Post-harvest challenges cause substantial loss of agricultural products during its handling, transportation and storage. To control the same, various synthetic chemical agents are used which ultimately result in serious health and environmental problems. Hence the application of efficient biological methods is essential to minimise the use of hazardous synthetic agents for post-harvest applications. Several microbial biocontrol agents have the potential to be applied against the pathogenic and spoilage causing microorganisms for post-harvest protection (Droby et al. 2022).
Microbial infestation of fruits and vegetables during the harvesting is a serious issue which will be intensified during its transportation and storage. Here comes the relevance of biocontrol agents which can provide resistance to agricultural products by acting as a barrier to pathogenic microorganisms through diverse mechanisms. Such protective effects of food items can significantly ensure the quality of the food materials used further for consumption (Carmona-Hernandez et al. 2019). Hence, the identification of beneficial microorganisms from novel source is essential to explore its application for post-harvest protection. Even the cell free supernatant of such beneficial organisms can have the potential to be applied as coating material for post-harvest applications.
The present study has focused on the isolation, identification, characterization and analysis of microorganisms from the leaves of Musa acuminata. The leaves of M. acuminata have already been reported for the anti-inflammatory, antioxidant and anti-diabetic properties (Sonibare et al. 2018). The ethanolic extract of M. acuminata leaves has also been reported to have inhibitory activity against methicillin-resistant and susceptible Staphylococcus aureus (Sivasamugham et al. 2021). Due to the waxy coating and the hydrophobic nature of leaf surface of M. acuminata and also because of its continuous interaction with fungi and bacteria, it can be well expected to have the presence of beneficial microorganisms with biocontrol properties. Various species of Bacillus, Pseudomonas and Serratia are well characterised for their antifungal mechanisms executed through diverse metabolites and enzymes (Thomashow & Weller, 1996; Sansinenea, 2019). The Pseudomonas spp. have wide environmental distribution which indicate its diverse metabolic adaptations and are generally considered as promoters of plant elongation with significant impact on plant disease protection. The rhamnolipids produced by Pseudomonas spp. are also known to act as microbe associated molecular pattern which could activate the plant defense by binding to pattern recognition receptors (PRR). In this study, out of 26 bacterial isolates screened, Pseudomonas sp. MP 11 was found to have promising antifungal activity against F. oxysporum, P. aphanidermatum, P. infestans, and S. rolfsii in both dual culture and VOC mediated assays which might be due to the broad range of antagonistic mechanisms present in MP 11 against plant pathogens. In a recent study, P. fluorescens has been reported to have the antifungal activity against the Macrophomina phaseolina which could infect soybeans (Castaldi et al. 2021). In another study, P. fluorescens BNM296 isolated from the rhizosphere has been reported to have antifungal activity against Pythium ultimum (León et al. 2009). Besides, P. chlororaphis subsp. aureofaciens SPS-41 has been reported to have antifungal activity against Ceratocystis fimbriata through the release of VOCs which contained the antifungals like 3-methyl-1-butanol, phenylethyl alcohol, and 2-methyl-1-butanol (Zhang et al. 2021). Furthermore, P. putida BP25 isolated from the black pepper has been demonstrated to have VOCs mediated inhibition of pathogens like P. capsici, P. myriotylum, R. solani, C. gloeosporioides, A. rolfsii, G. moniliformis, M. oryzae, and R. pseudosolanacearum. These results indicate that the Pseudomonas spp. to have broad range antagonistic principles against plant pathogens and have already been characterised from diverse parts of plants. In the current study, the MP 11 was also found to have the presence of rhlAB gene coding for antifungal rhamnolipids. As the rhamnolipids are already known to have activity against diverse plant pathogens, the bacteria selected in the study can consider to have the promises for biocontrol applications. In a previous study, rhizospheric Pseudomonas sp. isolated from Cyclea peltate was reported to have antifungal activity against P. myriotylum due to the production of rhamnolipids (Jishma et al. 2021). In the study, the MP 11 was observed to have the produced rhamnolipid as identified by LC-MS analysis where the compound with M + H+ (503.17) correspond to rhamnolipid. By combining the results of PCR and sequencing of rhlAB gene of MP 11 along with the LC-MS result, the isolate can consider to have rhamnolipid as a key compound to be responsible for its antagonism against selected plant pathogens. In a previous study, mechanistic basis of antifungal activity of rhamnolipid has been demonstrated to be due to the direct interaction with membrane lipids and thereby affecting the integrity of plasma membrane. This could further induce the targeted cell death of the pathogenic microorganisms very quickly (Botcazon et al. 2022; Crouzet et al. 2020). The isolate MP 11 has also been described for the volatile mediated antifungal activity. This could be due to its HCN production also with the activity of blend of VOCs like propane-1,2-dimethoxy-, methyl pyruvate dimethyl acetal, and 9-decenoic acid. as identified by GC-MS analysis. In a previous study, Hexadecanoic acid, methyl ester has already been described for the antimicrobial activity as observed in the current study (Shaaban et al., 2021). The demonstrated antifungal properties of MP 11 is clear indication of its application for post-harvest protection which is an emerging area of application of plant beneficial microorganisms.
The isolate MP 11 was also found to be positive for various plant growth promoting properties like phosphate and zinc solubilisation, IAA production, and ACC deaminase activity. The ability of transforming insoluble to soluble phosphate is one of the important characteristics for improving the crop productivity as the phosphorus is considered to be a limiting factor for plants (Sah et al. 2021). Zn deficiency is a major problem in the agriculture field and it could severely affect the growth, development, maturity and productivity of plants. Hence the identification and application of zinc solubilising microorganisms has already been considered to have a huge impact on the plant growth and yield. Various bacterial genera including, Bacillus and Pseudomonas have already been reported to solubilise insoluble zinc to soluble forms extensively (Hashemnejad et al. 2021). Moreover, the capability for IAA production could be beneficial for the host plant for better uptake of nutrients and thereby the overall plant growth. It could have determining effects on the cell enlargement, cell division, root initiation, and apical dominance in plants (Ahemad & Khan, 2012). Many Pseudomonas spp. have previously been reported to express the ACC deaminase production property which hence could confer the tolerance to plants against stress factors by lowering the ethylene level (Win et al. 2018). As the bacteria isolated from this study, Pseudomonas sp. MP 11 has also been described for plant beneficial traits, it can also have the promising application in agricultural fields to promote plant growth and productivity.
The isolate MP 11 has also found to have protective effect on rice kernels infected with P. aphanidermatum and S. rolfsii. Pseudomonas strains isolated from the rhizosphere have already been described for in vitro antibiosis against Xanthomonas oryzae and R. solani under normal and saline conditions (Rangarajan et al. 2003). In another study, P. fluorescens PfALR2 was found to have inhibitory activity against R. solani under field trial when applied as a pet-based formulation (Rabindran & Vidhyasekaran, 1996). Moreover, Pseudomonas spp. have been reported for their post-harvest protective effect against various fungal species. For instance, Pseudomonas sp. isolated from the rhizospheric soil have described for the potential inhibitory activity against Penicillium digitatum infecting citrus fruit during the post-harvest (Qessaoui et al. 2022). Also, rhizospheric P. aeruginosa BRp3 has demonstrated for biocontrol activity against X. oryzae in Basmati rice due to the production of secondary metabolites including rhamnolipids (Yasmin et al. 2017). Hence the protective activity exhibited by MP 11 supernatant against P. aphanidermatum and S. rolfsii in rice seeds might be due to the presence and activity of antifungal metabolites especially rhamnolipids.
The present study also focused on the isolation and identification of potential antifungal metabolites from MP 11. Because, Pseudomonas sp. MP 11 was found to have significant inhibitory activity against phytopathogens. Here, the GC-MS and LC-MS analyses revealed the presence of major antifungal metabolites including rhamnolipids. The amplification of genes rhlAB and pqqC further confirmed the ability of MP 11 to produce metabolites with antifungal mechanisms and plant growth promotion potential. Eventhough various Pseudomonas spp. have already been isolated from various resources, the isolation of MP 11 is the first report on the isolation of Pseudomonas sp. from M. acuminata which highlights the importance of the study. Also, the post-harvest protective effect of Pseudomonas sp. against P. aphanidermatum and S. rolfsii is the first report in this field. Therefore, Pseudomonas sp. MP 11 isolated in the study can have the promises to develop as a microbial formulation to apply on various economically important crops, during post-harvest stage targeting different fungal pathogens.