Veerubommu Shanmugam

Extracellular Chitinases of Fluorescent Pseudomonads Antifungal to Fusarium oxysporum f. sp. dianthi Causing Carnation Wilt

Vascular wilt of carnation caused by Fusarium oxysporum f. sp. dianthi (Prill. & Delacr.) W. C. Synder & H.N. Hans inflicts substantial yield and quality loss to the crop. Mycolytic enzymes such as chitinases are antifungal and contribute significantly to the antagonistic activity of fluorescent pseudomonads belonging to plant-growth-promoting rhizobacteria. Fluorescent pseudomonads antagonistic to the vascular wilt pathogen were studied for their ability to grow and produce chitinases on different substrates. Bacterial cells grown on chitin-containing media showed enhanced growth and enzyme production with increased anti-fungal activity against the pathogen. Furthermore, the cell-free bacterial culture filtrate from chitin-containing media also significantly inhibited the mycelial growth. Both the strains and their cell-free culture filtrate from chitin-amended media showed the formation of lytic zones on chitin agar, indicating chitinolytic ability. Extracellular proteins of highly antagonistic bacterial strain were isolated from cell-free extracts of media amended with chitin and fungal cell wall. These cell-free conditioned media contained one to seven polypeptides. Western blot analysis revealed two isoforms of chitinase with molecular masses of 43 and 18.5 kDa. Further plate assay for mycelial growth inhibition showed the 43-kDa protein to be antifungal. The foregoing studies clearly established the significance of chitinases in the antagonism of fluorescent pseudomonads, showing avenues for possible exploitation in carnation wilt management.

Genetic relatedness of Trichoderma isolates antagonistic against Fusarium oxysporum f.sp. dianthi inflicting carnation wilt

Twenty-eight isolates of Trichoderma belonging to four different species were screened in vitro for their antagonistic ability against Fusarium oxysporum f.sp. dianthi causing carnation wilt. Three different levels of antagonism observed in dual plate assay were further confirmed by cell-free culture filtrate experiments. Isolates showing class I level of antagonism produced maximum lytic enzymes, chitinases and β-1,3-glucanases. Genetic variability of 25 selected isolates was assessed by random amplified polymorphic DNA technique and the amplified products were correlated for their level of antagonism. Unweighed pair-group method with arithmetical averages cluster analysis revealed prominent inter-and intraspecific genetic variation among the isolates. Based on their genetic relationship, the isolates were mainly distributed into 3 major groups representing T. atroviride, T. pseudokoningii and T. harzianum, with 20–35% interspecific dissimilarity. However, the polymorphism shown by the isolates did not correlate to their level of antagonism.

Purification and characterization of an extracellular 24 kDa chitobiosidase from the mycoparasitic fungus Trichoderma saturnisporum.

A Trichoderma saturnisporum Hamill isolate GITX‐Panog (C) exhibiting strong chitinolytic and antifungal activity against Fusarium oxysporum f.sp. dianthi, the causal agent of vascular wilt in carnation was used to purify extracellular chitobiosidase using Czapek‐Dox broth amended with the fungal mycelium as the carbon source. The protein was purified by precipitation with ammonium sulphate, followed by DEAE‐Cellulose anion‐exchange and Sephacryl S‐200 high resolution gel filtration chromatography. The purity of the enzyme was determined by SDS‐PAGE, with an estimated molecular mass of 24 kDa. In native gel assay with 4‐methylumbelliferyl ‐N,N ′ diacetyl‐β‐D‐chitobioside (4‐Mu‐(GluNAc)2, the purified chitobiosidase was visualized as single fluorescent band. Enzyme activity towards short oligomeric natural substrates indicated that the enzyme has properties that are characteristic to exochitinases. The enzyme was active up to 60 °C and at pH 4.0, and displayed maximum stability at 50 °C. Mn2+ and Zn2+ stimulated the enzyme activity by 63% and 41%, respectively. The Km and Vmax values of the purified enzyme for 4‐Mu‐(GluNAc)2were 338.9 μM ml–1 and 0.119 μM ml–1 min–1, respectively. This appears to be the first report of characterization of a chitobiosidase from antagonistic Trichoderma saturnisporum. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Cloning and characterization of a bifunctional glycosyl hydrolase from an antagonistic Pseudomonas putida strain P3(4)

A fluorescent pseudomonad strain P3(4) showing chitinolysis on chitinase detection agar and antagonism against Fusarium oxysporum f.sp dianthi causing vascular wilt of carnation was isolated from pea rhizosphere soil. PCR primers specific for glycosyl hydrolase family 5 (GH5) of Pseudomonas putida isolate KT2440 amplified a 947 bp fragment of the GH5 gene from P3(4). Cloning of this gene into Escherichia coli M15 using an expression vector pQE‐30UA and screening on chitin and chitosan detection agar identified one positive clone (Pchi+). Sequence analysis of the cloned insert revealed an open reading frame of 947 nucleotides corresponding to a protein of 315 amino acids with a predicted molecular mass of 38.0 kDa. The deduced amino acid sequence of the open reading frame (gene product/GH) showed 83–84% homology to the GH5 of P. putida strains F1 and KT2440, respectively. The purified enzyme was homogenous, as examined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and was visualized as single fluorescent band in native gel assay with 4‐methylumbelliferyl‐N ‐acetyl‐β;‐D‐glucosaminide and glycol chitosan, respectively. For hydrolysis of 4‐nitrophenyl‐N ‐acetyl‐β;‐D‐glucosaminide (pNP‐(GlcNAc) and colloidal chitosan, the enzyme had an optimal temperature of 40 °C, and was stable within the temperature range of 10 °C to 40 °C. The enzyme showed an optimal pH of 3.5, with maximum stabilities at 5.0 and 5.5 for hydrolysis of pNP‐(GlcNAc) and colloidal chitosan, respectively. Fe3+ and Cu2+ stimulated chitinase and chitosanase activities by 74.2 and 51.4%, respectively. The purified GH displayed 70 and 45% inhibition of spore germination of the pathogenic fungi, Fusarium oxysporum f.sp. dianthi and Alternaria solani, respectively. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Bacterial diversity and soil enzyme activity in diseased and disease free apple rhizosphere soils

A culture-independent survey of the bacterial diversity in rhizosphere soils of diseased (scab) and disease-free apple trees was conducted to assess the role of bacteria in disease suppression. Community DNA was extracted from soil samples and amplified by PCR using primers specific for bacterial 16S rRNA gene sequences. Clone libraries were constructed with the PCR products and analysed based on amplified rDNA restriction analysis (ARDRA) patterns. The phylotypes and their frequency distribution in both libraries indicated that the phylotype did not represent a single group. Rarefaction curve, and Shannon and Simpson diversity indices exhibited insignificant diversity differences between the samples in terms of bacterial community composition, whereas more chitinase and β-1,3 glucanase activities were recorded in samples from disease-free trees than from diseased trees. Based on the operational taxonomic units identified in ARDRA, 80 representative clones were selected from the libraries and partially sequenced. Sequence similarity searches with the resulting sequences identified the dominance of uncultured bacteria to the extent of 70% and 72.5% in disease free and diseased rhizospheres, respectively. The foregoing studies conclude a possible role for enhanced microbial activity in terms of enzyme production in tree (apple) health, although no distinct partitioning of composition or significant diversity of bacterial communities inhabiting diseased and disease-free rhizosphere soils was observed at any given time.

Unraveling the multilevel aspects of least explored plant beneficial Trichoderma saturnisporum isolate GITX-Panog (C)

Species of Trichoderma offer an eco-friendly and economical solution to the management of plant pathogens in agricultural crops. The secretion and spectrum of different bioactive molecules by Trichoderma spp. is known to follow adaptive and species/strain behavior, depending upon the edaphic conditions. In present study, GITX-Panog (C), an antagonistic isolate of Trichoderma saturnisporum against Fusarium oxysporum was screened for the production of different glycosyl hydrolases and secondary metabolites. The evaluation of antagonistic isolates for lytic enzymes and secondary metabolites revealed their significance as plant probiotics. Profiling of lytic enzymes such as chitinases and β-glucanases secreted by T. saturnisporum GITX-Panog (C) in the presence of autoclaved mycelium of F. oxysporum and colloidal chitin or laminarin showed increased enzyme activities. Production of bioactive secondary metabolites such as siderophore, peptaibols, 6-pentyl-α-pyrone, and the known potential of these compounds in mineral acquisition and suppression of plant F. oxysporum, revealed the potential significance of Trichoderma saturnisporum isolate GITX-Panog C in agricultural application against biotic and abiotic stresses. The antagonistic isolate also showed significant increases in fresh biomass and seed production in Arabidopsis thaliana in the greenhouse compared to controls. Thus, the current study highlights the multiple attributes of an antagonistic isolate of T. saturnisporum for future agricultural applications as a plant probiotic agent.