Antifungal Activity and Molecular Docking of Phenol, 2, 4-Bis (1, 1-Dimethylethyl) - Produced By Plant Growth-Promoting Mangrove Actinobacteria; Kutzneria Sp. Strain TSII

The present study reveals the plant growth-promoting (PGP) potentials and characterizes the antifungal metabolites of Kutzneria sp. strain TSII isolated from mangrove sediment soil through in vitro and in silico studies. In this study, Kutzneria sp. strain TSII was screened for PGP activities and the antifungal activities against Pithomyces atro-olivaceous, a leaf spot associated pathogen in groundnut plants. The ethyl acetate extract of Kutzneria sp. strain TSII was puried using column chromatography, and the presence of various antimicrobial compounds was studied by Gas Chromatography-Mass Spectrometry (GC-MS) analysis. In silico modeling and docking were carried out to evaluate the antifungal potent of bioactive compound. Kutzneria sp. strain TSII produced Proteases, Phosphatases, Ammonia, Siderophores, Cellulases, Indole Acetic Acid (IAA), Lipases, and Amylases, indicating its ability to enhance the growth of plants. The ethyl acetate extract of Kutzneria sp strain TSII was found to be a potent inhibitor of fungal mycelial growth in the potato dextrose agar (PDA) plates. The GC-MS spectral study showed 24 antimicrobial compounds belonging to ve chemical groups: Phenolics, Phthalates, Fatty acid methyl esters (FAME), Spiro, and Fatty alcohols. In silico docking studies showed that Phenol, 2, 4-Bis (1, 1-Dimethylethyl) – effectively attaches with the active site of mitochondrial F 1 F 0 Adenosine triphosphate synthase enzymes of Pithomyces atro-olivaceous. Hence, it is clear that these antifungal compounds shall be formulated shortly to treat many plant fungal diseases in an eco-friendly manner.


Introduction
Groundnut (Arachis hypogaea L.) is one among the most important oilseed crops. Groundnut is ranked 13 th among the most critical food crops. Leaf spot disease is almost co-existent with the plants and contributes to a signi cant yield loss throughout the world (Meena 2010). Massive application of chemical fungicides adversely affects the resident microbiota and also causes severe health hazards to society. Besides, systemic use of these pesticides in uences the fungicide resistance in phytopathogens (Mishra et al. 2008). Hence, the effective alternative method suggested was biocontrol agents such as bacteria, fungus, and actinobacteria. These bene cial microbes control the soil-borne pathogens either directly by inducing the plants' immune system or indirectly by producing ammonia, HCN, cell wall degrading enzymes, siderophores, and antibiotics (Kohl et al. 2019).
Actinomycetes are gram positive, lamentous spore-forming bacteria belonging to the order Actinomycetales. They are isolated from the terrestrial and aquatic ecosystem, mainly involved in the decomposition of organic matter, there by facilitating the soil's nutrient recycling process. Actinobacteria, especially Streptomyces species, are reported for the massive array of bioactive compounds like antibiotics, bio-pesticides, enzymes, which could nd applications in agricultural and pharmaceutical industries (Genilloud 2017). Recently, researchers report the plant-growth-promoting and antagonistic traits in some actinobacteria species; their inoculation improved the biomass yield and in uenced the synthesis of defense-related compounds in legume plants (Sathya et al. 2017). However, the formulation and commercialization of the bioactive metabolites from actinobacteria with relevance to the biocontrol of plant diseases are less than those from bacteria, owing to its slow growth. Mangrove is the outstanding muddy ecosystem support the growth of microbes, including actinobacteria. These microbes are well adapted to this salty habitat to produce a wide range of extracellular metabolites for their survival. Hence, we described here the antifungal metabolites of a rare unexplored mangrove actinobacteria Kutzneria sp. strain TSII for the biocontrol of Pithomyces atro-olivaceous, a leaf spot disease-causing novel fungus in groundnut crops identi ed in this study.
In a previous study by Vijay et al. (2020), GC-MS/MS identi ed antifungal metabolites from tomatoassociated rhizobacteria were docked with short-chain dehydrogenase/reductase enzymes in Fusarium oxysporum and regarded to be the potential target for the observed antifungal effects. Similarly, we address the mitochondrial ATP synthase subunit 'A' found in the F 0 complex as the potential target for the fungicide leads from actinobacteria identi ed in the study using GC-MS/MS analysis. Because this subunit plays a crucial role in proton channeling across the inner mitochondrial membrane, which offers ATP synthesis in the mitochondrial matrix of Pithomyces atro-olivaceous (Carbajo et al. 2005), we performed an in silico targeting of subunit 'A' with mangrove sediment-associated actinobacterial ligands. Hence these compounds would probably attenuate ATP synthesis and substantiate the observed in vitro growth inhibitory effects on the novel leaf spot disease-causing pathogen Pithomyces atro-olivaceous in groundnuts.

Characterization of direct biocontrol molecules, the antifungals
Ethyl acetate extract of bioactive actinobacterium was fractionated through silica gel GF254 thin layer chromatography (TLC) followed by active spot identi cation in an autobiography assay on PDA (Vijay et al. 2020). Active fractions were collected using silica gel column chromatography with mesh size between 200 and 400 at a linear gradient (% v/v) of Dichloromethane and Benzene. The pooled active fraction was again subjected to the linear gradients of chloroform and ethyl acetate. Puri ed active fraction was further separated in High-performance liquid chromatography (HPLC) equipped with ODS/C18 column using the mobile phase of chloroform and ethyl acetate (95% / 5%, V/V) at ow rate of 0.5mL/min wherein Kirby Bauer disc diffusion assay was used to monitor the antifungal activity. In GC-MS, the active fraction was injected into capillary type column with injector port at a temperature of 250º C, temperature of detector port at 280º C, column oven at 50º C with constant increment of 6º C per min, and holding time of 2 min. The entire procedure lasted for 40.33 minutes obtaining a Gas chromatogram and corresponding mass spectrum for each compounds using Shimadzu GCMS/QP 2020. More recently, researchers are disclosing the direct or indirect mechanism of plant growth-promoting potentials of actinobacteria. Interestingly these bacteria secrets the phytohormones such as indole-3acetic acid (IAA), cytokinins, and gibberellins, solubilize the minerals such as phosphorus (P) and produce iron chelators-siderophores and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. They indirectly bene t the plants by protecting them from pathogens by synthesizing antagonistic substances and fungal cell wall degrading enzymes. Though several previous reports evidenced the PGP properities of actinobacteria, to the best of our knowledge, this is the rst attempt revealing the PGP traits and antagonistic potential of Kutzneria sp. strain TSII against P. atro-olivaceous isolated from the infected groundnut leaves ( demonstrated that the IAA produced by Streptomyces fradiae NKZ-259 increases the biomass of tomato seedlings in invivo conditions. The role of HCN in controlling the plant pathogen was proved by several researchers (Etminani et al. 2018; Kumar et al. 2012). In addition to biocontrol, HCN makes phosphorous and iron available, which enables the growth of bacteria and plants (Rijavec et al. 2016). In our experiment, the strain TS II was found to produce HCN in a moderate amount.

Plate Assay
Along with these growth regulating substances, TS II produced some extracellular enzymes such as protease, lipase, cellulase, and lipase. The role of these hydrolytic enzymes in biocontrol has already been proved (Jadhav et al. 2017) and strongly supports our ndings. In contrast, the chitinase enzyme test showed that TS II did not produce chitinase that is needed for the cleavage of the cell wall of fungi.
However, ethyl acetate extract effectively inhibited fungal growth. The possible reason might be due to the synthesis of various other antimicrobial compounds.

Characterization of indirect biocontrol molecules, the siderophores
Siderophores are produced by microbes to chelate ferric iron from the surrounding environment.Though these chelators has wider applications it will be more useful for the development of sustainable agriculture (Venkat Kumar et al. 2019). This study indicated that the strain TS II produces siderophores with distantly acting nature when compared to other siderophore producing actinobacteria studied, in an iron starved medium (Fig. 3A), thus able to accumulate iron in root proximity and thereby may improve the plant growth and yield. Further characterization showed the chemical nature of siderophores that belongs to Hydroxamate and Catecholates types based on Tetrazolium salt test and Arnow's test, respectively ( Table 2).

Antifungal activity
The pathogenic fungi were observed with microscopic characteristics of grey to the dark brownish colony (Fig. 3B). An expert taxonomist further con rmed the fungal isolate at Indian Agricultural Research Institute -Indian Type Culture Collection (ITCC) with identi cation no. 11,167.19. Cell-free culture supernatant of strain TSII has effectively inhibited the growth of P. atro-olivaceous, a pathogenic fungal strain, by agar well diffusion assay (Fig. 3C). The reason might be due to the diffusion of antimicrobial metabolites produced by the actinobacteria (Qi et al. 2019).

Characterization of direct biocontrol molecules, the antifungals
The antimicrobial compound present in ethyl acetate extracts of strain TSII exhibiting excellent antifungal activity was fractionated through thin layer chromatography (TLC) silica gel GF254 using Benzene: Dichloromethane: Methanol (6:2:2) as the solvent system and UV/iodine vapor as detection system and the active spot (Rf value: 0.83) was identi ed by persistent antifungal activity in an autobiography assay on PDA (Fig. 3D). An antifungal fraction collected using a silica gel column with a linear gradient of Benzene and Dichloromethane at 6:4 was pooled and further eluted in chloroform: ethyl acetate at 9:1.
This partially puri ed fraction was also separated into its isomeric forms in HPLC equipped with an ODS/C18 column with linear gradients of chloroform and ethyl acetate. GC-MS pro ling of HPLC puri ed active fractions of strain TSII was performed in Shimadzu GCMS/QP2020 and resulted in 24 bioactive metabolites whose m/z ratio, retention time, and molecular formula were matched with compounds available with Wiley Library (Version 8.0) ( Table 3 and Fig. 4). These bioactive compounds are categorized into ve major groups: Phenolics, Phthalates, Fatty acid methyl esters (FAME), Spiro, and 3.6. Sequence retrieval, homology modeling and docking

Conclusion
Many researchers spoke about the antimicrobial activity of these biologically synthesized compounds against the human pathogen. Only a few were attempted to demonstrate their e ciency against plant pathogens. Most chemical fungicides causes severe health issues to humans and bene cial microbes, ultimately leading to severe environmental pollution. Given this, we paid more attention to nd out an alternate method to replace and reduce the use of chemical fungicides. Besides being eco-friendly, microbial fungicides are more economical when compared to the former. At the outset of this study, we conclude that the Kutzneria sp. strain TSII alone or its product phenol, 2, 4-bis (1, 1-dimethylethyl) -may act as a promising biocontrol agent. This nding shall improve the crop productivity as well as the economic status of the farmer.           Shows the Root Mean Square Deviation and Fluctuation stability of interacted ligand receptor complex.