Fusarium species is a devastating pathogen in agriculture that cause severe loss of economically important plants such as wheat, maize, banana, tomato, sugarcane etc. It is also known to produce mycotoxins such as fumonisins, zearalenone, deoxynivalenol, fusaric acid and trichothecenes. Different species such as F. graminearum, F. proliferatum, F. tricinctum, F. moniliforme, F. verticillioides and F. foetens, known as the plant pathogens (Aoki et al. 2014). The F. foetens has been reported as a pathogen in ornamental crops, specifically Begonia plants (Schroers et al. 2004). It causes damping-off of rooibos seedlings and destructive vascular wilt disease that leads to the plant's death (Lamprecht et al. 2017). Recently, (González-Jartín et al. 2019) reported production of mycotoxins such as beauvericin and fusaric acid from F. foetens in the maize plant.
Despite having negative impact on humans and the environment, many chemical fungicides are being used to control Fusarium attacks. However, due to its excessive and frequent use, the phytopathogenic fungi are able to acquire resistance to the existing fungicides. In the sense of searching safe, eco-friendly and sustainable alternative, biocontrol bacteria and their active components are considered the best choice (Köhl et al. 2019). In the past few decades' extensive study has been conducted on terrestrial bacteria and their active compounds against various plant pathogenic fungi. Still, the discovery of potential organisms and their novel metabolites is diminishing. Oceans are the most diverse, adverse and competitive ecosystem. To survive in such a unique environment, marine bacteria have developed adaptation mechanisms to produce unique biomolecules. Consequently, marine bacteria can produce bioactive compounds generally not found in terrestrial environments (Imhoff et al. 2011, Dionisi et al. 2012).
In certain studies marine bacteria such as Paenibacillus sp. PNM200, B. marinus B9987 and
P. aeruginosa were reported against various plant pathogenic fungi (Vinchira-Villarraga et al. 2021, Zhang et al. 2010, Manwar et al. 2004). The dominant genera Bacillus, Pseudomonas, Streptomyces and Serratia are well known for their biocontrol potential against several plant pathogenic fungi. In the current study, the marine isolate BKACT showed significant antifungal activity by reducing over 50 per cent mycelial growth against all tested Fusarium spp. The highest 75.56 ± 0.80 per cent of mycelial growth inhibition was observed against F. foetens NCIM 1330. To date there are no reports highlighting use of biocontrol bacteria against F. foetens. To the best of our knowledge, this is the first study to feature the antagonistic activity of marine S. marcescens against F. foetens.
The biocontrol potentials of S. marcescens has mainly focused on its indigenous chitinase producing ability (Ordentlich et al. 1988, Someya et al. 2001 and Dhar Purkayastha et al. 2018). A recent study by Hover et al. (2016) reported the chitinase mutant S. marcescens which retained fungal killing ability and suggested that antifungal compounds, along with chitinase enzyme together helping the antifungal activity. In the present study, isolation, purification and characterization of the antifungal compound from marine S. marcescens BKACT is carried out. The antifungal compound was characterized as 2, 4- DTBP by GC-MS and NMR spectroscopy. The results were validated using TLC and HPLC by comparing the retention time and Rf value of purified compound with the standard reference compound (Sigma). Additionally, GC-MS analysis of the crude extract from strain BKACT also ascertained the compound to be 2, 4- DTBP. Several previous studies (Dharni et al. 2014, Varsha et al. 2015 and Wang et al. 2021) have reported 2, 4- DTBP from Pseudomonas monteilii, Lactococcus sp. and Bacillus subtilis CF-3 for the control of plant pathogenic fungi. However, there are no reports detailing the purification and characterization aspects of 2, 4-DTBP from marine Serratia marcescens. To the best of our knowledge this is the first study to purify, characterize, and experimentally validate in-vitro and in-vivo antifungal activity of 2, 4-DTBP from the marine S. marcescens.
Regardless of antifungal and antioxidant activity, the 2, 4-DTBP has great volatile property. The microbial, volatile organic compounds (VOCs) have a significant role in disease management, especially for controlling the plant pathogenic fungi. VOCs are generally effective at minimal concentration, and they are capable of spreading in the atmosphere over the large distances. VOCs exert their inhibitory activity without direct physical contact with target pathogens (Schmidt et al. 2015). In the recent study 2, 4-DTBP at one mole per liter reported as the effective volatile concentration against Colletotrichum gloeosporioides (Wang et al. 2021). However, in our present study, we found that 2, 4-DTBP from S. marcescens showed a great volatile antifungal activity against F. foetens NCIM 1330 at minimal concentration of 0.53 mM as compared to reported values. Mycelial growth inhibition was significantly higher when compared to control, even at lower concentrations. The highest 86.6 ± 2.0% mycelial growth was inhibited at 0.53 mM concentration. At the same concentration, 100% spore inhibition was also observed. A significant difference in the inhibitory concentration could be attributed to the 2, 4-DTBP produced by different microorganisms and the solvent variation of DMSO (Wang et al. 2021) and methanol (used in the present study) for dissolution of 2, 4-DTBP.
Varsha et al. (2015) in their study coated a 25 mg/ml concentration of 2, 4-DTBP on wheat seed which identified to protect from A. niger, F. chlamydosporum and F. moniliforme infections. Considering its antifungal activity and volatile property, we believe that, this molecule could control F. foetens on the wheat seeds. To date there is only one report highlighting F. foetens to produce mycotoxin such as beauvericin and fusaric acid in the cereal like maize (González-Jartín et al. 2019). Here for the first time we observed in the absence of compound F. foetens infect wheat seed and grew easily at above 90% relative humidity. At 0.53 mM concentration of 2, 4-DTBP, percent seed contamination index (PSCI) was significantly lowered when compare to control. Hundred percent controls of F. foetens NCIM 1330 was observed at 1.0 mM concentration and it was identified as the effective treatment. Interestingly, the compound has not shown any adverse effect on the germination of wheat seeds at 1.0 mM concentration.