Efficacy of various organic inputs against seed mycoflora of okra cultivar

Introduction

Okra (Abelmoschus esculentus L. Moench) is the most commonly grown vegetable crops across the different zones of world. It belongs to family Malvaceae. Okra is usually consumed for its green tender fruits as a vegetable in different ways. The mature okra seeds are the good source of oil and protein has been known to have superior nutritional quality. Its mature fruit and stems contain crude fiber, which is used in the paper industry. Okra bast fibre possess an excellent quantity of cellulose, which can be used in cellulose-based industries (Kumar et al., 2013) [7] . In paper industries, the stem of okra plants is used for fibre purposes (Qayyum, 1990; Mithal, 2006). For the clarification of sugarcane juice from which gur or brown sugar is prepared, the roots and stems of okra plants are used (Chauhan, 1972) [5] . Medicinal value of okra has also been reported in curing ulcers and relief from hemorrhoids (Adams, 1975) [2] . Raw fruits and seeds are also rich in mucilage, serves a good source of antioxidants. The seeds of okra when roasted and grounded can be used as coffee additive or as its substitute (Moekchantuk and Kumar, 2004) [9] . Thus, it is indeed an economically important crop. Fungal pathogens severely threaten okra cultivation worldwide, causing heavy crop losses regardless of regional weather conditions. Seeds of okra affected by various fungi including; Alternaria alternata, Aspergillus niger, Curvularia lunata, Fusarium oxysporum, F. solani, F. moniliforme, Macrophomina phaseolina, Rhizoctonia solani, Stemphylium botryosum, Penicillium digitatum, Pythium aphanidermatum (Al-Kassim and Monawar, 2000; Odofin, 2010) [4,11] . Although chemical fungicides successfully target these pathogens, they leave behind hazardous residues that damage human health and ecosystems. To make matters worse, these fungi are evolving into new, resistant strains that make traditional chemical sprays increasingly useless. This highlights the critical need for alternative control methods. To succeed, these new solutions must be affordable, consistent, biodegradable, and biologically derived. Implementing organic approaches such as priming seeds, applying angiosperm leaf extracts, and using cow-based products offers a practical and eco-friendly way forward. It is an ecological approach using either bacteria or selected fungal antagonists against the soil and seed-borne pathogens (Rai and Basu, 2014) [13] . Nwangburuka et al. (2012) [10] studied the effects of moringa leaf extracts on seed germination, seedling growth rate and fungal abundance in okra. Abdulhassan (2020) [1] evaluated the efficacy of some plant extracts (Cuminum cymanum, Zinziber oficinale and Citrullus colocynthis) against seed rotting and seedling death causing fungus Macrophomina phaseolina and found that the alcoholic extract of Cuminum cyminum had a significant effect on the inhibition of the radial growth and dry weight of M. phaseolina followed by Zingiber officinale and Citrullus. Hence, this research was conducted for evaluating the efficacy of various organic inputs against seed mycoflora of okra cultivar.

Materials and Methods

Experiment details

An in vitro experiment conducted for evaluation of various organic inputs viz, Beejamrut, cow urine and phytoextracts (moringa and neem leaf extract) for the management of seed mycoflora of okra cultivar i.e., Gujarat Anand Okra-5 (GAO-5) by using agar plate method at Department of Plant Pathology, B. A. College of Agriculture, Anand Agricultural University, Anand, Gujarat, India.

Methodology

The seeds of okra cultivar (GAO-5) were pre-soaked for 30 minutes with the various organic inputs of different concentrations (3, 5 and 10%) into four repetitions, each of 100 seeds (As per the treatment details of Table 1). Presoaked seeds were placed on the agar plates and the growth of various fungi was recorded accordingly.

Preparation of beejamrut

Beejamrut was prepared by suspending 5 kg of cow dung (collected from Livestock Research Station, AAU, Anand) in 20 L of water for 12 hours. The cloth was then squeezed to release all soluble ingredients into the suspension, which was subsequently mixed with a separate solution of 50 g of lime in 1 L of water, 5 L of cow urine, and a handful of virgin soil. After incubating for 8-12 hours, the mixture was filtered. The resulting filtrate was diluted with water to formulate 3%, 5%, and 10% concentrations, which were used for a 30-minute pre-soaking seed treatment.

Preparation of phytoextracts

Fresh neem and moringa leaves were washed with distilled water, surface-sterilized with 1% sodium hypochlorite, and homogenized in a mixer grinder. The crude extracts were filtered through a single layer of sterile muslin cloth to serve as 100% stock solutions. These were diluted with water to obtain 3%, 5%, and 10% concentrations, which were then used as a 30-minute pre-soaking seed treatment.

Collection of Cow urine

Cow urine was obtained from the Livestock Research Station, AAU, Anand, and utilized as the 100% standard stock solution. This stock was subsequently diluted with the requisite volume of water to prepare the target treatment concentrations of 3%, 5%, and 10%. The resulting formulations were used to pre-soak the seeds for a duration of 30 minutes. Beejamrut @ 3% 500 ml T2

Neem leaf extact @ 3% 500 ml T8 Neem leaf extact @ 5% 500 ml T9 Neem leaf extact @ 10% 500 ml T10 Moringa leaf extract @ 3% 500 ml T11 Moringa leaf extract @ 5% 500 ml T12 Moringa leaf extract @ 10% 500 ml T13

Untreated check (Control) -

Bioassay of the Dominant Seed Mycoflora against Various Organic Inputs Experiment details

An in vitro bioassay was conducted using the poisoned food technique to evaluate the treatments (As per the treatment details of Table 1) against the dominant mycoflora isolated from the okra cultivars at Department of Plant Pathology, B.

A. College of Agriculture, Anand Agricultural University, Anand, Gujarat, India. Observation of the radial growth was recorded from 24 hrs. of the incubation to the complete growth of test pathogens in control plates obtained. The per cent growth inhibition over control was calculated using formula given by Vincent (1947) [15] .

Where, PGI= Per cent growth inhibition DC = Mean diameter of mycelial colony in control treatment (mm) DT = Mean diameter of mycelial colony in treated set (mm)

Methodology

In Vitro Evaluation via Poisoned Food Technique

To evaluate the organic inputs in vitro, specific quantities of each treatment were individually incorporated into conical flasks containing melted, sterile Potato Dextrose Agar (PDA) to achieve the target concentrations (e.g., 3 mL of input in 97 mL of PDA for a 3% concentration). The poisoned media were thoroughly agitated to ensure uniform distribution before 20 mL portions were poured into sterile Petri plates. Control plates were prepared simultaneously using unamended PDA medium. Upon solidification, each plate was centrally inoculated with a 5 mm diameter culture disc, cut aseptically using a cork-borer from a 7-10 days old pure culture of the dominant seed mycoflora isolated from okra cultivars. Each treatment was maintained in triplicate.

The inoculated plates were incubated in a BOD incubator at 25 + 2 °C until the test pathogen reached full growth in the control plates, with colony diameters recorded periodically.

Results

Significant variation was recorded in the percentage of seeds infected by seed-borne mycoflora across different organic inputs (Table 2). Application of cow urine @ 3% (T 4 ) proved most effective in suppressing overall mycoflora incidence, recording the minimum seed infestation of 5.72%, which was closely followed by cow urine treatments at 5% (7.10%) and 10% (7.28%). In contrast, the highest seed mycoflora prevalence (12.24%) was observed in seeds treated with Beejamrut @ 3% (T 1 ). Qualitative evaluation of seeds treated with cow urine @ 10% (T 6 ) revealed the presence of only four predominant fungal taxa: Aspergillus flavus (2.43%), A. fischeri (2.08%), Fusarium oxysporum f. sp. vasinfectum (1.45%), and A. niger (1.33%). Seed treatment with cow urine @ 10% (T 6 ) completely suppressed the growth of Macrophomina phaseolina, Colletotrichum dematium, Aspergillus terreus, and A. tamarii. Similarly, cow urine @ 3% (T 4 ) achieved 100% inhibition of C. dematium, A. terreus, and A. tamarii. Furthermore, Beejamrut @ 10% (T 3 ) exhibited complete efficacy (100% inhibition) against Aspergillus fischeri and A. tamarii. (Plate 1) Significant variation was observed in the antifungal efficacy of the evaluated phytoextracts (Plate 2). Moringa leaf extract @ 10% (T 12 ) recorded the lowest seed infection rate (6.74%) and the lowest species richness (4 species), followed by Moringa leaf extract @ 5% (7.03%), Neem leaf extract @ 10% (9.43%), and Moringa leaf extract @ 3% (9.93%).

Complete inhibition (100%) of C. dematium, A. flavus, A. terreus, and A. tamarii was observed in seeds treated with Moringa leaf extract @ 10% (T 12 ). Similarly, treatment with Neem leaf extract @ 10% (T 9 ) completely eliminated the growth of M. phaseolina, A. niger, and A. terreus.

Bioassay of the Dominant Seed Mycoflora against Various Organic Inputs In vitro evaluation of organic inputs

To assess the antifungal potential of various organic inputs against the principal seed mycoflora of okra (Fusarium oxysporum f. sp. vasinfectum), an in vitro assay was conducted using the poisoned food technique. The treatments exhibited significant variation in suppressing mycelial growth.

Evaluation of treatments against the test pathogen demonstrated significant variation in mycelial growth inhibition (Table 3). Among all evaluated treatments, Moringa leaf extract @ 10% (T 12 ) exhibited superior antifungal activity, achieving the highest growth inhibition at 44.81%. This was followed by Neem leaf extract @ 10% (T 9 ) (37.41%) and Moringa leaf extract @ 5% (T 11 ) (36.67%). (Plate 3)

Discussion

The present study highlights the significant biological potential of traditional organic inputs and botanical extracts in suppressing seed-borne mycoflora and controlling major phytopathogenic fungi in okra. Evaluation of organic treatments (Beejamrut and cow urine) alongside botanical extracts (Moringa oleifera and Azadirachta indica) across varying concentrations (3%, 5%, and 10%) demonstrated concentration-dependent antifungal efficacy under in vitro conditions. Among the organic inputs, cow urine proved highly effective in restricting both total mycoflora incidence and fungal species richness. Treatment with cow urine @ 3% recorded the lowest overall seed infestation (5.71%), while cow urine @ 10% restricted species diversity to just four persistent fungal taxa: Fusarium oxysporum f. sp. vasinfectum (1.45%), Aspergillus fischeri (2.08%), A. niger (1.33%), and A. flavus (2.43%). The broad-spectrum fungicidal action of cow urine; evidenced by the complete inhibition of Macrophomina phaseolina, Colletotrichum dematium, A. terreus, and A. tamarii can be attributed to its bio-active constituents. Cow urine naturally contains phenols, uric acid, hippuric acid, nitrogen, and essential salts, which create an unfavorable micro-environment for fungal spore germination and mycelial proliferation (Randhawa, 2010) [14] . Regarding botanical inputs, Moringa leaf extract @ 10% demonstrated superior antifungal performance compared to neem extract, exhibiting the lowest species richness (4 species) and minimum total seed mycoflora incidence (6.74%). Furthermore, Moringa leaf extract @ 10% completely suppressed (100% inhibition) key storage and field pathogens, including C. dematium, A. flavus, A. terreus, and A. tamarii. Neem leaf extract @ 10% also showed potent selective efficacy, achieving complete eradication of M. phaseolina, A. niger, and A. terreus. The strong antifungal properties of Moringa oleifera are primarily linked to its rich profile of secondary metabolites, particularly glucosinolates, isothiocyanates, and polyphenolic compounds, which disrupt fungal cellular membranes and inhibit enzymatic pathways essential for hyphal expansion (Adewole, 2024; Kubheka et al., 2020) [3,6] . When evaluated against the dominant seed-borne pathogen of okra, F. oxysporum f. sp. vasinfectum, using the poisoned food technique, Moringa leaf extract @ 10% outperformed all other treatments, recording the highest mycelial growth inhibition (44.81%). These findings underscore the potential of Moringa leaf extract as an eco-friendly bio-fungicide and seed-dressing agent, offering a sustainable, non-chemical alternative to synthetic fungicides for managing seed-borne vascular wilt pathogens in okra cultivation.

Conclusion

In conclusion, the present study demonstrates the significant potential of organic inputs and botanical extracts as sustainable, eco-friendly alternatives to chemical fungicides for managing seed mycoflora in okra.

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