MSc

Trichoderma spp, one of the widely exploited biocontrol agents in the arena of crop disease
management, are known for promoting growth and inducing abiotic stress tolerance in plants.
However, direct application in the field limits their efficiency due to several adverse factors.
Hence, they should be adequately formulated to escalate the efficacy in field application.
Eventhough, a number of such formulations have been developed in the recent past, many of
them are bulky in nature, having reduced shelf life with a high risk of contamination and
desiccation. Thus, a study was conducted to develop a novel formulation of T. viride, with
sodium alginate, a biodegradable polymer as the encapsulating carrier material.
Evaluation of the effect of additive substances viz., mannitol (2%), trehalose (15 mM),
polyvinyl pyrrolidone (1%), polyethylene glycol (0.25%), carboxymethyl cellulose (0.5%),
liquid paraffin (1%) and tween 80 (0.5%) in eight combination on the shelf life of T. viride was
carried out. After seven months of evaluation, a combination of trehalose, PVP, CMC and tween
80 outperformed the other treatments. Beads were prepared using 0.75, 1.5, 2.5, 3.0 and 3.5 per
cent sodium alginate and 2.5, 3.0 and 3.5 per cent CaCl2 solution with and without additives.
Without additives, no beads were formed at 0.75 and 1.5 per cent sodium alginate and spherical
beads were formed at 2.5, 3.0 and 3.5 per cent. When additives were amalgamated, 0.75 and 1.5
per cent sodium alginate yielded spherical beads, while beads were not formed at 2.5, 3.0 and
3.5 per cent. Hence, the concentration of sodium alginate was standardized as 0.75 and 1.5 per
cent. The impact of height of air column between the orifice of the separating funnel and the
level of CaCl2 bath on the bead shape was evaluated at different heights where 2 and 4 cm
yielded tailed beads while, rest of the heights yielded spherical beads. Hence, based on the shape
of the bead, 8 - 12 cm was selected as the optimum height of air column.
Previously standardized parameters along with 2.5, 3.0 and 3.5 per cent CaCl2 solution
was adopted to prepare beads by employing ionotropic gelation and cross linking technique in 31
treatments. The beads were dried for 48 h at room temperature and stored in air tight containers.
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Properties like bead weight, diameter, yield and number of beads formed per ml sodium alginate
solution was estimated. A maximum bead weight of 96.20 ± 1.8 mg and bead diameter of 2.42 ±
0.13 mm was recorded in control (T31-sodium alginate 2.5% and CaCl2 2.5%). Maximum per
cent yield was documented in T19 (trehalose, PVP, CMC, tween 80, sodium alginate 1.5%, CaCl
2
2.5%) (69.09%) and a maximum of 26.91 beads were formed from each ml of sodium alginate in
T9 while, it was 8.0 in control. Shelf life evaluation was carried out for six months, where T21
(trehalose, PVP, CMC, tween 80, sodium alginate 1.5%, CaCl2 3.5%) outperformed the other
treatments even after six months hence, T21 was selected as the best treatment. Degree of
contamination was estimated at 1, 3 and 6 months after preparation, where T1 - T6 and T19 - T30
remained free of contamination even after six months of preparation.
Per cent of potentially infective beads were estimated where cent per cent germination
was recorded at 72 h after incubation. Moisture content of the beads were assessed where the
beads retained its intact shape at 48 h and therefore was selected as the optimum period of
drying. Swelling behaviour of microbeads were estimated at pH 7.4 in phosphate buffer and at
pH 1.0 in 0.1M HCl. A gradual increase in weight of beads at pH 7.4 confirmed the swelling
behaviour while, reduction in weight at pH 1.0 revealed its shrinking nature. Time of gelation
was standardized as 60 min as the bead weight declined until this time and attained stability after
this time. The effect of pH on the selected bead (T21) was evaluated at pH 5.09 and 8.91 in soil
under in vitro conditions where the release in former was sustained while a sudden release was
observed in latter. Observations on biometric parameters, yield and the per cent disease
incidence from pot culture experiments revealed that sodium alginate bead based formulation @
5.0 g plant-1 performed superior to talc based and liquid formulations. The study on degradation
revealed that the beads were biodegradable in nature.
Thus, the present investigation succeeded in formulating T. viride using sodium alginate
as the encapsulating carrier material, which would help to compensate the drawbacks associated
with presently available formulations. However, the study should be complimented with
multilocational trials to confirm its efficacy under field conditions.