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The study area has tropical moist deciduous type of climate and major vegetation types, Champion and Seth, 1968 [1]. Therefore, the climate of this district is suitable for the growth of IAPs. The northern part of the district is covered by dense natural forests such as Chakia, Sujauli, Nishangara, Mihinpurwa, Bichhia and Baghauli. Mihinpurwa block of the northern of the district has dense forest and there are forest villages where there is a good population of Tharu tribes, who reside in forest villages named- Phakeerpuri, Balaigaon, Harraiya, Bhaishahi, Ranapur, Jaliya, etc. These tribes are very well acquainted with the different ethnomedicinal plants and they heal various ailments with the help of different parts of the plant. Hence, they are the real informers regarding the ethnomedicinal properties of the plants.

IAPs are dominating in this area and threatening not only to the biodiversity and ecosystem service but also to economic development and human welfare, Ricciardi et al., 2000 [2]. Instead of harmful impacts of invasive plants, most of the IAPs are traditionally used for the treatment of various ailments. Thus, the medicinal plants are regarded as a gift of nature for curing various diseases like diuretic, anti-helminthes, neurological disorders, inflammation, jaundice, dysentery, typhoid, etc. Due to poverty and lack of modern medicines, people inhabiting in different rural as well as tribal areas are completely dependent on the local medicinal plants for their primary healthcare and food. During the study, a total of 27 medicinally important IAPs belonging to 16 families were evaluated for antifungal activity against phytopathogenic fungi. We studied the inhibitory effect of extracts of 27 IAPs on mycelial growth of five phytopathogenic fungi.  

According to Hill in 1952, medicinal plants have been used as sources of medicine since time immemorial [3]. According to Pushpangadan, 1995, more than 43 percent of all flowering plants in India are medicinally important [4]. Rao and Shanpru noted in 1981 that, due to their constant association with forests, ethnic groups have immense plant lore, which they inherit and pass on from generation to generation just through oral conversation [5]. India is provided with a rich wealth of medicinal plants, being perhaps the largest producer and rightly acclaimed as the "Botanical Garden of the World" by Dubey et al., 2004 [6].

Bajwa et al., (2001) [7] found inhibitory potential in aqueous extracts of three asteraceous allelopathic species against Aspergillus niger. The shoot and root extracts of two asteraceous plants, such as Parthenium hysterophorus L. and Ageratum conyzoides L. were tested for antifungal activity against Macrophomina phaseolina (Tassi) by Bajwa et al., (2007) [8]. They found that the lowest concentration of 2% root and shoot extracts of P. hysterophorus and 4% A. conyzoides was most effective in suppressing M. phaseolina biomass production.

Yanar et al., (2011) used the radial growth technique to test the antifungal activities of 26 plant extracts against Phytophthora infestans [9].

In vitro antifungal activity of extracts of different plant species against Alternaria spp. was screened by Dellavalle et al., 2011 [10].

Das and Devkota (2018) tested the antifungal activities of Ageratina adenophora and Ipomoea carnea ssp. fistulosa of Nepal against five phytopathogenic fungi such as Alternaria brassicae, Botrytis cinerea, Fusarium oxysporum, Phytophthora capsici, and Sclerotium rolfsii [11].

Eman and Hanan (2021) investigated the antifungal activity of aqueous and ethanol extracts of cinnamon, garlic, ginger and guava against five isolates of Aspergillus fumigatus. The results showed that effect of ethanol and aqueous extracts of cinnamon exhibited equal effect on all the five isolates of Aspergillus fumigatus. Minimum inhibitory concentration (MIC) of ethanol extract of garlic and cinnamon was 2 mg/ml whereas aqueous extract of garlic and cinnamon was 40 mg/ml and 10 mg/ml, respectively [12]. 

I. Collection of IAPs:

The field survey of the different agricultural land areas of different villages, viz. Raniya Pur, Balai Gaon, Rama Pur, Fakirpuri, Aama of district particularly wheat, sugarcane, and paddy fields as well as forest wise done during 2018-2020. Discussion and interview held by the local farmers, old persons and tribals of different age groups randomly. The aim of the interview and conversation related to IAPs was to collect the information regarding local name of the plants and their ethnomedicinal uses. Such plants were collected, brought to department and herbarium was made by using herbarium technique of Jain and Rao, 1977 [13]. The plants have been identified by using relevant floras and standard literatures, Duthie, 1903-1922 [14]; Panigrahi et al., 1969 [15]; Saini 2005a, 2005b [16], and by seeking help of authorities of field. The herbarium was put in department for record and reference. Ethnobotanical information of IAPs was collected by using a semi-structured questionnaire developed by Jain, 1989 [17]. Brief description of 27 IAPs are listed below in Table1.1.

II.  Extract preparation

For the preparation of extracts of IAPs, leaves are collected and thoroughly washed in running tap water, then with a 2% aqueous sodium hypochlorite solution, and finally with sterile distilled water. It was then air-dried and powdered. In the case of aqueous extract preparation, 10 g of each powdered material was soaked separately in 100 ml of distilled water (1:10 w/v) at room temperature for 24 hours.  In the case of solvent extract preparation, 10 g of each powdered material was soaked separately in 100 ml of chloroform and methanol (1:10 w/v) at room temperature for 24 hours. The mixtures were shaking occasionally. After 24 hours, each mixture is filtered by muslin clothes, and the supernatant is filtered by Whatman No. 1 filter paper. Each extract was separately concentrated by evaporating to dryness under reduced pressure. The yields were dissolved in methanol to a final concentration of 100 mg/ml. The prepared extracts were adjusted to pH 6.8 with 1.0 M HCl and stored in closed containers at 4 °C for evaluation of antifungal activities by Nasrine Salhi et al.,, 2017 [18].

III.  Isolation, Identification, and Purification of Fungal Test Organisms:

Infected parts of the potato, tomato, onion, and sugarcane are collected and cut into small pieces. After washing the tissues thoroughly in sterile water, the fungal pathogens are isolated from plant tissues exhibiting clear symptoms. Using flame-sterilized forceps, infected tissues are cut into small pieces and transferred to sterile petridishes containing 0.1% mercuric chloride solution for surface sterilization of plant tissues. The plant parts were transferred to Potato Dextrose Agar (PDA) plates and incubated for 5-7 days for the complete growth of fungal pathogens. The fungal pathogens were purified by using the hyphal tips technique and a single sporing method on agar medium, followed by subcultures of each isolated fungus on slant medium for future studies. PDA media is prepared by suspending potato infusion (200 g) and 20 g of dextrose in a glass beaker containing distilled water. Dissolve the ingredients by using a magnetic stirrer. The pH of the medium was adjusted to 5.6 by using 0.1N HCl and 0.1N KOH. Now add 15 g of agar-agar and adjust the final volume of medium with distilled water to make up to 1000 ml, and then stir the mixture using a magnetic plate. The mixture was then autoclaved at 15 psi for 25–30 minutes before being allowed to cool under the laminar flow hood. Pour the medium into sterile petridishes. Add ampicillin (50µg/ml) in combination with 10% tartaric acid before pouring the media into petridishes to avoid bacterial contamination. Tartaric acid decreases the pH of the mixture to about 3.5, thus inhibiting bacterial growth. Finally, the medium was cotton-plugged and sterilized at 121 °C for 20 minutes before being stored at 2 °C in the dark. To maintain purity, the fungi were cultured several times on a PDA plate with a 6 mm diameter disc. These fungi were identified according to cultural characteristics described by Gilman in 1957 [19], Barnett and Hunter in 1972 [20], and Nelson et al., in 1982 [21]. There are a total of five fungal phytopathogens that are isolated from their respective host plants. These fungal test organisms are listed below in Table 1.2.

IV.  Antifungal activity assay

The “poisoned food technique” is used to assess the antifungal activity of different extracts of 27 IAPs against test fungi in vitro, Grover and Moore, 1962 [22]. PDA plates were prepared with the desired concentration of plant extracts, and a 6 mm-diameter disc of a 7-day-old fungal culture was inoculated in the centre of the plate and incubated at 30 °C for 7 days. PDA plates with respective solvents instead of extracts serve as controls. After 7 days of incubation, the inhibition zones were measured by mycelial growth inhibition and calculated as per the formula of Pandey et al., 1982 [23].

Percentage of mycelial growth inhibition = (dc- dt)/dc x 100

Where,  

dc = Fungal colony average increase (in mm) in control

dt = Average increase in fungal colonies (in mm) during treatment

^*All tests were performed in triplicates.

Three replicates were analyzed using one-way analysis of variance (ANOVA) in the SPSS program Ver. 20. The mean of the replicates is used to calculate the standard deviation (SD). 

A total of 27 aqueous extracts and 54 solvent extracts from two solvents (methanol and chloroform) are prepared from aerial parts of 27 plant species.  In vitro antifungal activity of a total 81 extracts was evaluated against 5 different phytopathogenic fungi using the poisoned food technique.

(I) Evaluation of Antifungal activity of aqueous extracts of IAPs against test fungi:

A total of 27 aqueous extracts prepared from aerial parts of 27 IAPs evaluated for in vitro antifungal activity against 05 phytopathogenic fungi. The corresponding results are presented in Table 3.1. A total of 11 aqueous extracts out of 27, exhibited in vitro antifungal activity against all the test fungi selected. The percentage of inhibition recorded by aqueous extracts of Ageratum conyzoides L., Argemone mexicana L., Calotropis  procera (Ait.) R.Br., Datura metel L., Hyptis suaveolens (L.) Poit., Ipomoea fistulosa Mart DC., Lantana camara L. Parthenium hysterophorous L., Ricinus communis Linn.,  Solanum nigrum L. and Tridax procumbens L. against all the test fungi selected, were ranging in between 22.80% to 95.70%.

The maximum percentage of inhibition recorded from aqueous extracts of Ageratum conyzoides L. against Rhizoctonia solani (95.70%) and Fusarium oxysporum f. sp. lycopersici (80%) followed by aqueous extract of Cannabis sativa L. against Alternaria solani (83.50%), Parthenium hysterophorous L. against Colletotricum fulcatum (79.70%), and Aspergillus niger (77.40%).

Aqueous extracts of five IAPs such as Blumea eriantha DC., Opuntia elatior Mill., Salvia hispanica L., Sida acuta Burm.f., and Urena lobata L. did not exhibit any growth inhibitory potential against any of the fungi selected.

(I) Evaluation of Antifungal activity of Solvent extracts of IAPs against test fungi:

A total of 54 solvent extracts are prepared from two solvents (methanol and chloroform) and aerial parts of 27 IAPs are evaluated for in vitro antifungal activity against 05 phytopathogenic fungi using poisoned food techniques. The corresponding results are presented in Table 3.2 and 3.3. Among 27 methanolic extracts, 09 extracts exhibited in vitro antifungal activity against all the test fungi selected. The percentage of inhibition recorded by methanolic extracts of Ageratum conyzoides L., Argemone mexicana L., Cannabis sativa L., Clerodendrum splendens G. Don, Datura metel L., Lantana camara L., Parthenium hysterophorous L., Ricinus communis Linn., and Tridax procumbens L. against all the test fungi selected, were ranging in between 12.55% to 91.70%.

The maximum percentage of inhibition is recorded from methanolic extracts of Ageratum conyzoides L. against Rhizoctonia solani (91.70%), followed by Parthenium hysterophorous L. against Aspergillus niger (90.40%), Cannabis sativa L. against Alternaria solani (85.50%), Cuscuta reflexa Roxb. against Fusarium oxysporum f. sp. lycopersici (82.40%) and Ipomoea fistulosa Mart DC. against Colletotricum fulcatum (71.50%).

Methanolic extracts of six IAPs such as Blumea eriantha DC., Eichhornia crassipes (Mart.) Solms,  Opuntia elatior Mill., Salvia hispanica L., Sida acuta Burm.f. and Urena lobata L. did not exhibit any growth inhibitory potential against any of the fungi selected.

Among 27 chloroform extracts, 13 extracts exhibited in vitro antifungal activity against all the test fungi selected. The percentage of inhibition recorded by chloroform extracts of Ageratum conyzoides L., Argemone mexicana L., Cassia occidentalis L., Chenopodium album L., Clerodendrum splendens G. Don, Datura metel L., Hyptis suaveolens (L.) Poit., Lantana camara L., Nicotiana  plumbaginifolia Viv., Parthenium hysterophorous L., Ricinus communis Linn., Solanum nigrum L., and Tridax procumbens L. against all the test fungi selected, were ranging in between 06% to 73.55%.

The maximum percentage of inhibition is recorded from chloroform extract of Cuscuta reflexa Roxb against Fusarium oxysporum f. sp. lycopersici (89.50%) followed by Cannabis sativa L. against Alternaria solani (86.70%), Clerodendrum splendens G. Don against Aspergillus niger (73.55%), Argemone mexicana L. against Colletotricum fulcatum (73.20%) and Calotropis  procera (Ait.) R.Br., against Rhizoctonia solani (65.50%).

Chloroform extracts of six IAPs such as Blumea eriantha DC., Eichhornia crassipes (Mart.) Solms,  Opuntia elatior Mill., Salvia hispanica L., Sida acuta Burm.f., and Urena lobata L. did not exhibit any growth inhibitory potential against any of the fungi selected.

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