Mukesh Meena

Comparative Evaluation of Biochemical Changes in Tomato (Lycopersicon esculentum Mill.) Infected by Alternaria alternata and Its Toxic Metabolites (TeA, AOH, and AME)

In the present study, we have evaluated the comparative biochemical defense response generated against Alternaria alternata and its purified toxins viz. alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA). The necrotic lesions developed due to treatment with toxins were almost similar as those produced by the pathogen, indicating the crucial role of these toxins in plant pathogenesis. An oxidative burst reaction characterized by the rapid and transient production of a large amount of reactive oxygen species (ROS) occurs following the pathogen infection/toxin exposure. The maximum concentration of hydrogen peroxide (H2O2) produced was reported in the pathogen infected samples (22.2-fold) at 24 h post inoculation followed by TeA (18.2-fold), AOH (15.9-fold), and AME (14.1-fold) in treated tissues. 3,3′- Diaminobenzidine staining predicted the possible sites of H2O2 accumulation while the extent of cell death was measured by Evans blue dye. The extent of lipid peroxidation and malondialdehyde (MDA) content was higher (15.8-fold) at 48 h in the sample of inoculated leaves of the pathogen when compared to control. The cellular damages were observed as increased MDA content and reduced chlorophyll. The activities of antioxidative defense enzymes increased in both the pathogen infected as well as toxin treated samples. Superoxide dismutase (SOD) activity was 5.9-fold higher at 24 h post inoculation in leaves followed by TeA (5.0-fold), AOH (4.1-fold) and AME (2.3-fold) treated leaves than control. Catalase (CAT) activity was found to be increased upto 48 h post inoculation and maximum in the pathogen challenged samples followed by other toxins. The native PAGE results showed the variations in the intensities of isozyme (SOD and CAT) bands in the pathogen infected and toxin treated samples. Ascorbate peroxidase (APx) and glutathione reductase (GR) activities followed the similar trend to scavenge the excess H2O2. The reduction in CAT activities after 48 h post inoculation demonstrate that the biochemical defense programming shown by the host against the pathogen is not well efficient resulting in the compatible host-pathogen interaction. The elicitor (toxins) induced biochemical changes depends on the potential toxic effects (extent of ROS accumulation, amount of H2O2 produced). Thus, a fine tuning occurs for the defense related antioxidative enzymes against detoxification of key ROS molecules and effectively regulated in tomato plant against the pathogen infected/toxin treated oxidative stress. The study well demonstrates the acute pathological effects of A. alternata in tomato over its phytotoxic metabolites.

Mannitol metabolism during pathogenic fungal-host interactions under stressed conditions

Numerous plants and fungi produce mannitol, which may serve as an osmolyte or metabolic store; furthermore, mannitol also acts as a powerful quencher of reactive oxygen species (ROS). Some phytopathogenic fungi use mannitol to stifle ROS-mediated plant resistance. Mannitol is essential in pathogenesis to balance cell reinforcements produced by both plants and animals. Mannitol likewise serves as a source of reducing power, managing coenzymes, and controlling cytoplasmic pH by going about as a sink or hotspot for protons. The metabolic pathways for mannitol biosynthesis and catabolism have been characterized in filamentous fungi by direct diminishment of fructose-6-phosphate into mannitol-1-phosphate including a mannitol-1-phosphate phosphatase catalyst. In plants mannitol is integrated from mannose-6-phosphate to mannitol-1-phosphate, which then dephosphorylates to mannitol. The enzyme mannitol dehydrogenase plays a key role in host–pathogen interactions and must be co-localized with pathogen-secreted mannitol to resist the infection.

Alternaria Toxins: Potential Virulence Factors and Genes Related to Pathogenesis.

Alternaria is an important fungus to study due to their different life style from saprophytes to endophytes and a very successful fungal pathogen that causes diseases to a number of economically important crops. Alternaria species have been well-characterized for the production of different host-specific toxins (HSTs) and non-host specific toxins (nHSTs) which depend upon their physiological and morphological stages. The pathogenicity of Alternaria species depends on host susceptibility or resistance as well as quantitative production of HSTs and nHSTs. These toxins are chemically low molecular weight secondary metabolites (SMs). The effects of toxins are mainly on different parts of cells like mitochondria, chloroplast, plasma membrane, Golgi complex, nucleus, etc. Alternaria species produce several nHSTs such as brefeldin A, tenuazonic acid, tentoxin, and zinniol. HSTs that act in very low concentrations affect only certain plant varieties or genotype and play a role in determining the host range of specificity of plant pathogens. The commonly known HSTs are AAL-, AK-, AM-, AF-, ACR-, and ACT-toxins which are named by their host specificity and these toxins are classified into different family groups. The HSTs are differentiated on the basis of bio-statistical and other molecular analyses. All these toxins have different mode of action, biochemical reactions and signaling mechanisms to cause diseases. Different species of Alternaria produced toxins which reveal its biochemical and genetic effects on itself as well as on its host cells tissues. The genes responsible for the production of HSTs are found on the conditionally dispensable chromosomes (CDCs) which have been well characterized. Different bio-statistical methods like basic local alignment search tool (BLAST) data analysis used for the annotation of gene prediction, pathogenicity-related genes may provide surprising knowledge in present and future.

Expression of chickpea CIPK25 enhances root growth and tolerance to dehydration and salt stress in transgenic tobacco.

Calcium signaling plays an important role in adaptation and developmental processes in plants and animals. A class of calcium sensors, known as Calcineurin B-like (CBL) proteins sense specific temporal changes in cytosolic Ca2+ concentration and regulate activities of a group of ser/thr protein kinases called CBL-interacting protein kinases (CIPKs). Although a number of CIPKs have been shown to play crucial roles in the regulation of stress signaling, no study on the function of CIPK25 or its orthologs has been reported so far. In the present study, an ortholog of Arabidopsis CIPK25 was cloned from chickpea (Cicer arietinum). CaCIPK25 gene expression in chickpea increased upon salt, dehydration, and different hormonal treatments. CaCIPK25 gene showed differential tissue-specific expression. 5′-upstream activation sequence (5′-UAS) of the gene and its different truncated versions were fused to a reporter gene and studied in Arabidopsis to identify promoter regions directing its tissue-specific expression. Replacement of a conserved threonine residue with an aspartic acid at its catalytic site increased the kinase activity of CaCIPK25 by 2.5-fold. Transgenic tobacco plants overexpressing full-length and the high active versions of CaCIPK25 displayed a differential germination period and longer root length in comparison to the control plants. Expression of CaCIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK25 was required for these functions. Expressions of the abiotic stress marker genes were enhanced in the CaCIPK25-expressing tobacco plants. Our results suggested that CaCIPK25 functions in root development and abiotic stress tolerance.

Investigation of genes encoding calcineurin B-like protein family in legumes and their expression analyses in chickpea (Cicer arietinum L.)

Calcium ion (Ca2+) is a ubiquitous second messenger that transmits various internal and external signals including stresses and, therefore, is important for plants’ response process. Calcineurin B-like proteins (CBLs) are one of the plant calcium sensors, which sense and convey the changes in cytosolic Ca2+-concentration for response process. A search in four leguminous plant (soybean, Medicago truncatula, common bean and chickpea) genomes identified 9 to 15 genes in each species that encode CBL proteins. Sequence analyses of CBL peptides and coding sequences (CDS) suggested that there are nine original CBL genes in these legumes and some of them were multiplied during whole genome or local gene duplication. Coding sequences of chickpea CBL genes (CaCBL) were cloned from their cDNAs and sequenced, and their annotations in the genome assemblies were corrected accordingly. Analyses of protein sequences and gene structures of CBL family in plant kingdom indicated its diverse origin but showed a remarkable conservation in overall protein structure with appearance of complex gene structure in the course of evolution. Expression of CaCBL genes in different tissues and in response to different stress and hormone treatment were studied. Most of the CaCBL genes exhibited high expression in flowers. Expression profile of CaCBL genes in response to different abiotic stresses and hormones related to development and stresses (ABA, auxin, cytokinin, SA and JA) at different time intervals suggests their diverse roles in development and plant defence in addition to abiotic stress tolerance. These data not only contribute to a better understanding of the complex regulation of chickpea CBL gene family, but also provide valuable information for further research in chickpea functional genomics.

Structural and Functional Insights into WRKY3 and WRKY4 Transcription Factors to Unravel the WRKY–DNA (W-Box) Complex Interaction in Tomato (Solanum lycopersicum L.). A Computational Approach

The WRKY transcription factors (TFs), play crucial role in plant defense response against various abiotic and biotic stresses. The role of WRKY3 and WRKY4 genes in plant defense response against necrotrophic pathogens is well-reported. However, their functional annotation in tomato is largely unknown. In the present work, we have characterized the structural and functional attributes of the two identified tomato WRKY transcription factors, WRKY3 (SlWRKY3), and WRKY4 (SlWRKY4) using computational approaches. Arabidopsis WRKY3 (AtWRKY3: NP_178433) and WRKY4 (AtWRKY4: NP_172849) protein sequences were retrieved from TAIR database and protein BLAST was done for finding their sequential homologs in tomato. Sequence alignment, phylogenetic classification, and motif composition analysis revealed the remarkable sequential variation between, these two WRKYs. The tomato WRKY3 and WRKY4 clusters with Solanum pennellii showing the monophyletic origin and evolution from their wild homolog. The functional domain region responsible for sequence specific DNA-binding occupied in both proteins were modeled [using AtWRKY4 (PDB ID:1WJ2) and AtWRKY1 (PDBID:2AYD) as template protein structures] through homology modeling using Discovery Studio 3.0. The generated models were further evaluated for their accuracy and reliability based on qualitative and quantitative parameters. The modeled proteins were found to satisfy all the crucial energy parameters and showed acceptable Ramachandran statistics when compared to the experimentally resolved NMR solution structures and/or X-Ray diffracted crystal structures (templates). The superimposition of the functional WRKY domains from SlWRKY3 and SlWRKY4 revealed remarkable structural similarity. The sequence specific DNA binding for two WRKYs was explored through DNA-protein interaction using Hex Docking server. The interaction studies found that SlWRKY4 binds with the W-box DNA through WRKYGQK with Tyr408, Arg409, and Lys419 with the initial flanking sequences also get involved in binding. In contrast, the SlWRKY3 made interaction with RKYGQK along with the residues from zinc finger motifs. Protein-protein interactions studies were done using STRING version 10.0 to explore all the possible protein partners involved in associative functional interaction networks. The Gene ontology enrichment analysis revealed the functional dimension and characterized the identified WRKYs based on their functional annotation.

Antagonistic assessment of Trichoderma spp. by producing volatile and non-volatile compounds against different fungal pathogens

Abstract Trichoderma spp. are well-known biological agents that have significant antagonistic activity against several plant pathogenic fungi. In the present study, Trichoderma spp. were tested in vitro for their antagonistic activity against different spp. of Fusarium and Alternaria viz. Alternaria alternata, A. brassicae, A. solani, Fusarium oxysporum and F. solani using dual plate assay and by the production of volatile and non-volatile compounds. The results obtained revealed that Trichoderma harzianum and T. viride effectively inhibited the growth and spore production of different spp. of Fusarium and Alternaria. The highest growth inhibition was found in A. alternata 62.50% and 60.00% by non-volatile compounds of T. harzianum and T. viride, respectively. Similarly, the volatile compounds inhibit the maximum growth of A. alternata 40.00% and 35.00% by T. harzianum and T. viride, respectively. Volatile and non-volatile compounds of Trichoderma spp. were analysed by GC-MS technique and the properties of distinguished compounds showed antifungal, antimicrobial and antibiotic activities. Volatile compounds of T. harzianum and T. viride showed highest percent abundance for glacial acetic acid (45.32%) and propyl-benzene (41.75%), respectively. In case of non-volatile compounds, T. harzianum and T. viride showed D-Glucose, 6-O-α-D-galactopyranosyl- (38.45%) and 17-Octadecynoic acid (36.23%), respectively. The results of present study confirmed that T. harzianum can be used as a promising biological control agent against Alternaria and Fusarium spp. that cause diseases in various vegetables and crops.

Improvement Strategies, Cost Effective Production, and Potential Applications of Fungal Glucose Oxidase (GOD): Current Updates

Fungal glucose oxidase (GOD) is widely employed in the different sectors of food industries for use in baking products, dry egg powder, beverages, and gluconic acid production. GOD also has several other novel applications in chemical, pharmaceutical, textile, and other biotechnological industries. The electrochemical suitability of GOD catalyzed reactions has enabled its successful use in bioelectronic devices, particularly biofuel cells, and biosensors. Other crucial aspects of GOD such as improved feeding efficiency in response to GOD supplemental diet, roles in antimicrobial activities, and enhancing pathogen defense response, thereby providing induced resistance in plants have also been reported. Moreover, the medical science, another emerging branch where GOD was recently reported to induce several apoptosis characteristics as well as cellular senescence by downregulating Klotho gene expression. These widespread applications of GOD have led to increased demand for more extensive research to improve its production, characterization, and enhanced stability to enable long term usages. Currently, GOD is mainly produced and purified from Aspergillus niger and Penicillium species, but the yield is relatively low and the purification process is troublesome. It is practical to build an excellent GOD-producing strain. Therefore, the present review describes innovative methods of enhancing fungal GOD production by using genetic and non-genetic approaches in-depth along with purification techniques. The review also highlights current research progress in the cost effective production of GOD, including key advances, potential applications and limitations. Therefore, there is an extensive need to commercialize these processes by developing and optimizing novel strategies for cost effective GOD production.

Isolation, characterization and toxicological potential of Alternaria- mycotoxins (TeA, AOH and AME) in different Alternaria species from various regions of India

Alternaria species produce various sorts of toxic metabolites during their active growth and causes severe diseases in many plants by limiting their productivity. These toxic metabolites incorporate various mycotoxins comprising of dibenzo-α-pyrone and some tetramic acid derivatives. In this study, we have screened out total 48 isolates of Alternaria from different plants belonging to different locations in India, on the basis of their pathogenic nature. Pathogenicity testing of these 48 strains on susceptible tomato variety (CO-3) showed 27.08% of the strains were highly pathogenic, 35.41% moderately pathogenic and 37.5% were less pathogenic. Phylogenetic analysis showed the presence of at least eight evolutionary cluster of the pathogen. Toxins (TeA, AOH and AME) were isolated, purified on the basis of column chromatography and TLC, and further confirmed by the HPLC-UV chromatograms using standards. The final detection of toxins was done by the LC-MS/MS analysis by their mass/charge ratio. The present study develops an approach to classify the toxicogenic effect of each of the individual mycotoxins on tomato plant and focuses their differential susceptibility to develop disease symptoms. This study represents the report of the natural occurrence and distribution of Alternaria toxins in various plants from India.

Beneficial Microbes for Disease Suppression and Plant Growth Promotion

Plant growth-promoting microorganisms (PGPMs) constitute the microbes that are intricately associated with the plant system and may consist of rhizospheric bacteria, fungi, mycorrhiza, endophytic fungi, actinomycetes, or those having the mutualistic relationship or nonsymbiotic relationship with plants. One of the most remarkable features of these microbes is the adoption of certain ecological niches or may be occupied with multiple niches at a time in the soil ecosystem that makes way for other species to establish the mutual interactions (physical or biochemical) with other microbes (bipartite) or with plants (tripartite). The plant growth promotion by these microbes involves common mechanisms such as nitrogen fixation, siderophore production, phytohormone production, solubilization of mineral phosphates and secretion of novel secondary metabolites having positive effect on plant health. Some beneficial fungi have been found to promote plant growth through increased photosynthetic rate with improved mineral use efficiency and nutrient uptake, as inoculating these microbes with plants lead into increased chlorophyll content and biomass. These indigenous microbes have been also reported to counteract the different abiotic and biotic stress conditions. The mutual interaction observed between beneficial fungi and pathogenic microbes has been investigated at microscopic level which involves certain physical changes such as coiling of beneficial hyphae around the pathogenic hyphae and some cellular changes such as dissolution of host cytoplasm or secretion of antimicrobial compounds or lytic enzymes in the nearby localities that check the growth and reproduction of pathogenic species. The comprehensive knowledge of the functional mechanism of plant growth promotion by these microbes will help to develop strategies against damages covered by various abiotic and biotic stress conditions, and therefore will help in increasing the agricultural production at a global scale.