Muthukrishnan Sathiyabama

Biological preparation of chitosan nanoparticles and its in vitro antifungal efficacy against some phytopathogenic fungi

The aim of the present study was to prepare Chitosan nanoparticles through biological method with high antifungal activities. Chitosan nanoparticles were prepared by the addition of anionic proteins isolated from Penicillium oxalicum culture to chitosan solutions. The formation of chitosan nanoparticles was preliminary confirmed by UV-vis spectrophotometric analysis. The physico-chemical properties of the chitosan nanoparticles were determined by size and zeta potential analysis, FTIR analysis, HRTEM and XRD pattern. The chitosan nanoparticles were evaluated for its potential to inhibit the growth of phytopathogens viz., Pyricularia grisea, Alternaria solani, Fusarium oxysporum. It is evident from our results that chitosan nanoparticles inhibit the growth of phytopathogens tested. Chitosan nanoparticle treated chickpea seeds showed positive morphological effects such as enhanced germination%, seed vigor index and vegetative biomass of seedlings. All these results indicate that chitosan nanoparticle can be used further under field condition to protect various crops from the devastating fungal pathogens as well as growth promoters.

Preparation of Chitosan nanoparticles and its effect on detached rice leaves infected with Pyricularia grisea.

The aim of the present study was to prepare chitosan nanoparticles to evaluate their effect on protection of rice plants from blast fungus. Nanoparticles were prepared using the ionic gelation method by the interaction of Chitosan and sodium tripolyphosphate. The particle size, polydispersity index, zetapotential and structure was confirmed by DLS, FTIR, TEM and XRD. The Chitosan nanoparticle was evaluated for suppression of rice blast fungus (Pyricularia grisea) under the detached leaf condition. It is evident from our results that chitosan nanoparticle have potential in suppressing blast disease of rice which can be used further under field condition to protect rice plants from the devastating fungus.

Preparation of β-D-glucan nanoparticles and its antifungal activity.

This work demonstrates the preparation of β-D-glucan (isolated from the cell wall of Pythium aphanidermatum) nanoparticles through the addition of 2% (w/v) sodium hydroxide to β-D-glucan solution with constant stirring at 90°C. Addition of sodium tripolyphosphate (TPP) aids the stable formation of nanoparticles. Fourier transform infrared (FTIR) spectroscopy confirmed phosphoric groups of TPP linked with OH group of β-D-glucan in the nanoparticles. The formation of nanoparticles was observed by the peak at 386 nm using UV-vis spectroscopy. The average size of nanoparticle as determined by Zetasizer was about 60 nm, while the zeta potential was negative. Scanning electron microscope image showed spherical, smooth and almost homogenous structure for nanoparticles with size ranging from 20 to 50 nm. Further analysis by TEM, indicated polydispersity with an average size of 20-30 nm. The XRD analysis confirmed the crystalline structure of β-glucan nanoparticle. The prepared nanoparticles exhibited antifungal activity against P. aphanidermatum, a devastating fungus which affects major crop plants.

Foliar application of β-D-glucan nanoparticles to control rhizome rot disease of turmeric.

The soilborne Oomycete Pythium aphanidermatum is the causal agent of rhizome rot disease, one of the most serious threats to turmeric crops. At present, effective fungicides are not available. Researches on nanoparticles in a number of crops have evidenced the positive changes in gene expression indicating their potential use in crop improvement. Hence, experiments were carried out to determine the effect of β-D-glucan nanoparticles (nanobiopolymer) in protection of turmeric plants against rot disease by the way of products that reinforce plants own defense mechanism. Foliar spray of β-D-glucan nanoparticles (0.1%, w/v) elicited marked increase in the activity of defense enzymes such as peroxidases (E.C.1.11.1.7), polyphenol oxidases (E.C.1.14.18.1), protease inhibitors (E.C.3.4.21.1) and β-1,3-glucanases (E.C.3.2.1.39) at various age levels. Constitutive and induced isoforms of these enzymes were investigated during this time-course study. β-D-glucan nanoparticles (GNPs) significantly reduced the rot incidence offering 77% protection. Increased activities of defense enzymes in GNPs-applied turmeric plants may play a role in restricting the development of disease symptoms. These results demonstrated that GNPs could be used as an effective resistance activator in turmeric for control of rhizome rot disease.

Fungal cell wall polymer based nanoparticles in protection of tomato plants from wilt disease caused by Fusarium oxysporum f.sp. lycopersici.

Cell wall polymer (chitosan) was isolated from Fusarium oxysporum f.sp. lycopersici. They were cross linked with sodium tripolyphosphate (TPP) to synthesize nanoparticles (CWP-NP). The nanoparticles were characterized by FTIR, DLS, SEM, XRD and NMR analyses. The isolated CWP-NP exhibit antifungal activity under in vitro condition. The foliar application of the CWP-NP to tomato plants challenged with F. oxysporum f. sp. lycopersici showed delay in wilt disease symptom expression and reduce the wilt disease severity. Treated plants also showed enhanced yield. These results suggested the role of the CWP-NP in protecting tomato plants from F. oxysporum f.sp. lycopersici infection.

Gymnemagenin-producing Endophytic Fungus Isolated from a Medicinal Plant Gymnema sylvestre R.Br

Gymnema sylvestre is a plant containing the triterpenoid gymnemagenin, which is used in the pharmaceutical industry as an antidiabetic agent. The objective of this study was to determine whether endophytic fungi, isolated from G. sylvestre, produce gymnemagenin. We isolated an endophytic fungal strain from the leaves of G. sylvestre which produces gymnemagenin in the medium. The fungus was identified as Penicillium oxalicum based on morphological and molecular methods. The strain had a component with the same TLC Rf value and HPLC retention time as authentic gymnemagenin. The presence of gymnemagenin was further confirmed by FTIR, UV, and 1H NMR analyses.

Chitosan nanoparticle induced defense responses in fingermillet plants against blast disease caused by Pyricularia grisea (Cke.) Sacc.

The in vitro antifungal properties of chitosan nanoparticle and its role in protection of fingermillet plants from blast disease were evaluated. Chitosan nanoparticle inhibited the radial growth of Pyricularia grisea indicating the antifungal property. Application of chitosan nanoparticle delayed blast symptom expression on fingermillet leaves for 25days while it was on 15day in control plants. Chitosan naoparticle was able to induce the reactive oxygen species and the level of peroxidase actvitiy in leaves of fingermillet, which might be the reason for delayed symptom. The treated plants showed reduced disease incidence when compared to untreated control plants. These results suggested the role of chitosan nanoparticle in protecting fingermillet plants from P. grisea infection.

Production, partial purification and characterization of protease from a phytopathogenic fungi Alternaria solani (Ell. and Mart.) Sorauer

An alkaline serine protease producing strain Alternaria solani was optimized for its enzyme production under submerged conditions. The maximum production of protease by A. solani was achieved by using sodium nitrate at the optimum concentration of 0.2% w/v. A. solani produced higher quantities (3.75 [unit/mg of protein]) of an inducible extracellular proteases on day 9 after incubation in czapeks dox broth medium amended with 1% casein as an inducer at pH 8.5, temperature 27 °C and 3% sucrose as carbon source. Extracellular proteases were precipitated by ammonium sulphate saturation (80%) method and purified on Sephadex G-100 column chromatography. The molecular mass of SDS-PAGE and Sephadex G-100 Column Gel permeation chromatography purified protease was estimated to 42 kDa. In addition, trypsin digestion of 42 kDa protein band was carried out and analyzed by MALDI-TOF for the identification of protease. The sequence IKELATNGVVTNVK (378-391) segment of the alkaline serine protease was found by using MS/MS spectrum at 1485 m/z from the purified fraction. It showed optimal activity at 50 °C and pH 9-10 and broad pH stability between pH 6-12. The protease activity was inhibited by phenyl methyl sulfonyl fluoride (PMSF), all the results indicated that the presence of a serine residue in the active site and is thus most likely a member of the serine protease family. This may function as a virulence protein during pathogenesis by A. solani. The results suggested that the presence of appreciable extracellular proteolytic activity in filamentous fungi may serve as a marker of their phytopathogenicity.

Lovastatin-producing endophytic fungus isolated from a medicinal plant Solanum xanthocarpum

Lovastatin is a potent drug for lowering blood cholesterol. An endophytic fungus Phomopsis vexans was isolated from the healthy leaf tissues of Solanum xanthocarpum, a medicinal plant, and screened for lovastatin production. The fungus was identified by their characteristic cultural morphology and molecular analysis. The strain had a component with the same TLC Rf value and HPLC retention time as authentic lovastatin. The presence of lovastatin was further confirmed by FT-IR, UV, 1H, 13C NMR and LC–MS analyses. The amount of lovastatin produced by this endophytic fungus was quantified to be 550 mg/L, and thus the fungus can serve as a potential material to improve the production of lovastatin.

Proteases from phytopathogenic fungi and their importance in phytopathogenicity

Phytopathogenic fungi, causal agents of some of the world’s most serious plant diseases, can significantly reduce yields during large-scale agricultural production. Among the numerous hydrolytic enzymes they produce for nutritional and/or pathogenicity purposes, hydrolases and proteases are required for their growth and survival. The present review focuses on extracellular and/or secretory proteases from phytopathogenic fungi. Several extracellular proteases have been identified that contribute to fungal growth, infection structure formation, cell wall degradation, proteolytic processing of pathogenesis-related proteins and that act as elicitors of defense responses. In this review, the positive correlation between protease secretion and disease aggressiveness and/or necrosis is highlighted. The involvement of various fungal proteases in pathogenic mechanisms makes them potential targets for designing protease inhibitors that may provide an improved way to combat plant diseases, which in turn will reduce dependence on fungicides.