DEVELOPMENT OF BIO-FORMULATIONS USING NATIVE BIO-CONTROL AGENTS (BCAs) FOR THE BIOLOGICAL MANAGEMENT OF FALSE SMUT AND STEM ROT DISEASES OF RICE
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Date
2023-09-26
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PROFFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY
Abstract
Rice stem rot and false smut diseases are caused by Sclerotium hydrophilum and
Ustilaginoidea virens are major threat to the rice cultivation in almost all the major rice
growing regions of the country. Detailed investigations were carried out on
symptomatology, cultural and morphological characters of the pathogen, and their
molecular identity through ITS primers and sequences were subjected to sanger
sequencing and submitted to NCBI revealed similarity with Sclerotium hydrophilum
SHPS1 and Ustilaginoidea virens UVPS1 (Accession number: OP480227, OP480796).
Rhizosphere microflora were isolated and studied cultural, morphological and
biochemical parameters and tested their antagonistic activity against the test pathogens
under in-vitro and glass house conditions in susceptible rice varietiey TN1. Among
fourteen isolates, P14 showed maximum production of IAA (7.37 μg/ ml), PS (67.26
μg/ml), siderophores (49.02 % siderophore units), biofilm production (1.35 OD600nm), and
HCN (0.047 OD620nm). Molecular identification of potential isolate, nucleotide
sequencing, NCBI BLAST, and phylogenetic analysis confirmed the identity of the
bioagent as Pseudomonas otitidis POPS 1 (Accession number: ON782043).
The antagonistic activity of isolated potential Pseudomonasisolate P14 along with
standard bioagents viz., Trichoderma isolate (TAIK1) Bacillus isolate (BIK3) and
Pseudomonas isolate (PIK1) collected from ICAR-IIRR culture collections were tested
for their efficacy against S. hydrophilum and U. virens, their interaction was observed
using scanning electron microscope (SEM). Phytohormones released from the bioagents
were analysed using LC-ESI MS/MS. Potential isolate POPS1 along with TAIK1, BIK3
and PIK1 were screened for phytohormones secretion into their growth media.
The selected potential bioagents (POPS1, TAIK1, BIK3, and PIK1) were tested
against S. hydrophilum and U. virens under glass house conditions in rice susceptible
variety TN1. The results showed that among different treatments, combination of four
bioagents consortia was most effective in promoting plant growth viz., shoot length and
root length in TN1 variety at two seasons (Rabi 2020-21 and Kharif 2021-22).
Furthermore, secondary metabolites of PIK1 were extracted and characterized. A
novel compound, 2-Methyl Pyrazine (2MP), was identified and confirmed using Nuclear
magnetic resonance (NMR) spectroscopy and High-resolution mass spectrometry (HR MS). The purified 2MP and 6-Pentyl Pyrone (6PP) from TAIK1 were tested for their bio
efficacy against S. hydrophilum and U. virens under in vitro conditions.
To enhance the efficacy of the bioagents and metabolites, micro and nano encapsulated formulations were developed using extrusion, emulsification, and spray
drying methods. The most efficient method was standardized, resulting in
microencapsulated spores of TAIK1 and bacterial cells of PIK1, as well as
nanoencapsulated 6PP and 2MP. The encapsulated formulations were then tested under
in-vitro and in-vivo conditions against stem rot and false smut diseases using rice variety
TN1 in a glasshouse.
The stability and long-term storage ability of the microencapsulated formulations
were demonstrated, with minimal decrease in population density over 90 days. The
combination of microencapsulated TAIK1 and PIK1 exhibited the highest plant growth
promotion and yield parameters. Nanoencapsulated 6PP showed strong inhibition of
pathogen growth.
The study also investigated changes in physiological parameters, soil enzymes,
and chemical properties upon bioformulations treatment. Significant improvements were
observed in plants treated with microencapsulated formulations, and there was a positive
correlation between soil properties and plant growth promotion, and a highly negative
relation with PDI percent of stem rot disease, indicated that change in soil properties upon
encapsulated formulations has an impact on pathogen development and disease severity.
Moreover, the research explored the impact of false smut disease and
microencapsulated bioagent treatment on grain quality and panicle topology. The
combination of MET + MEP treatment showed the most significant changes in these
parameters.
The whole-genome sequencing of PIK1 provided insights into its multifaceted
activities and identified genes associated with various functions, including phytohormone
production,secondary metabolite secretion, CRISPR/Cas genes, adhesion, detoxification,
virulence-related, some volatiles and toxins, and antibiotic resistance.
In conclusion, TAIK1 and PIK1 show promising potential as biocontrol agents
against S. hydrophilum and U. virens in rice. The encapsulation of these bioagents
enhances their efficacy, with nanoencapsulated 6PP being particularly effective in disease
suppression. However, further research is needed for the commercial production and
application of nano-encapsulated metabolite formulations at the farmer level. This thesis
contributes valuable knowledge to the field of agricultural biocontrol and opens new
avenues for sustainable disease management in rice cultivation