Evaluation of bioagents, plant extracts and fungicides for the management of Alternaria leaf spot of okra caused by Alternaria alternata: An in vitro study

Introduction

Okra (Abelmoschus esculentus L. Moench), belonging to the family Malvaceae, is one of the most important vegetable crops cultivated in tropical and subtropical regions of the world (Naveed et al., 2009; Oyelade et al., 2003) [12,13] . It is valued for its tender green pods, which are rich in vitamins, minerals, dietary fibre and antioxidants and contribute significantly to nutritional security and farmers' income. India is the largest producer of okra in the world, accounting for more than 70 per cent of the global production (Anonymous, 2025). Among the major okra-growing states, Gujarat occupies a leading position, with Surat, Tapi and Navsari districts being the principal okra-producing regions of South Gujarat (Anonymous, 2024-25). Despite its economic importance, okra production is severely constrained by several biotic and abiotic stresses. Among the fungal diseases, Alternaria leaf spot caused by Alternaria alternata (Fr.) Keissler is one of the most destructive foliar diseases of okra. The disease initially appears as small light brown lesions on leaves, which later enlarge into dark brown concentric necrotic spots, leading to premature defoliation, reduction in photosynthetic area and poor fruit yield. Under favourable environmental conditions, the disease may cause yield losses ranging from 30 to 50 per cent or even higher (Hafiz, 1986 [5,7,14,19] . Alternaria alternata is a Ubiquitous fungal pathogen capable of infecting a wide range of cultivated crops and surviving on infected plant debris and seeds. The pathogen proliferates rapidly under warm and humid conditions and produces abundant airborne conidia, which facilitate rapid disease spread. Because of its broad host range, efficient dissemination and adaptability to diverse environmental conditions, management of Alternaria leaf spot remains a major challenge in okra cultivation (Maheshwari et al., 2000; Dube, 2013; Singh, 2015) [11] . Recent studies have also emphasized that A. alternata remains one of the major foliar pathogens of okra and highlighted the need for continuous evaluation of effective management strategies due to its persistent occurrence and economic impact (Choudhary and Panchouli, 2024) [3] . Management of Alternaria leaf spot largely depends on fungicidal application. However, indiscriminate and repeated use of chemical fungicides may lead to the development of fungicide resistance, environmental pollution and residual toxicity. Therefore, the use of biological control agents and plant-derived extracts, either alone or in combination with effective fungicides, has emerged as an environmentally safe and sustainable approach for disease management. Evaluation of these management options under in vitro conditions is an essential prerequisite for identifying effective treatments before their validation under field conditions. Recent investigations have demonstrated that integrating fungicides with biological agents and botanicals provides effective suppression of A. alternata while promoting sustainable disease management in okra (Mahanty and Zacharia, 2024) [10] .

Material and methods

Isolation and pathogenicity

The culture of A. alternata used in the present investigation was isolated from infected okra leaves collected from the RHRS (Regional Horticultural Research Station) field at Navsari Agricultural University, Navsari. For isolation, infected leaf samples were cut along with a small portion of healthy tissue and surface sterilized with 0.1 per cent mercuric chloride or 1 per cent sodium hypochlorite solution for one minute, followed by three washings with sterile distilled water. The sterilized bits were transferred aseptically to Potato Dextrose Agar (PDA) medium under laminar airflow. The inoculated plates were incubated at 25 ± 2 °C for 7-10 days. The isolated fungus was identified as A. alternata based on cultural and morphological characteristics using standard literature. The fungal growth developing around the tissue bits was transferred to PDA slants for sub-culturing. The isolate was purified by the hyphal tip technique, maintained on PDA slants by periodic sub-culturing and stored at 5 °C until further use (Tuite, 1969) [20] . For pathogenicity, a spore suspension of A. alternata (2 × 10⁶ spores ml⁻¹) was prepared from the pure culture and sprayed on 30 day old healthy okra plants grown in earthen pots under glasshouse conditions. Control plants were sprayed with sterile distilled water. The inoculated and uninoculated plants were covered with polyethene bags for 48 h to maintain high humidity. The pathogen was reisolated from the inoculated plants for confirmation of Koch's postulates.

In vitro evaluation of bio-agents by dual culture method

The efficacy of different bio-agents against A. alternata was evaluated by the dual culture technique described by Foldes et al. (2000). The pathogen and test bio-agents were grown separately on PDA medium. A 5 mm diameter mycelial disc from the periphery of a 10 day old culture of the bio-agent was placed at one end of a sterilized Petri plate containing PDA medium and a similar disc of the pathogen was placed on the opposite side of the plate, approximately 70 mm away. Plates inoculated with the pathogen alone served as the control. The inoculated plates were incubated at 25 ± 2 °C until the control plates were completely covered with the mycelial growth of the pathogen. Each treatment was replicated four times. Radial mycelial growth of the pathogen was recorded and per cent growth inhibition over the control was calculated by using the formula suggested by Vincent (1947).

Where, PGI = Per cent growth inhibition C = Average diameter (mm) of mycelial colony in control plate T = Average diameter (mm) of mycelial colony in treated plate

In vitro evaluation of phytoextracts by poisoned food technique

Aqueous leaf extracts of the test phytoextracts were prepared by grinding healthy fresh leaves (50 g) with an equal volume (50 ml) of sterile distilled water using a grinder. The homogenate was filtered through doublelayered muslin cloth and centrifuged at 2500 rpm for 10 min to obtain the crude (100%) extract. The efficacy of different phytoextracts against A. alternata was evaluated by the poisoned food technique. Required quantities of the crude extract were incorporated into sterilized PDA medium to obtain 10 and 20 per cent concentrations, mixed thoroughly and poured into sterilized Petri plates (20 ml per plate). PDA plates without phytoextract served as the control. After solidification of the medium, the plates were inoculated aseptically with a 5 mm diameter mycelial disc taken from the periphery of a seven day old culture of A. alternata and incubated at 25 ± 2 °C. Each treatment was replicated three times. Radial mycelial growth was recorded after seven days of incubation and per cent growth inhibition over the control was calculated by using the formula suggested by Vincent (1947) mentioned above.

In vitro evaluation of fungicides by poisoned food technique

Poisoned food technique (Nene and Thapliyal, 1979) was used to evaluate the efficacy of systemic, non-systemic and ready-mix fungicides against A. alternata. PDA medium was prepared and dispensed at the rate of 100 ml in 250 ml conical flasks and sterilized in an autoclave. Before solidification of the medium, the required quantity of fungicides at their respective concentrations was incorporated aseptically into the sterilized PDA and mixed thoroughly. About 20 ml of poisoned medium was poured into each sterilized Petri plate. PDA plates without fungicide served as the control. After solidification, the plates were inoculated aseptically with a 5 mm diameter mycelial disc cut from the periphery of a seven day old culture of A. alternata and incubated at 25 ± 2 °C. Each treatment was replicated three times. Observations on radial mycelial growth were recorded after seven days of incubation and per cent growth inhibition over the control was calculated by using above formula.

Results and Discussion

In vitro evaluation of bio-agents against A. alternata

Five bio-agents were evaluated against the mycelial growth of A. alternata along with an untreated control by the dual culture technique. The observations on mycelial growth (mm) and per cent growth inhibition (PGI) were recorded seven days after inoculation and are presented in Table 1, Fig. 1 (2017) [6] , who reported Trichoderma spp. to be more effective than bacterial antagonists against A. alternata. Similar observations were reported by Shingne et al. (2020) [18] , who found Trichoderma spp. to be superior over Pseudomonas fluorescens and Bacillus subtilis in suppressing the mycelial growth of A. alternata. Kharbadkar et al. (2022) [9] also reported that T. harzianum exhibited maximum inhibition of the pathogen among the bio-agents tested. Likewise, Sahu et al. (2025) [15] confirmed the superior antagonistic potential of Trichoderma spp. against A. alternata, thereby supporting the results obtained in the present study. 2, Fig. 2 and Photo 2. All the plant extracts tested significantly inhibited the mycelial growth of A. alternata over the untreated control. The maximum mycelial growth (90.00 mm) was recorded in the untreated control. Among the plant extracts, Neem leaf extract proved to be the most effective and recorded the minimum mycelial growth of 35.50 and 22.17 mm with the highest per cent growth inhibition of 60.56 and 75.37 per cent at 10 and 20 per cent concentrations, respectively. The next best treatment was Eucalyptus leaf extract, which recorded mycelial growth of 34.50 and 22.50 mm with 61.67 and 75.00 per cent growth inhibition, respectively, followed by Datura leaf extract with mycelial growth of 33.17 and 29.50 mm and corresponding inhibition of 63.15 and 67.22 per cent at 10 and 20 per cent concentrations. Aak leaf extract also showed appreciable inhibition, whereas Bougainvillea, Tulsi, Karanj, Marigold and Barmasi leaf extracts were comparatively less effective in suppressing the mycelial growth of the pathogen. The present findings are in close agreement with Sharma et al. (2021b) [17] , who reported that garlic extract was the most effective, followed by neem leaf extract, against A. alternata. Similar observations were made by Kharbadkar et al. (2022) [9] , who found neem leaf extract among the most effective botanicals in inhibiting the mycelial growth of A. alternata. Ghule et al. (2023) [4] also reported significant inhibition of A. alternata by different botanicals, while Kansotia et al. (2024) observed that neem leaf extract recorded maximum inhibition of mycelial growth among the phytoextracts tested. These findings support the effectiveness of botanicals, particularly neem and eucalyptus leaf extracts, observed in the present investigation.

In vitro evaluation of fungicides against A. alternata

The experiment was conducted to evaluate the efficacy of different fungicides against Alternaria alternata by the poisoned food technique as described under materials and methods. The fungicides were grouped into systemic, nonsystemic and ready-mix categories and tested at different concentrations based on active ingredient. Systemic fungicides were evaluated at 250, 500 and 1000 ppm, nonsystemic fungicides at 1000, 1500 and 2000 ppm, while ready-mix fungicides were tested at 500, 1000 and 1500 ppm.

Evaluation of systemic fungicides

All the systemic fungicides tested significantly inhibited the mycelial growth of A. alternata over the untreated control. The maximum mycelial growth (90.00 mm) was recorded in the control. Among the fungicides, Propiconazole 25% EC proved to be the most effective by recording the minimum mycelial growth of 20.50, 14.00 and 13.67 mm with the highest per cent growth inhibition of 77.22, 84.44 and 84.25 per cent at 250, 500 and 1000 ppm, respectively. The next effective fungicide was Azoxystrobin 25% SC, which recorded mycelial growth of 30.33, 26.50 and 24.50 mm with 66.30, 70.56 and 86.85 per cent inhibition, respectively, followed by Hexaconazole 5% EC, which recorded 34.00, 29.83 and 24.00 mm mycelial growth with 62.22, 66.85 and 73.33 per cent inhibition at the corresponding concentrations (Table 3, Fig. 3 andPhoto 3). The present findings are in conformity with those of Pansambal et al. (2015) [14] , who reported complete inhibition of Alternaria spp. by Propiconazole and Hexaconazole. Similar results were reported by Kadam et al. (2018) [8] , where Propiconazole exhibited maximum inhibition followed by Hexaconazole against A. alternata. Likewise, Kharbadkar et al. (2022) [9] observed Propiconazole to be the most effective systemic fungicide against A. alternata, while Uniyal et al. (2023) [21] and Choudhary and Panchouli (2024) [3] also reported superior efficacy of Propiconazole under in vitro conditions. More recently, Sahu et al. (2025) [15] confirmed complete inhibition of A. alternata by Propiconazole even at lower concentrations, thereby supporting the present investigation.

Evaluation of non-systemic fungicides

All the non-systemic fungicides significantly reduced the mycelial growth of A. alternata compared with the untreated control. The untreated control recorded maximum mycelial growth (90.00 mm). Among the fungicides tested, Copper oxychloride 50% WP was the most effective and recorded the minimum mycelial growth of 25.50, 19.17 and 14.00 mm with corresponding inhibition of 71.67, 78. 70 [18] , where Copper oxychloride proved highly effective against A. alternata. Kharbadkar et al. (2022) [9] also reported that Copper oxychloride was superior to Chlorothalonil in suppressing mycelial growth of the pathogen, which closely agrees with the present results. Furthermore, Sahu et al. (2025) [15] demonstrated that Copper oxychloride remained one of the effective nonsystemic fungicides against A. alternata under laboratory conditions.

Evaluation of ready-mix fungicides

All the ready-mix fungicides significantly inhibited the mycelial growth of A. alternata over the untreated control. The untreated control recorded maximum mycelial growth (90.00 mm). Among the fungicides, Azoxystrobin 18.2% + Difenoconazole 11.4% SC and Tebuconazole 50% + Trifloxystrobin 25% WG completely inhibited the mycelial growth of the pathogen and recorded cent per cent growth inhibition at all the tested concentrations (500, 1000 and 1500 ppm), proving significantly superior to all other treatments. Metiram 55% + Pyraclostrobin 5% WG ranked next by recording mycelial growth of 22.50, 21.00 and 9.00 mm with corresponding inhibition of 75.00, 76.67 and 90.00 per cent at 500, 1000 and 1500 ppm, respectively (Table 5, Fig. 5 [4] , who reported complete inhibition of A. alternata with Azoxystrobin + Difenoconazole and complete inhibition by Tebuconazole + Trifloxystrobin at higher concentrations. Sharma et al. (2022) [16] also observed cent per cent inhibition of A. alternata with Tebuconazole + Trifloxystrobin at 500 ppm. Likewise, Sahu et al. (2025) [15] reported complete inhibition of the pathogen by Azoxystrobin + Difenoconazole under in vitro conditions, thereby confirming the superiority of these ready-mix fungicides observed in the present study.

Conclusions

In vitro evaluations of biocontrol agents, plant extracts and fungicides revealed the two most effective biocontrol agents, viz., T. harzianum and T. asperellum, recorded minimum mycelial growth and highest per cent growth inhibition (31.25 mm and 65.28%) and (36.62 mm and 59.31%), respectively, against A. alternata under in vitro conditions. Among the plant extracts, Neem leaf extract and Eucalyptus leaf extract at 20 per cent concentration were found most effective with minimum mycelial growth and highest per cent growth inhibition (22.17 mm and 75.37%) and (22.50 mm and 75.00%), respectively. Among the ready-mix fungicides, Azoxystrobin 18.2% + Difenoconazole 11.4% SC and Tebuconazole 50% + Trifloxystrobin 25% WG recorded cent per cent mycelial growth inhibition against A. alternata at 500, 1000 and 1500 ppm concentrations under in vitro conditions.

Acknowledgement

AcknowledgementsThe authors are grateful to the Navsari Agricultural University, Navsari for providing all the resources in this research work.

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