Development of next-generation formulation against Fusarium oxysporum and unraveling bioactive antifungal metabolites of biocontrol agents.

Biocontrol agents serve as a sustainable means of controlling wilt caused by the widespread plant pathogen, Fusarium oxysporum f. sp. lycopersici. The present study aimed to develop water dispersible granules (WDG) using response surface methodology (RSM) for Bacillus subtilis MTCC 2274 and Trichoderma harzianum MTCC 3928, and to compare their antifungal efficacy with other formulations. Further, characterization of the bioactive metabolites responsible for biocontrol was performed. A new microbial formulation, WDG, was developed in the present study with talcum powder (substrate), alginic acid (dispersing agent) and acacia gum (wetting agent) (suspensibility 82.23%; wetting time 2.5 min; dispersion time 10.08 min) that fulfilled the guidelines of Collaborative International Pesticides Analytical Council (CIPAC). In planta study demonstrated that WDG of B. subtilis showed maximum reduction in disease incidence (48%) followed by talc formulation of B. subtilis (44%) and WDG of T. harzianum (42%) with profound effect on plant growth promotion. B. subtilis and T. harzianum demonstrated protease (929 and 846 U ml-1 min-1), chitinase (33.69 and 154 U ml-1 min-1), and ß-1,3-glucanase (12.69 and 21.47 U ml-1 min-1) activities. Culture filtrates of B. subtilis and T. harzianum exhibited significant inhibition against mycelial growth of pathogen. The compounds present in the culture filtrates were identified with GC-MS as fatty acids, alkanes, phenols, benzene, pyran derivatives etc. The major non-volatile compounds in bioactive antifungal fraction were identified as derivatives of morpholine and piperdine for T. harzianum and B. subtilis, respectively. The findings propose a multivariate biocontrol mechanism against phytopathogen by production of hydrolytic enzymes, volatile and non-volatile compounds, together with development of an efficient next-generation formulation.

Differential response of photosynthetic apparatus towards alkaline pH treatment in NIES-39 and PCC 7345 strains of Arthrospira platensis.

Alkaline stress is one of the severe abiotic stresses, which is not well studied so far, especially among cyanobacteria. To affirm the characteristics of alkaline stress and the subsequent adaptive responses in Arthrospira platensis NIES-39 and Arthrospira platensis PCC 7345, photosynthetic pigments, spectral properties of thylakoids, PSII and PSI activities, and pigment-protein profiles of thylakoids under different pH regimes were examined. The accessory pigments showed a pH-mediated sensitivity. The pigment-protein complexes of thylakoids are also affected, resulting in the altered fluorescence emission profile. At pH 11, a possible shift of the PBsome antenna complex from PSII to PSI is observed. PSII reaction center is found to be more susceptible to alkaline stress in comparison to the PSI. In Arthrospira platensis NIES-39 at pH 11, a drop of 68% in the oxygen evolution with a significant increase of PSI activity by 114% is recorded within 24 h of pH treatment. Alterations in the cellular ultrastructure of Arthrospira platensis NIES-39 at pH 11 were observed, along with the increased number of plastoglobules attached with the thylakoid membranes. Arthrospira platensis NIES-39 is more adaptable to pH variation than Arthrospira platensis PCC 7345.

Synergistic effect of oilseed cake and biocontrol agent in the suppression of Fusarium wilt in Solanum lycopersicum.

The antagonistic efficacy of a biocontrol agent in combination with oilseed cake against fungal phytopathogens has been sparsely explored. The present study aimed to evaluate the antifungal activity of a biocontrol agent (Trichoderma harzianum MTCC 3928) formulated with oilseed cake (OSC) against Fusarium oxysporum f. sp. lycopersici responsible for causing vascular wilt in Solanum lycopersicum. In in vitro studies, OSC of mustard (Brassica juncea) exhibited significant mycelial inhibition against the pathogen. The volatile plate assay showed mycelial inhibition of 70 and 40% with unautoclaved and autoclaved mustard cakes, respectively. The aqueous extract (10% v/v) of the mustard cake was the most effective with 47.3% mycelial inhibition of pathogen over control. In addition, volatiles and aqueous extract of mustard cake subjected to GC-MS analysis revealed a range of antifungal bioactive compounds with hexanedioic acid, dioctyl ester (16.57%), and oleic acid trimethylsilyl ester (12.41%) being predominant compounds. In in vitro studies, it was noticed that the T. harzianum strain was compatible with mustard cake, and hence used as a growth substrate for its mass multiplication. SEM analysis revealed no distortion in spores and mycelium of T. harzianum grown on the mustard cake. Further, seed germination assay suggested the optimum concentration of mustard cake (10%) supporting the germination rate and economics of formulation development. In in planta assay, the combination of biocontrol agent and mustard cake showed 48% disease reduction, and ~ 40% with T. harzianum alone in comparison to untreated control. Also, the combination of mustard cake and T. harzianum significantly enhanced the growth parameters of S. lycopersicum. The findings of the current study identified an environmentally friendly alternative for mitigation of Fusarium wilt, thereby providing a sustainable option for mitigation of wilt disease and enhancement of plant health.

Analysis of seven putative Na+/H+ antiporters of Arthrospira platensis NIES-39 using transcription profiling and in silico studies: an indication towards alkaline pH acclimation.

Na+/H+ antiporters mediated pH regulation is one of the known mechanism(s), which advocates a possible role of the antiporters in the alkaline pH tolerance of Arthrospira platensis NIES-39. Seven putative Na+/H+ antiporters have been reported in A. platensis NIES-39. Based upon the in silico analysis, the seven putative antiporters were characterized into two different superfamilies, where A1, Q2, L2, and L6 belonged to the CPA1 family whereas C5, D5 and O6 belonged to CPA2 family. The orientation of functionally important residues in both CPA1 and CPA2 subfamily are conserved in modeled Q2 and C5 antiporters. Conserved domain analysis of the seven putative antiporters indicated the presence of nine different kinds of domains. Out of these nine domains, six domains function as monovalent cation-proton antiporters and two as the universal stress protein (Usp) category. Transcription profile of these seven antiporters was also generated at three different pH (7, 9 and 11) and time frames which showed a significant difference in the mRNA levels along with a temporal pattern of the expression profile. The in silico and the real-time PCR analysis put together, suggest the active participation of these seven putative Na+/H+ antiporters in alkaline pH homeostasis of this cyanobacterial strain where CPA1 subfamily antiporters play a major role.