Appa Rao Podile

Plant Growth Promoting Rhizobacteria (PGPR): the bugs to debug the root zone.

Interaction of plant growth promoting rhizobacteria (PGPR) with host plants is an intricate and interdependent relationship involving not only the two partners but other biotic and abiotic factors of the rhizosphere region. Survival and establishment of PGPR in the rhizosphere is a major concern of agricultural microbiologists. Various factors that play a determining role include the composition of root exudates, properties of bacterial strain, soil status, and activities of other soil microbes. This review focuses on the different components that affect root colonization of PGPR and the underlying principles behind the success of these bugs to tide over the unfavorable conditions.

Biological Control of Late Leaf Spot of Peanut (Arachis hypogaea) with Chitinolytic Bacteria.

ABSTRACT Late leaf spot (LLS), caused by Phaeoisariopsis personata, is a foliar disease of groundnut or peanut (Arachis hypogaea) with high economic and global importance. Antifungal and chitinolytic Bacillus circulans GRS 243 and Serratia marcescens GPS 5, selected among a collection of 393 peanut-associated bacteria, were applied as a prophylactic foliar spray and tested for control of LLS. Chitin-supplemented application of B. circulans GRS 243 and S. marcescens GPS 5 resulted in improved biological control of LLS disease. Supplementation of bacterial cells with 1% (wt/vol) colloidal chitin reduced lesion frequency by 60% compared with application of bacterial cells alone, in the greenhouse. Chitinsupplemented application of GRS 243 and GPS 5 also resulted in improved and stable control of LLS in a repeated field experiment and increased the pod yields by 62 and 75%, respectively, compared with the control. Chitin-supplemented application of GPS 5 was tested in six onfarm trials, and the increase in pod yields was up to 48% in kharif (rainy season). A 55-kDa chitinase was purified from the cell-free culture filtrate of GPS 5 by affinity chromatography and gel filtration. Purified chitinase of S. marcescens GPS 5 (specific activity 120 units) inhibited the in vitro germination of P. personata conidia, lysed the conidia, and effectively controlled LLS in greenhouse tests, indicating the importance of chitinolysis in biological control of LLS disease by GPS 5.

Biotechnological approaches to develop bacterial chitinases as a bioshield against fungal diseases of plants

Fungal diseases of plants continue to contribute to heavy crop losses in spite of the best control efforts of plant pathologists. Breeding for disease-resistant varieties and the application of synthetic chemical fungicides are the most widely accepted approaches in plant disease management. An alternative approach to avoid the undesired effects of chemical control could be biological control using antifungal bacteria that exhibit a direct action against fungal pathogens. Several biocontrol agents, with specific fungal targets, have been registered and released in the commercial market with different fungal pathogens as targets. However, these have not yet achieved their full commercial potential due to the inherent limitations in the use of living organisms, such as relatively short shelf life of the products and inconsistent performance in the field. Different mechanisms of action have been identified in microbial biocontrol of fungal plant diseases including competition for space or nutrients, production of antifungal metabolites, and secretion of hydrolytic enzymes such as chitinases and glucanases. This review focuses on the bacterial chitinases that hydrolyze the chitinous fungal cell wall, which is the most important targeted structural component of fungal pathogens. The application of the hydrolytic enzyme preparations, devoid of live bacteria, could be more efficacious in fungal control strategies. This approach, however, is still in its infancy, due to prohibitive production costs. Here, we critically examine available sources of bacterial chitinases and the approaches to improve enzymatic properties using biotechnological tools. We project that the combination of microbial and recombinant DNA technologies will yield more effective environment-friendly products of bacterial chitinases to control fungal diseases of crops.

Ethyl Methanesulfonate Mutagenesis–Enhanced Mineral Phosphate Solubilization by Groundnut-Associated Serratia marcescens GPS-5

Twenty-three bacterial isolates were screened for their mineral phosphate–solubilizing (MPS) ability on Pikovskaya and National Botanical Research Institute’s phosphate (NBRIP) agar. The majority of the isolates exhibited a strong ability to solubilize hydroxyapatite in both solid and liquid media. The solubilization in liquid medium corresponded with a decrease in the pH of the medium. Serratia marcescens GPS-5, known for its biocontrol of late leaf spot in groundnut, emerged as the best solubilizer. S. marcescens GPS-5 was subjected to ethyl methanesulfonate (EMS) mutagenesis, and a total of 1700 mutants, resulting after 45 minutes of exposure, were screened on buffered NBRIP medium for alterations in MPS ability compared with that of the wild type. Seven mutants with increased (increased-MPS mutants) and 6 mutants with decreased (decreased-MPS mutants) MPS ability were isolated. All seven increased-MPS mutants were efficient at solubilizing phosphate in both solid and liquid NBRIP medium. Among the increased-MPS mutants, EMS XVIII Sm-35 showed the maximum (40%) increase in the amount of phosphate released in liquid medium compared with wild-type S. marcescens GPS-5, therefore, it would be a useful microbial inoculant in groundnut cultivation. EMS III Sm W, a nonpigmented mutant, showed the lowest solubilization of phosphate among the 6 decreased-MPS mutants.

Synthesis of Long-Chain Chitooligosaccharides by a Hypertransglycosylating Processive Endochitinase of Serratia proteamaculans 568

We describe the heterologous expression and characterization of a 407-residue single-domain glycosyl hydrolase family 18 chitinase (SpChiD) from Gram-negative Serratia proteamaculans 568 that has unprecedented catalytic properties. SpChiD was optimally active at pH 6.0 and 40 °C, where it showed a K(m) of 83 mg ml(-1), a k(cat) of 3.9 × 10(2) h(-1), and a k(cat)/K(m) of 4.7 h mg(-1) ml(-1) on colloidal chitin. On chitobiose, the K(m), k(cat), and k(cat)/K(m) were 203 μM, 1.3 × 10(2) h(-1), and 0.62 h(-1) μM(-1), respectively. Hydrolytic activity on chitooligosaccharides (CHOS) and colloidal chitin indicated that SpChiD was an endo-acting processive enzyme, with the unique ability to convert released chitobiose to N-acetylglucosamine, the major end product. SpChiD showed hyper transglycosylation (TG) with trimer-hexamer CHOS substrates, generating considerable amounts of long-chain CHOS. The TG activity of SpChiD was dependent on both the length and concentration of the oligomeric substrate and also on the enzyme concentration. The length and amount of accumulated TG products increased with increases in the length of the substrate and its concentration and decreased with increases in the enzyme concentration. The SpChiD bound to insoluble and soluble chitin substrates despite the absence of accessory domains. Sequence alignments and structural modeling indicated that SpChiD would have a deep substrate-binding groove lined with aromatic residues, which is characteristic of processive enzymes. SpChiD shows a combination of properties that seems rare among family 18 chitinases and that may resemble the properties of human chitotriosidase.

Warriors at the gate that never sleep: Non-host resistance in plants

The native resistance of most plant species against a wide variety of pathogens is known as non-host resistance (NHR), which confers durable protection to plant species. Only a few pathogens or parasites can successfully cause diseases. NHR is polygenic and appears to be linked with basal plant resistance, a form of elicited protection. Sensing of pathogens by plants is brought about through the recognition of invariant pathogen-associated molecular patterns (PAMPs) that trigger downstream defense signaling pathways. Race-specific resistance, (R)-gene mediated resistance, has been extensively studied and reviewed, while our knowledge of NHR has advanced only recently due to the improved access to excellent model systems. The continuum of the cell wall (CW) and the CW-plasma membrane (PM)-cytoskeleton plays a crucial role in perceiving external cues and activating defense signaling cascades during NHR. Based on the type of hypersensitive reaction (HR) triggered, NHR was classified into two types, namely type-I and type-II. Genetic analysis of Arabidopsis mutants has revealed important roles for a number of specific molecules in NHR, including the role of SNARE-complex mediated exocytosis, lipid rafts and vesicle trafficking. As might be expected, R-gene mediated resistance is found to overlap with NHR, but the extent to which the genes/pathways are common between these two forms of disease resistance is unknown. The present review focuses on the various components involved in the known mechanisms of NHR in plants with special reference to the role of CW-PM components.

Biotechnological approaches for field applications of chitooligosaccharides (COS) to induce innate immunity in plants

Abstract Plants have evolved mechanisms to recognize a wide range of pathogen-derived molecules and to express induced resistance against pathogen attack. Exploitation of induced resistance, by application of novel bioactive elicitors, is an attractive alternative for crop protection. Chitooligosaccharide (COS) elicitors, released during plant fungal interactions, induce plant defenses upon recognition. Detailed analyses of structure/function relationships of bioactive chitosans as well as recent progress towards understanding the mechanism of COS sensing in plants through the identification and characterization of their cognate receptors have generated fresh impetus for approaches that would induce innate immunity in plants. These progresses combined with the application of chitin/chitosan/COS in disease management are reviewed here. In considering the field application of COS, however, efficient and large-scale production of desired COS is a challenging task. The available methods, including chemical or enzymatic hydrolysis and chemical or biotechnological synthesis to produce COS, are also reviewed.

Plant growth-promoting chitinolytic Paenibacillus elgii responds positively to tobacco root exudates.

Bacterial strains from chitin/chitosan-rich soils, from two industries, were screened for their chitinolytic, antifungal, and mineral phosphate solubilization abilities. The isolate SMA-1-SDCH02, positive for all three properties, was selected and identified as Paenibacillus elgii based on morphological and biochemical characters and supported by 16S rRNA gene sequence analysis. P. elgii enhanced the growth of groundnut in terms of shoot height, root length, total chlorophyll, and fresh and dry weight when applied alone or in combination with chitosan. The plant growth-promoting activity of P. elgii was seen in tobacco in a specially designed gnotobiotic setup indicating its capability to promote growth of at least groundnut and tobacco. Metabolite changes in the bacteria, studied using attenuated total reflectance-infrared (ATR-IR) spectroscopy, revealed split bands of amide I at the 1659- and 1636-cm−1 regions when grown in minimal media amended with tobacco root exudates. The difference in ATR-IR bands in the presence of tobacco root exudates indicated production of compounds with differences in functional groups.

Differential interactions and structural stability of chitosan oligomers with human serum albumin and α-1-glycoprotein

Chitosan is a naturally occurring deacetylated derivative of chitin with versatile biological activities. Here, we studied the interaction of chitosan oligomers with low degree of polymerization such as chitosan monomer (CM), chitosan dimer (CD), and chitosan trimer (CT) with human serum albumin (HSA) a major blood carrier protein and α-1-glycoprotein (AGP). Since, HSA and AGP are the two important plasma proteins that determine the drug disposition and affect the fate of distribution of drugs. Fluorescence emission spectra indicated that CM, CD, and CT had binding constants of KCM = 6.2 ± .01 × 105 M−1, KCD = 5.0 ± .01 × 104 M−1, and KCT = 1.6 ± .01 × 106 M−1, respectively, suggesting strong binding with HSA. However, binding of chitooligomers with AGP was insignificant. Thermodynamic and molecular docking analysis indicated that hydrogen bonds and also hydrophobic interaction played an important role in stabilizing the HSA-chitooligomer complexes with free energies of −7.87, −6.35, and −8.4 Kcal/mol for CM, CD, and CT, respectively. Further, circular dichroism studies indicated a minor unfolding of HSA secondary structure, upon interaction with chitooligomers, which are supported with fluctuations of root mean square deviation (RMSD) and radius of gyration (Rg) of HSA. Docking analysis revealed that all three chitooligomers were bound to HSA within subdomain IIA (Site I). In addition, RMSD and Rg analysis depicted that HSA-chitooligomer complexes stabilized at around 4.5 ns. These results suggest that HSA might serve as a carrier in delivering chitooligomers to target tissues than AGP which has pharmacological importance.

Chitinase A from Stenotrophomonas maltophilia shows transglycosylation and antifungal activities.

Stenotrophomonas maltophilia chitinase (StmChiA and StmChiB) genes were cloned and expressed as soluble proteins of 70.5 and 41.6 kDa in Escherichia coli. Ni-NTA affinity purified StmChiA and StmChiB were optimally active at pH 5.0 and 7.0, respectively and exhibited broad range pH activity. StmChiA and StmChiB had an optimum temperature of 40°C and are stable up to 50 and 40°C, respectively. Hydrolytic activity on chitooligosaccharides indicated that StmChiA was an endo-acting enzyme releasing chitobiose and StmChiB was both exo/endo-acting enzyme with the release of GlcNAc as the final product. StmChiA showed higher preference to β-chitin and exhibited transglycosylation on even chain length tetra- and hexameric substrates. StmChiA, and not StmChiB, was active on chitinous polymers and showed antifungal activity against Fusarium oxysporum.