3.1.Isolation and identification of actinobacteria
The mangrove actinobacteria was identified as Kutzneria sp strain TSII based on Morphological, Biochemical, and 16S rRNA sequencing (Table 1). The 16S rRNA sequence was submitted to the Genbank database of NCBI and obtained the accession number MN 565961. The identified strain TSII was found to be pale yellow colored, stable, branched, cottony aerial mycelium. Scanning Electron Microscopy observation showed the arrangements of rod-shaped cells of Kutzneria sp. strain TSII at the maximum resolution of 5000 x in the field view of 10 µm with energy of electrons at 10.0 keV (Fig. 1). Evolutionary analysis (using MEGA X software) showed the isolate's phylogenetic relationship based on 16S gene sequence homology of 100 and 99.91% with Kutzneria chonburiensis strain SMC 256 and Kutzneria buriramensis A-T 1846, respectively (Fig. 2).
3.2.PGP traits of Actinobacteria
3.2.1. Tube Assay
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-3-acetic 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 benefit 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 first attempt revealing the PGP traits and antagonistic potential of Kutzneria sp. strain TSII against P. atro-olivaceous isolated from the infected groundnut leaves (Table 1). IAA plays a vital role in plant development and physiological processes, including embryogenesis, organogenesis, vascular differentiation, root and shoot development, trophic growth, and fruit development. Examination for the production of IAA indicates that the strain TS II was a strong producer and hence undoubtly the growth and development of the crop plant can be achieved through direct inoculation of this actinobacteria. A previous investigation by Myo et al. (2019) 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.
3.2.2. 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 findings. 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.
3.3. 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).
3.4. Antifungal activity
The pathogenic fungi were observed with microscopic characteristics of grey to the dark brownish colony (Fig. 3B). An expert taxonomist further confirmed the fungal isolate at Indian Agricultural Research Institute - Indian Type Culture Collection (ITCC) with identification 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).
3.5. 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 identified 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 purified 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 profiling of HPLC purified 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 five major groups: Phenolics, Phthalates, Fatty acid methyl esters (FAME), Spiro, and Fatty alcohols. Eicosane (C20H42) and dibutyl phthalate (C16H22O4) were shown for their antifungal property against Rhizoctonia solani AG-3 strain KX85246 (Ashan et al. 2017). The previous study by Qi et al. (2019) demonstrated that phenolics, pyrrolizidine, hydrocarbons, esters, and acids in the crude extract of Streptomyces sp. SCA3-4 was responsible for antimicrobial activity against phytopathogens. Similarly, Pyrrolo (1, 2-a) pyrazine -1, 4-dione and aminocoumacin, fungichromin, N- acetyl-D, L- Phenylalanine, and rampamycin from Streptomyces sp. UPMRS4 inhibited the growth of Pyricularia oryzae, a causative agent of blast disease in rice (Awla et al. 2016).
3.6. Sequence retrieval, homology modeling and docking