of the A. porri causing purple blotch disease of onion was observed due to Difenoconazole 25% EC and Tebuconazole 50% + Trifloxystrobin 25% WG at 0.1 per cent. Agale et al. (2014) [1] studied in vitro efficacy of various different fungicides against purple blotch disease of onion incited by A. porri and resulted that Propiconazole 25% EC (0.1 %) completely inhibited the mycelial growth of A. porri. Difenoconazole 25% EC (0.1 %), Tebuconazole 25.9% EC (0.1 %) and combi fungicide Carbendazim 12% + Mancozeb 63% WP (0.2 %), showed 88.00, 85.77 and 84.66 per cent inhibition, respectively. Wanggikar et al. (2014) [24] reported that cent per cent (100.00 %) inhibition of mycelial growth of A. porri inciting purple blotch disease of onion was observed in plate with Hexaconazole 5% EC followed by Difenoconazole 25% EC (83.91%), which was followed by Mancozeb 75% WP (63.58%). Priya et al. (2015) [18] studied in vitro efficacy of different fungicides against A. porri and found that maximum inhibition of mycelial growth of pathogen due to Propiconazole 25% EC (92.5%) followed by Hexaconazole 5% EC (91.1%), Difenoconazole 25% EC (86.36%) and Tebuconazole 25.9% EC (80.30%). Yadav et al. (2017) [25] also reported that Tebuconazole 25.9% EC, Trifloxystrobin 25% + Tebuconazole 50% WG and Propiconazole 25% EC proved the most effective among the tested fungicides as they completely (100%) inhibited the mycelial growth of test pathogen. Dangi (2018) also reported that Tebuconazole 25.9% EC, Hexaconazole 5% EC and Propiconazole 25% EC provided complete mycelial growth inhibition of A. porri at 1000 ppm concentration level. Swain et al. (2021) [21] reported that Propiconazole 25% EC (0.2%), Tebuconazole 25.9% EC (0.15%) and Hexaconazole 5% SC (0.2%) recorded complete (100%) mycelial growth inhibition of the A. porri inciting purple blotch disease of onion. [10] .

Conclusion

Soapnut rind extract was most effective plant extract with highest inhibition (75.73%) of mycelial growth of A. porri in vitro. Among the different bioagents evaluated in vitro Pseudomonas fluorescens and Trichoderma longibrachiatum were found most effective in inhibiting the mycelial growth of A. porri with 78.88 and 77.58% inhibition, respectively. Among the different systemic fungicides evaluated, Tebuconazole 25.9% EC, Hexaconazole 5% EC and Propiconazole 25% EC (each @ 0.1% conc.) and contact and combi fungicides viz., Tebuconazole 50% + Trifloxystrobin 25% WG @ 0.05% and Copper oxychloride 50% WP @ 0.25% were the most significantly effective and caused complete inhibition of A. porri.

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