Terpinen-4-ol from Trachyspermum ammi is a potential and safer candidate molecule for fungicide development against Alternaria solani

1 Introduction

Thevplants produce defense molecules as secondary metabolites (Hancock et al., 2015). More than 30,000 of these secondary metabolites are essential oils which have antifungal and antimicrobial properties (Beeby et al., 2020). These plant essential oils (PEOs) have been used in traditional medicines for many decades (Turek & Stintzing, 2013).

The hazardous effects of synthetic pesticides led to the concept and use of biological control methods in agroecosystems. Important agents of biological control are predators, parasitoids, microorganisms, plant semiochemicals, algae, animals (Ravensberg, 2015). Plant essential oils (PEO) are categorized as plant originated natural compounds which can be used as biological control agents. Due to their botanicals origin and antifungal and antibacterial properties PEO became an important component of IPM program (Lamichhane et al., 2016). The use of botanicals as substitute for synthetic chemicals have been highly supported in Europe by the directive 2009/128/CE. The ultimate goal of this approach is to minimize the use of chemical pesticides and encourage the application of natural products and agricultural inputs more in line with sustainable agriculture development. http://data.europa.eu/eli/dir/2009/128/oj

The phytopathogenic fungi are the reason for almost 30% of all agricultural crop diseases (Jain et al., 2019) and cause about 38% yield losses by affecting them in field and/or after harvest (Ons et al., 2020). Plant essential oils have shown significant activities against such plant pathogenic fungi (Tabassum & Vidyasagar, 2013).

Early blight is one of the dominant diseases of the genus Alternaria and causes 32–57% yield losses (Nisa et al. 2015). Alternaria can tolerate adverse climatic conditions and thrives a wide range of temperature and atmospheric moisture (Tomazoni et al., 2017). It causes serious economic losses in tomato production around the world.

Computer Aided Drug Design (CADD) approaches are used in pesticide development research from the last few years (Chu et al., 2012). The CADD approaches are designed to perform the drug development tasks computationally. A lead molecule can be identified by screening compound libraries containing millions of synthetic and natural compounds (Speck-Planche et al., 2011). Speedy and convenient methods of lead molecule identification by using CAPD approaches provide base line information about novel and safer pesticide molecules (Yousafi et al., 2019). With the advancement in computational methods, we might expect dramatic growth for a number of pesticides in the market with new, presumably safer, modes of action.

Keeping in view the efficacy of PEO against pathogenic fungi and non-hazardous nature they can be used for fungicide development industry.. The current study was designed to detect antimicrobial effectiveness of five medicinal PEOs against Alternaria solani. The findings of our study will provide a base line knowledge and data for novel and safer fungicide development and new target site identification to avoid fungicide resistance.

2 Materials and methods

3 Results

3.1

3.1 Laboratory evaluation of PEO

3.1.1

3.1.1 Minimum inhibitory concentration (MIC)

The significantly largest inhibition zone (37.3 mm) was found for T. ammi (25µl/ml) followed by significantly smaller (25.3 mm) for T. ammi (10µl/ml). The inhibition zone formed by T. ammi (10µl/ml) was significantly larger than all other treatments, except that of C. cyminum (25µl/ml) (Fig. 1).

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Fig. 1

Minimum inhibitory concentration of different concentrations of plant essential oils against Alternaria solani. (p < 0.05).

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3.1.2

3.1.2 Minimum fungicidal concentration (MFC)

Three PEOs of T. ammi, C. cyminum and A. indica were tested for their fungicidal potential. The minimum fungicidal concentration was evaluated for three concentrations (25,50 and 75 µl/ml) of PEOs. The experiment set was replicated three times. After 72 h, PEO of T. ammi at 50 µl/ml, C. cyminum at 75 µl/ml, and A. indica at 75 µl/ml concentration, showed no fungal colonies growth (Fig. 2).

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Fig. 2

Minimum fungicidal concentration activity different plant essential oils against Alternaria solani.

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3.2

3.2 Field trials

The percent disease incidence after first application was the lowest (2.2) for EO of T. ammi at 80 µl/ml concentration, which was not significantly different from that of Ridomil Gold. After four PEO foliar applications only T. ammi at 80 µl/ml concentration showed a significantly lower percent disease incidence than the commercial fungicide (Fig. 3). All other treatments were found not significantly different among each other for percent A. solani incidence. Disease incidence from different application intervals was not significantly different across the application dates.

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Fig. 3

Effect of foliar application of different concentrations of plant essential oils on percent disease incidence of Alternaria solani in tomato ((p < 0.05).

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3.3

3.3 Computer Aided Fungicide Design (CAFD)

3.3.1

3.3.1 Three dimensional protein model prediction

The Expassy ProSiteScan predicted only one 170 aa long domain in the solanapyrone synthase (UniProt ID: D7UQ40) i.e., FAD-binding domain, PCMH-type. The predicted 3D structure of domain was qualified by VERIFY 3D, 90.40% of the residues have averaged 3D-1D score >= 0.2. The quality factor by ERRAT was 83.64. More than 90% of the residues in structure were found in most favored region and 9.2% in additional allowed region of Ramachandran Plot. The refined and evaluated 3D structure was saved in pdb format for docking analysis.

3.3.2

3.3.2 Molecular docking

Ten non-toxic chemical compounds from T. ammi were selected for docking with protein FAD-binding domain. The complexes with lower binding energies and hydrogen bonds were selected (Table 1). The binding energy of the selected complexes ranged from −6.0 (Terpinen-4-ol) to −5.1 ((E)-Octadec-2-enoic acid). Two hydrogen bond interactions and lowest binding energy was observed for Terpinen-4-ol (Fig. 4).

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Fig. 4

Terpinen-4-ol-polyketide synthase interaction complex in Alternaria solani.

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3.3.3

3.3.3 Molecular dynamics (MD) simulation

RMSD was fluctuated at the start with a steep rise initially but after 25 ns a stable RMSD (1 nm) was recorded (Fig. 5a). The RMSF peaks were observed at Gly105 for 0.7 nm height with the highest peak (0.8 nm) observed at Gly35 and Ala 137 (Fig. 5b). Radius of gyration was 1.6 nm at the start with a peak of 1.8 after 5 ns, then started dropping and ended up at 1.4 nm at 50 ns. A very little drop in radius of gyration was observed (Fig. 5c).

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Fig. 5

Results for MD simulation of Terpinen-4-ol-polyketide synthase complex (a: RMSD, b:RMSF, c: Radius of Gyration)

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4 Discussion

Phytopathogenic fungi are a serious threat to agriculture s worldwide (Fisher et al. 2012). Indiscriminate use of fungicides, especially on vegetables and fruits is very deleterious for human health (Hakala et al., 2011). Moreover, it causes environmental pollution and pesticide resistance (Boxall et al., 2009). These issues are less addressed for fungicides than insecticides (Zubrod et al., 2019). The fungicidal properties of PEO have been reported in different research findings (Jiménez-Reyes et al., 2019). The basic advantage of using plant essential oils in fungicide development is that they are biodegradable, so are least toxic to mammals and cause no environmental pollution (Raja, 2014).

In the current study we tested the efficacy of five plant essential oils (PEOs) viz. Azadirachta indica, Eucalyptus grandis, Citrus sinensis, Cuminum cyminum, and Trachyspermum ammi, against Alternaria solani in tomato. The testing of essential oils was done in three trials, i.e., in vitro, in field and computational analysis. In the laboratory, five PEOs were tested by investigating their minimum fungicidal concentration (MFC) and minimum inhibitory concentration (MIC). In our study T. ammi EO had the lowest MIC and MFC against A. solani. Our results confirmed the finding of Khan and Jameel (2018), who found antifungal potential of T. ammi seed oil, by using disk diffusion method, against different fungal species. Current findings also confirmed the results of Sharifzadeh et al. (2015), who reported 50% MFC of T. ammi essential oil against Candida albicans.

Three qualified PEOs, i.e., of T. ammi, C. cyminum and A. indica, from the laboratory experiment were selected to be tested against A. solani in tomato under field conditions. The lowest percent disease incidence and severity were observed for T. ammi PEO. This is a good parameter for testing the efficacy of a chemical against the target (Naziya et al., 2020). Trachyspermum ammi belongs to the family Apiaceae, commonly known as ajwain. The seeds and leaves extracts of T. ammi have antimicrobial properties and has been effectively used as medicines (Dwivedi et al., 2012).

The best PEO (T. ammi) was selected for Computer Aided Fungicide Design (CAFD). Computer aided drug designing (CADD) has become a significant approach for novel pharmaceutical drug designing (Khan et al., 2019). But this approach is lacking in agricultural pesticide development. However, the pharmacodynamics and tools used in CADD can be used for computer aided pesticide designing (CAPD) except pharmacokinetic considerations (Tice, 2001). The number of known experimental findings and infrastructure of targets sites and new drug molecules identification is very least studied in the field of pesticide chemistry (Bordas et al., 2003). Little work has been done for CAPD for phytopathogenic fungi (Jiménez-Reyes et al., 2019). If we adopt these approaches for pesticide development we can develop safer and effective pesticides speedily in a least expensive manner.

In this study, we selected the active compounds in T. ammi PEO from the literature. The target enzyme of A. solani, bifunctional solanapyrone synthase, was selected to be inhibited. It is a polyketide synthase responsible for biosynthesis of crucial phytotoxin, solanapyrone, which causes early blight infection in potato and tomato (Kasahara  et al., 2010). A protein domain is a region in a protein's polypeptide chain which controls its function independently and a conserved region which is self-stabilizing . To be more precise in inhibiting toxin production we chose the toxin producing domain to be blocked by an efficient and non-toxic inhibitor molecule from T. ammi. FAD-binding domain, PCMH-type, a toxin producing domain, (Fraaije & Mattevi, 2000) of solanapyrone synthase was selected to be blocked.

The top compound, Terpinen-4-ol, with lowest binding affinity (−6.0 kcal/mol) and 2 hydrogen bonds with target, was selected for further confirmation of its binding and stability with the target site. Molecular Dynamics (MD) simulations technique was used for this purpose. This analysis of MD simulation is used to evacuate the movements of the highly complex macromolecular systems (Phillips et al., 2005). The estimation of structural fluctuations of the macromolecule is the most crucial feature of this analysis (Karplus et al., 2002), which reflects the stability and flexibility in the ligand receptor complex. For the present study, Root-Mean-Square-Fluctuations (RMSF) and Root-Mean-Square-Deviation (RMSD) are most important parameters to be analyzed (Humphrey et al., 1996). The value of RMSD reflects the stability interaction profile. In this study, the average ligand-receptor RMSD was 1 nm, indicating that the system was stable (Faraj et al., 2016). The Root Mean Square Fluctuation (RMSF) is an estimation of the displacement of a specific atom, or group of atoms, relative to the reference structure, averaged over the number of atoms (Martínez, 2015). RMSF values in our study reflect stable complex conformation.

Results of the current study indicated Terpinen-4-ol from T. ammi EO had antifungal activities as reported by different studies (Morcia et al., 2012). Terpinen-4-ol from Thymus villosus essential oil was found effective for fungicide resistance management in Candida, Cryptococcus and Aspergillus species (Pinto et al., 2013). The compounds nontoxic to humans were selected for docking against toxin producing domain in A. solanim, for its inhibition. The predicted lead molecule, Terpinen-4-ol, in the current study is not only non-toxic to humans but has been aiso reported to have therapeutic properties (Mondello et al., 2006) especially against cancer (Shapira et al., 2016). Therefore, this compound can be used in fungicide formulation as an effective and safe active ingredient. Terpinen-4-ol molecule can be used alone or in combination with other suitable molecules for better and safer management of A. solani.