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.