Alginate based encapsulation of pseudomonas fluorescens for management of soil borne pathogens

Abstract

Biological control, an eco-friendly and cost effective approach for plant disease management in agriculture has been practiced for several decades. Pseudomonas fluorescens, one such biocontrol agent is used to combat many phytopathogens. For commercial use, microbial inoculum should be supported by an appropriate formulation preventing a rapid decline of introduced microorganisms and extending their shelf-life. Various formulations available in the market are powder, liquid and granular formulations where such carrier based inoculants which generally faces problems like poor shelf life, high chances of contamination, bulk sterilization problem, unpredictable field performance and sometimes unavailability of good carrier materials. In order to overcome the disadvantages of these formulations, microencapsulation is one such alternate viable option prepared by using sodium alginate as a polymer which results in extended shelf-life and controlled microbial release from the formulation thus, enhancing their application efficacy. Hence, the present study was undertaken in the Department of Plant Pathology, College of Horticulture, Vellanikkara to develop alginate based formulation of Pseudomonas fluorescens for the management of soil borne pathogens. P. fluorescens, the reference culture of KAU was used for preparation of alginate beads. To improve the shelf life of alginate beads, King’s B broth was enriched with adjuvants viz., sugar source (mannitol and trehalose), wetting agent (PVP and PEG), adhesive (CMC and liquid paraffin) and surfactant (tween-80) in nine different treatments and was evaluated at monthly intervals. After nine months of observation, maximum population of P. fluorescens was recorded in treatments T1 (mannitol +PVP +CMC +tween-80) (1.33 x 108 cfu ml-1) and T3 (mannitol +PEG +CMC +tween-80) (1.66 x 108 cfu ml-1) compared to control which were selected for the preparation of beads. The beads were prepared from the above selected treatments as per the protocol of Bashan et al. (2002) with modifications. The beads were prepared in three different batches viz., beads from alginate alone, beads from alginate amended with skim milk and beads after secondary multiplication. Various parameters were standardised in order to prepare effective bead formulation. Beads from both alginate and alginate amended skim milk formulations produced from higher concentration of sodium alginate (3%) and calcium chloride (3.5% and 3.0%) solution with 60 min of curing time and 9 to 15 cm of falling distance produced perfectly spherical beads with maximum diameter of above 1.70 mm, higher bead weight of above 16.6 mg with more than 60 per cent bead yield. Such beads showed reduced swelling percentage which holds higher per cent of water content inside beads and lowest shrinkage percentage that facilitates higher survival and slow release of the bioagent for a longer period of time. Shelf life of P. fluorescens encapsulated in alginate beads alone prepared from two best treatments T1 (mannitol + PVP + CMC + tween-80) and T3 (mannitol + PEG + CMC + tween-80) showed a higher shelf life compared to alginate amended skim milk beads and beads after secondary multiplication. Higher bacterial entrapment were observed in alginate beads prepared from sodium alginate (3%) and calcium chloride (3.5% and 3.0%) respectively in treatments T3A12 (10.33 x 1020 cfu g-1) followed by treatments T3A11 (8.33 x 1020 cfu g-1), T1A6 (6.33 x 1020 cfu g-1) and T1A5 (5 x 1020 cfu g-1) respectively after four months of preparation. The alginate bead combinations B-1: T1 (mannitol + PVP + CMC + tween - 80) + sodium alginate (3%) + CaCl2 (3%) and B-2: T3 (mannitol + PEG + CMC + tween -80) + sodium alginate (3%) + CaCl2 (3%) were selected for in vitro evaluation studies against major soil borne pathogens and it was noticed that these formulations inhibited soil borne pathogens viz., Pythium aphanidermatum (100%) followed by Phytophthora nicotiana (72.22 and 77.77%) Ralstonia solanacearum (70.36 and 74.07%) and to some extent inhibition of Fusarium oxysporum (27.77 and 30.55%). However, no inhibition was observed on the growth of Sclerotium rolfsii and Rhizoctonia solani under in vitro conditions. Hence the study has clear by demonstrate a protocol to produce microbeads of P. fluorescens which are less bulky, non-toxic, biodegradable and enables slow and controlled release of the biocontrol agent and thus could maintain a bacterial population for a relatively longer period.