2.1. Botanicals, chemicals and reagents
Pongamia pinnata L. (Fabaceae) seeds were collected and taxonomically recognized from the local market. E. Merck India provided emulsifiers (such as Tween 20, Tween 80, nonylphenol ethoxylates, calcium alkylbenzene sulphonates, and dodecylbenzene sulphonate) and carrier solvents (such as aromatic hydrocarbon (C-9), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and methyl oleate). Without additional purification, the reagents were employed. As standard hard water of 342 ppm strength with an electrical conductivity (EC) value of 0.69 dSm-1, a solution of anhydrous CaCl2 (2.74 mM) and MgCl2.6H2O (0.68 mM) was produced in double-distilled water (1 liter) (CIPAC MT 18, 1995).
2.2. Hexane extract preparation
Pongamia pinnata seeds were washed carefully under a gentle flow of tap water to eliminate dust and other pollutants before being dried in the shade at room temperature. As reported in our earlier research paper (Purkait et al., 2019), air-dried seeds (1 kg) were ground to powder in a household grinder (Bajaj, Bravo Dlx 500) and extracted with hexane (4 liters) in a Soxhlet apparatus for 6 hours at 60 C. (Figure 1). The hexane extract was filtered and concentrated in a rotary vacuum evaporator (Buchi (R-3), Switzerland) at 40°C under decreased pressure (370 mbar) to yield (28.37 percent) of the desired extract, which was then stored at 4°C for further use.
2.3 Toxicity assessment of organic solvents
2.3.1. Isolation and maintenance of a pure culture
Phytophthora sp. and A. solani were isolated from infected potatoes using a single spore isolation technique, and identified by scientists from the Department of Plant Pathology, Bidhan Chandra Krishi Viswavidyalaya (BCKV), Mohanpur, West Bengal, India, based on colony morphology, morphometric characteristics of acervuli, seta, conidia, and conidiophores, as described by Prittesh (2016). Potatoes infected with the pathogen were chopped into little pieces along the edges of lesions (5 mm in diameter). The pieces were surface sterilized in a 0.1 percent w/v aqueous mercuric chloride solution, rinsed five times, dipped in streptomycin, and placed to a potato dextrose agar (PDA) growth plate. Mycelial bids were moved from culture plates to PDA slants afterwards and allowed to sporulate for 8–10 days.
The single spore isolation procedure developed by Alexopoulos et al. was used to establish pure cultures of Phytophthora sp. and A. solani (2002). At 4°C, a pure fungal culture was maintained on PDA media. Conidia from 10-day-old cultures grown on PDA on Petri dishes were combined to make pathogen inocula. Flooding the plates with sterile distilled water and gentle scraping with a sterile slide were used to remove conidia from the surface of the media. To eliminate mycelial pieces, the suspensions were filtered through a thin layer of absorbent cotton wool and the spore number in suspension was adjusted to 106 conidia-mL-1 using a hemocytometer. In in-vitro pathogenicity testing, this spore suspension was utilized to inoculate banana fingers and chili.
2.3.2. Bioassay in-vitro
Following the poison food technique, an in-vitro bioassay was used to screen out the toxicities of several organic carrier solvents (Jang and Kulk, 2018). According to the Clinical and Laboratory Standards Institute (CLSI) standard recommendations, the concentration of DMSO, C9, DMF, and methyl oleate in the final test solutions was 1% (v/v) (Hazen, 2013). At the moment of full radial growth (9 cm) of each control plate, i.e. 6 days following the incubation period, colony diameters of treatment plates were measured. All treatments and controls were duplicated three times and incubated at 28 1°C for the duration of the experiment. The following formula was used to compute the percentage inhibition of mycelial growth (Dutta et al. 2019):
Where dc is the test fungus's average radial growth (cm) on control plates, and dt is the test fungus's average radial growth (cm) in treatment plates. A statistically significant difference was defined as a difference of p 0.05.
2.4. Preparation of emulsifiable concentrate
With various adjustments, different emulsifiable concentrate (EC) formulations were created using the approach outlined in our earlier article (Purkait et al., 2019). From the toxicity assessment of organic solvents, seed extract (30 percent w/w) was dissolved in the least harmful solvent (Methyl oleate) (60 percent w/w) (Section 2.3). To prepare EC formulations, different emulsifiers in a blend (10%) were added to the extract solution and thoroughly swirled on a magnetic stirrer at 200 rpm at 50-60 C. (Supplementary Figure 1).
2.5. Physico-chemical characteristics
P. pinnata seed extracts were produced in thirteen distinct EC (30% w/w) formulations (EC-1 to EC-13), as indicated in Table 2. The generated formulations' physico-chemical properties were tested in triplicates according to CIPAC and Indian Standard (IS) requirements (BIS, 1997).
2.5.1. Emulsion stability
In a clean, transparent beaker, the prepared sample (2 mL) was poured (250 mL). At 32 degrees Celsius, standard hard water was poured on the sample at a rate of 15 to 20 mL min-1 to get the volume up to 100 mL and swirled constantly with a glass rod. The diluted emulsion was transferred to a clean and dry graduated cylinder (100 mL) with a stopper and left undisturbed for one hour to check for the formation of any creamy layer on top or deposition on the bottom (CIPAC MT 36.3, 2003).
2.5.2. Cold test
In ice-cold water, the formed sample (50 mL) was placed in a glass container with a stopper (-10 C). For 1 hour, the container was swirled at brief intervals and looked for turbidity, an oily layer, or both.
Abel's equipment was used to test the flashpoints of the produced EC formulations (Scavini, IP0170-110). Each formulation was carefully placed in the cup and slowly heated. At regular intervals, an external flame was directed at the cup, and the temperature at which the formulation lit was recorded. The formulation's flashpoint should be above 24.5 C, according to CIPAC MT12 (1995).
2.5.4. Storage stability evaluation
The formulations were stored at increased temperatures (4, 25, and 54 2C) for 14 days in three duplicated sets, equating to a two-year shelf life at ambient temperature (27 2C) (CIPAC MT46.3, 2000). After fourteen days of storage, the EC formulations were visually examined for phase separation or the creation of a creamy layer.
2.5.5. pH and specific gravity
A pH meter pre-calibrated at 25 1 C was used to measure the pH of the formed samples (1 percent aqueous solution) (Systronics, Model 335; Gujarat, India). Using a calibrated hydrometer (Fisher Scientific, 11-603-4F & 11-603-4G), the specific gravity of the produced formulations was also determined (CIPAC, 2000).
2.6. Bioassay of an emulsifiable concentrate (30 EC) formulation in vitro
The antifungal activity of the produced EC was assessed using an in-vitro bioassay based on the inhibition of A. solani and Phytophthora sp. mycelial radial growth. In conical flasks containing previously sterilized and cooled PDA medium, four doses (0.1, 0.25, 0.5, and 1.0 percent) of the selected formulation (EC-1) were created alongside a control (without formulation). 15 milliliters of medium were placed into disinfected petri dishes after thorough mixing (9 cm in diameter).
Five-day-old mycelial discs (7 mm in diameter) were removed and deposited separately in the center of PDA plates using aseptic techniques. All treatments and control plates were triple-duplicated and incubated at 28°C. At the moment of full radial growth (9 cm) of each control plate, i.e. 6 days following the incubation period, the colony diameters of the treatment plates were measured. Section 2.3.2 shows how to compute the percentage inhibition of mycelial development using equation (1). (Dutta et al. 2019). The logarithm of each concentration and the accompanying probit value for each inhibition percentage were used to compute the EC50 values of various concentrations.