In Vitro Evaluation of Botanicals Against Fusarium Oxysporum Causing Wilt of Cumin

Cumin ( Cuminum cyminum L.), known as Zeera or Jill, belongs to the Apiaceae family. Cumin is an essential seed herb in India. The most feared disease of cumin plants is Fusarium wilt caused by Fusarium oxysporum, and it is a significant production constraint for growing cumin crops in Gujarat as in India. Seven different plant species have been used for the environmentally friendly treatment of this disease. Crude plant extracts were prepared with water, acetone, and cow urine as solvents at different concentrations (5%, 10%, and 15%). The in vitro antifungal activity of these plant extracts was determined by the poisoned food method. The highest inhibition was recorded with plant extracts prepared in acetone followed by cow urine. All plants showed significant antifungal activity. The highest inhibition was found with Senna alexandrina (85.53%) extract with acetone followed by Azadirachtaindic (82.72%) extract with acetone and 77.70% by Psidium guajava prepared with cow urine (at p ≤ 0.01). The lowest inhibition was recorded for Aloe barbadensis miller (35.49%) and Asparagus racemosus 48.74% with water extract. This study might be the first report of Fusarium oxysporum as a causal agent of cumin wilt in the Saurashtra region of Gujarat in India. A plant extract prepared with cow urine may be the first report of this study


Introduction
Plants are a major source of food, fiber, fodder, medicines, and many other valuable products for humans.Individual parts of plants such as roots stem, leaves, fruits, flowers, and seeds are used for people's daily needs.A variety of insects, bacteria, viruses, fungi, and other pests invade plants at various stages of development, reducing productivity and causing great loss to humankind.More than 800 million people in developing countries lack adequate food supplies, with plant diseases causing at least 10% of their food loss.

Plant Description
Cumin seeds are mainly used as a spice in the form of essential ingredients in all mixed spices and curry powders for flavoring vegetables, pickles, soups, sausages, cheeses, and other dishes, bread, cakes, and biscuits.The chemical composition includes 2.5-4.5% volatile oils, 10% fixed oils, and proteins.Volatile oils are mainly composed of 30-50% cumin aldehyde, minor amounts of b-pinene, phellandrene, cumin alcohol, hydrated aldehydes, and hydrocumin, which are suitable for medicinal purposes.The aromatic oil of cumin seeds is also used as a flavoring in curries, liqueurs, and liqueurs and has excellent uses in the perfume industry.The residue left after precious oil extraction contains 17.2% protein and Fat.It is a seasonal crop grown during the Rabi period.It is also grown as a cover crop under rain-fed and irrigated conditions after caliph crops such as jowar, green gram, cowpea, and maize.Cumin is mainly grown in the two states of India, namely Gujarat, and Rajasthan. 2

Scenario and Status of Cumin Production in India
India is the world's leading producer (70% of global production), consumer, and exporter of cumin.Almost 80% of crops are grown in India. 3In 2021-2022, the cumin planted area was 151,849 hectares, with production and productivity of 81,700 tons and 6,700 tons/ha respectively.Cumin is only grown in Gujarat and Rajasthan.Gujarat was the leading state for cumin production in India from 2010 to 2019.Gujarat had the highest productivity in 2018 where he was 1.04 MT/ha. 4West Bengal, Punjab, Uttar Pradesh, and Andhra Pradesh contribute to India's production.Gujarat's core cumin-producing districts are Surendranagar, Banaskantha, Jamnagar, and Patan.Bama, Jalore, Jodhpur, and Nagpur in Rajasthan are major cumin-producing areas. 5

Cumin Diseases
Cumin is an important rabbi crop in parts of Saurashtra and parts of northern Gujarat and is mainly affected by three major diseases, blight (Alternaria burnsii) and wilt (Fusarium oxysporum f.Sp. cumini), and powdery mildew (Erysiphe polygoni).Cumin is an important rabi crop in the part of Saurashtra and North Gujarat and is affected mainly by three important diseases viz., blight (Alternaria burnsii), wilt (Fusarium oxysporum f.Sp.  cumini), and powdery mildew (Erysiphe polygoni).Wilt is the most destructive disease of cumin plants.Gaur (1949) first reported the existence of wilt disease caused by Fusarium species in Rajasthan, India.Plants are affected at all stages of growth, but the severity of wilting increases with plant age.When the plant height reaches 2.5cm to 5.0cm, it will die.In older plants, the leaf color changes from green to yellow, starting with the oldest leaves and moving to the younger leaves.In severe stages, the tops and leaves of the plant fall off, leading to complete plant death.Such plants are easy to pull out of the ground.The roots of diseased plants have dark brown spots.Sometimes only partial wilting is observed.If plants become infected during flowering, they remain sterile. 6The present study was conducted to evaluate the antifungal activity of selected plants against the growth of Fusarium oxysporum f.sp.Cumini causes cumin wilting.

Collection of Native Plants in and Around Rajkot District
Seven plant species were used in this study to evaluate antifungal activity (Table 1).Native plants were selected based on their antibacterial properties and abundant arable land.Nominated plants are well adapted to climatic conditions and popular with local residents for their medicinal properties. 7Selected plant leaves/flowers were collected from different villages in the Rajkot district.

Fungal Strains
Pure culture of Fusarium oxysporum f. sp.cumini was acquired from the Plant pathology department of Agriculture University, Junagadh (Figure1).

Preparation of Aqueous Extract
Fresh Leaves/flowers of each plant were washed theory with tap water followed by sterilized distilled water.100gm of these Leaves/ flowers were crushed in 100 ml of distilled water in a grinder.The extracts were strained through a muslin cloth to remove plant debris.The filtrate thus obtained was designated as stock solution.5%, 10%, and 15% plant extract were prepared from the stock solution of each plant. 8imilarly, each plant extract was prepared in acetone (95%) and cow urine(untreated).

Antifungal Activity Assay of Botanical Extracts by using Poison Food Technique
1 ml of each plant extract at different concentrations of 5%, 10%, and 15% was added to 15 ml of sterile molten potato dextrose agar in a sterile Petri dish.Fusarium oxysporum has grown at 7 days of age.A 5 mm diameter mycelial disc of the culture was placed in the center of the petri dish containing the extract of each plant.A plate without plant extract served as a negative control.Plates were incubated at 27±2°C.Triplicates were maintained for each treatment in this assay.Radial mycelial growth was measured after 6 days of incubation (Figure 2).Results were compared to negative controls. 9

Statistical Analysis
Experiments were repeated three times and the average of the three measurements was used for calculations.Percentage inhibition was determined using the average colony diameter and calculated using the following formula proposed by Vincent (1947).

L = [(C -T)/C] x 100
where L is the inhibition rate, C is the colony radius of the control plate, and T is the radial growth of Fusarium oxysporum in the presence of plant extracts. 10atistical analysis of results was performed using R language version 4. 1.1.1.One-way analysis of variance (ANOVA) at values of p ≤ 0.001, followed by Tukey's post hoc test at p ≤ 0.05, was used to determine significant differences between results obtained in each experiment.

Result
Extracts from seven different plants were tested for antifungal activity at concentrations of 5%, 10%, and 15%.Radial growth of Fusarium oxysporum f.sp. is measured, and the percent inhibition is calculated for each plant.(Tables 2, 3, and 4).At 5% concentration of cow urine, the most effective extract to inhibit mycelial growth was Psidium guajava (73.74%) followed by Triumfetta pilosa (72.32%).Other tested extracts such as Azadirachta indica, Asparagus racemosus, Senna alexandrina and Ocimumtenuiflorum showed potencies between 66.05% and 59.57%.A decrease in potency was recorded by Aloe barbadensis miller (43.88%).

Discussion
The antifungal activity of plant extracts may be due to the presence of some antifungal toxins.
The presence of antifungal compounds in higher plants is well-known and valuable in controlling plant diseases. 11Management of Fusarium wilt in cumin is primarily based on the application of chemical fungicides, the use of pathogenresistant cultivars, and crop rotation.The application of chemical fungicides has been the traditional method of controlling wilt disease caused by Fusarium oxysporum.Cumin is a major health hazard for which new eco-friendly fungicides need to be found. 12The results of this study showed that the plant extracts of Azadirachta indica, Psidium guajava, Senna alexandrina, and Triumfetta pilosa were most effective against Fusarium oxysporum causes cumin wilt.These results correlate with previous studies on the antifungal activity of different plant extracts, namely Azardiachta indicia, Artemessia Annua, Ocimum sanctum, and Rheum emodi, against the pathogen Fusarium solani at different concentrations, indicating a significant reduction in pathogen growth. 13.Azardiachta indica was also shown to be effective against A. solani. 14ycelial growth of different species of Fusarium was inhibited by different concentrations of Convolvulus arsinoides and C. plurlictis plant extracts 15 Allium cepa 16 Adhatodavasica Azadirachta indica Cinnamomum camphora and Ocimum sanctum 17 Ocimumbasilicum, Eucalyptus amygdalina, Ailanthus Excels und Lantana camera. 18In this study, extracts of all plant species belonging to different families were found to have significant levels of antifungal activity against Fusarium oxysporum.

Conclusion
Results of the present study indicated that all selected plant extracts showed antifungal activity against fusarium oxysporum f.sp.cumini.Extracts prepared with acetone and cow urine showed excellent antifungal activity and were able to inhibit the growth of Fusarium oxysporum f.sp.Cumini, which is responsible for cumin wilting.

Fig. 3 :Fig. 4 :Fig. 5 :
Fig. 3: In vitro evaluation of plant extracts in water at different concentration on PDA media Significant at p≤0.01 level according to Tukey's Post Hoc test

Table 2 : In vitro evaluation of plant extracts in the water against Fusarium oxysporum f.sp. cumini
*Mean of three replications, Significant at p≤0.01 level according to Tukey's Post Hoc test

Table 4 : In vitro evaluation of plant extracts in Cow urine against mycelial growth of Fusarium oxysporum f. sp. Cumini Treatment Colony Diameter(mm) *at
These preliminary results, obtained from in vitro experiments, may be supplemented by other more comprehensive studies in the open field (in vivo).From this research, it has been concluded that certain plant extracts are a good source of cost-effective and non-hazardous approach against Fusarium oxysporum f.sp.cumini.Plant extracts such as Psidium guajavam, Triumfetta Pilosa, Senna alexandrina, and Azadirachta indica have good antifungal efficacy, which may be used for formulating new, safer, and eco-friendly natural fungicides.This work has allowed selecting the best plant which may be used as a Phyto fungicide to control crop diseases, with the ultimate goal of developing a green alternative to synthetic fungicides.