MICRO RNA PROFILING, ANTIFUNGAL AND NANOFORMULATION CHARACTERIZATIONS OF MULTI STRESS TOLERANT Trichoderma FUSANTS FOR BIOCONTROL ACTIVITY AGAINST Sclerotium rolfsii Sacc. CAUSING STEM ROT IN GROUNDNUT 2522
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Date
2018-04
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JAU,JUNAGADH
Abstract
The soil borne fungal disease contributed 30-80% of average yield losses in
agricultural crops. Application of chemical fungicides as a chemical disease
management against fungal disease is widely used in farming system. The chemical
control produced environment and health hazards to human and also adversely affects
the beneficial microorganisms in soil and ecosystem. Trichoderma is one of the most
important filamentous fungi common in soil and root ecosystems and used as an
effective biocontrol agent against phytopathogens. There is an emerging need for
biocontrol Trichoderma strains that can tolerate chemical fungicides, which would be
a prerequisite for their application in an integrated disease management strategy
combining chemical and biological means of control. Protoplast fusion is an
imperative tool to develop Trichoderma inter-fusants having desire traits through
genetic manipulation.
The study designed to develop a unique and diverse inter specific protoplast
Trichoderma fusants from T. virens Tvs 12 (mycoparasitic) × T. koningii MTCC 796
(multistress tolerant) having a characteristic of multi stress tolerance (drought, salt
and fungicides) with enhancing antagonistic activity against Sclerotium rolfsii causing
stem rot in groundnut. Total 36 stable fusants were obtained and screened for
mycoparasitism, fungicides [Mancozeb 75 WP (3000 ppm a.i.) + Thiram 75 SD (1000 ppm
a.i.) + Tebuconazole 100 WP (500 ppm a.i.) + Carbendazim 50 WP (5 ppm a.i.)] and abiotic
stress [11.9 % of PEG (MW 6000) (-0.2 MPa or -2 bar osmotic stress for drought
tolerant) + 100 mM sodium chloride (NaCl) (-0.45 MPa or -4.5 bar water potential for
salt tolerant)] tolerance. The results indicated that 20 homozygous progenies showing
characteristic of either one parental strain and 14 heterozygous mutants depicting trait
of both parental strains (i.e. mycoparasitic and multi-stress tolerant). Trichoderma
fusants and parental strains were subjected to in vitro antagonism against Sclerotium
rolfsii (phytopathogen of stem rot of groundnut) up to 12 days after inoculation.
Novel concept of inhibition coefficient representing pathogen biology and biocontrol
related biophysics of Trichoderma fusants were estimated using growth related key
parameters. Results showed differential inhibition coefficient of tested pathogen and
highest inhibition coefficient (92.88 %) of S. rolfsii was observed by inter-stable
fusant Fu21 which also exhibited higher multi stress tolerant capacity. The codominant
SSR molecular analysis revealed highest observed heterozygosity (0.544),
coefficient of gene differentiation (0.526) and gene flow (0.387) by Fu21 indicating
better genetic exploitation of parental strains into that fusant with good genetic purity.
The study explained antagonist fusants microbiological, biochemical and molecular
mechanism thoroughly to restrain fungal test pathogen S. rolfsii. Some antifungal
secretome (31 metabolites: up-regulated), chitinase and β-1,3-glucanase was positive
correlated with inhibition coefficient of test pathogen during in vitro antagonism. Indepth
metabolome study (GC-MS) of antagonists demonstrated the biochemical
behavior and production of different bioactive compounds during biological
interactions.
Identification and characterization of miRNAs, their targeted and novel genes,
functional annotation and gene expression pattern are reported in this work, which
might be enhanced to understand miRNAs regulatory mechanism during antagonistic
activities of Trichoderma parental strains and derived fusants against S. rolfsii. The
study might be elucidated cellular, molecular and biological functions of miRNA and
designed biochemical pathway analysis for specific functions of these regulatory
miRNA for biocontrol activity of best Trichoderma fusant (Fu21). Total 56 unique
miRNA in FU21_CB (down expressed) and 66 unique miRNA in FU21_IB (up
expressed), 14 miRNA found to be down expressed during interaction with pathogen
S. rolfsii compared to normal growth (control) of antagonist FU21. Most of the
conserved miRNA families were predicted to target transcription factor genes; this
suggests that they may play a role in post transcriptional regulation and transcriptional
networks. Other miRNAs were predicted to target genes involved in diverse
physiological and metabolic processes, including the regulation of fungal metabolism,
transport, cell growth and maintenance, and stress responses (biotic and abiotic). Four
novel miRNAs (chi-mir-493, hme-miR-6309, efu-miR-9393 and mdo-miR-144)
found to be induced in Fu21_IB during interactions and correlated their expression
pattern with the pathway panel. The ggo-miR-320b responsible for down expression
in FU21_IB and elevate the pathway for protein phosphorylation, response to stress,
deoxyribonucleotide biosynthetic process, oxidation-reduction process. However, tcamiR-
3824 expressed in Fu21 during interaction (FU21_IB) and found against
response to salt by activating T cell receptor signaling pathway and aminobenzoate
degradation pathway.
The potential bioformulation (Fu21) developed having microbial load 1.83 x
108 cfu.g-1talc. The bioformulation reduced the stem rot disease incidence about 85%
in the field condition which is eco-friendly and cost-effective application. Nanobiotechnology
has immense potentials in agricultural uprising, high reactivity, better
bioavailability, bioactivity and the surface effect of nano-bioformulation for smart
protection of fungal diseases in plants. A novel nanoparticles based green
bioformulation prepared from diverse and potent Trichoderma fusant (Fu21) would be
efficient, eco-friendly and cost effective remedies to control the stem rot infection in
groundnut under adverse condition (climate change). The potent Fu21 was used for
green synthesis of silver nanoparticles (NPs). The morphology and uniformity of NPs
were investigated by using UV-visible spectrophotometer (λ max - 430 nm), particle
size analyzer (62.6 nm), zeta potential analysis (51.2 mv), scanning electron
microscopic (spherical shape). The interactions between protein and NPs were
analyzed by fourier transform infrared spectroscopy. The bioefficacy of Fu21 based
nanoformulations at minimum inhibitory concentration (20 μg Ag/ml green NPs) was
tested for smart protection of S. rolfsii (stem rot) infection in groundnut. The results
revealed that about 95% disease incidence reduced by green nanoformulation Fu21
under pathogen infestation compared to even better than fungicides treatment. Disease
severity index during the entire crop growth was found to be minimum with green
nano-formulation treatment (0.32) compared the pathogen infestation (1.34). The cost
effective green nano-bioformulation product developed as organic input which is
prime needs for promotion of organic cultivation under changing climate -drought,
salt and fungicides. The output of research presented a new business model fitting into
the criteria of green chemistry and sustainable agriculture.
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BIOTECHNOLOGY