S. Chandra Nayaka

Role of Rhizosphere Microflora in Potassium Solubilization

The K+ is not just the essential nutrient required to support optimal plant growth and yield, it is also an important signaling agent mediating a wide range of plant adaptive responses to abiotic and biotic stresses such as drought, salinity, oxidative stress, and apoptosis. The main source of K+ for plant is soil which is taken up by the plant roots through the epidermal and cortical cells and transported to the shoot and distributed to the leaves. Movement of K+ is facilitated by transport systems present in the cell membrane and the availability of which strongly determines crop yield. It is important to note that only a small percentage of the total K+ of the soil exists in a form available for plant uptake. The remainder is in complex with other elements and organic matter, making it unavailable and even intensive agricultural practices is adding to K+ deficiency in soil. It is already reported that large agricultural areas of world are deficient in K+ availability which is a major concern today. In this connection, efforts to understand the K+ uptake by plants and its solubilization from the K-bearing minerals such as waste muscovite, biotite, feldspars, orthoclase, illite, and mica have been undertaken. Recent investigations have shown that organic exudates of some microorganisms such as Pseudomonas spp., Burkholderia spp., Acidithiobacillus ferrooxidans, Bacillus mucilaginosus, B. edaphicus, B. megaterium, and Aspergillus spp., and even plant roots play a key role in releasing non-available K+ from the minerals. The list of rhizosphere microflora with potassium solubilization is increasing, and hence the present chapter discusses the mechanism of K+ solubilization and its role in signaling its uptake system in plants. Plant species effective in K+ uptake and K+-solubilizing microbial populations may be further key factors that control the K+ release from soil minerals.

Zearalenone induced toxicity in SHSY-5Y cells: The role of oxidative stress evidenced by N-acetyl cysteine.

Zearalenone (ZEN) is a mycotoxin from Fusarium species commonly found in many food commodities and are known to cause reproductive disorders, genotoxic and immunosuppressive effects. Although many studies have demonstrated the cytotoxic effects of ZEN, the mechanisms by which ZEN mediates its cytotoxic effects appear to differ according to cell type and route of exposure. Meantime, the available information on the neurotoxic effects of ZEN is very much limited. In the present study we evaluated the role of oxidative stress in ZEN mediated neurotoxicity in SH-SY5Y cells and investigated the possible underlying mechanism. ZEN induced ROS formation and elevated levels of MDA, loss of mitochondrial membrane potential (MMP) and increase in DNA damage in a dose dependent manner as assessed by COMET assay and agarose gel electrophoresis. However, there was no DNA damage by plasmid breakage assay at 6, 12 and 24h time points. DAPI staining showed apoptotic nuclei at 12 and 24h. Further, ZEN treated SH-SY5Y cells showed a marked suppressive effect on the neuronal gene expression. Use of an antioxidant N-acetylcysteine (NAC) reversed the toxin-induced generation of ROS and also attenuated loss of MMP. Collectively, these results suggest that ROS is the main upstream signal leading to increased ZEN mediated neurotoxicity in SH-SY5Y cells.

Rosmarinic acid mediated neuroprotective effects against H2O2-induced neuronal cell damage in N2A cells.

AIMS Oxidative stress plays a key role in several ailments including neurodegenerative conditions. The aim of the study was to demonstrate the effect of rosmarinic acid (RA) in preventing oxidative stress related death of neuronal cell lines. MAIN METHODS In the present study, we demonstrated direct neuroprotective effect of RA using H2O2-induced oxidative challenge in N2A mouse neuroblastoma cells. The mechanism of neutralization of H2O2-induced toxicity by RA was evaluated using MTT, lactate dehydrogenase, mitochondrial membrane potential (MMP), intracellular ROS, and comet assays. Up-regulation of brain neuronal markers at molecular level was performed by RT-PCR. KEY FINDINGS Results presented in the paper indicate that H2O2-induced cytotoxicity in N2A cells was suppressed by treatment with RA. Moreover, RA is very effective in attenuating the disruption of lactate dehydrogenase, mitochondrial membrane potential and intracellular ROS. Pretreatment with RA significantly prevents genotoxicity (3.7-fold, p<0.01) and promotes the up-regulation of tyrosine hydroxylase (TH) (4.5-fold, p<0.01), and brain-derived neurotrophic factor (BDNF) genes (5.4-fold, p<0.01) against H2O2-induced cytotoxicity in N2A cells. SIGNIFICANCE Our results revealed that N2A cells are suitable cellular models to evaluate neuroprotective effects of RA, and suggest that RA may potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress.

Seed biopriming with novel strain of Trichoderma harzianum for the control of toxigenic Fusarium verticillioides and fumonisins in maize

Fusarium verticillioides is one of the most important fungal pathogens in maize causing both pre- and post-harvest losses and also capable of producing Fumonisins. In the present study attempts have been made for screening potential T. harzianum from native rhizosphere and to study its effect on Fusarium ear rot disease, fumonisin accumulation in different maize cultivars grown in India. Eight isolates of T. harzianum were isolated and T. harzianum isolate Th-8 exhibited better antifungal activity than carbendizim. Th-8 was formulated in different solid substrates like wheat bran, paddy husk, talcum powder and cornstarch. Maize seeds of kanchan (moderately resistant), pioneer (resistant) and sweet corn (susceptible) were selected for laboratory and field studies and these seeds were treated with a conidial suspension of T. harzianum at the rate of 1 × 108 spore/ml and formulation at the rate of 10 g/kg. Treated seeds were subjected to evaluate F. verticillioides incidence, seed germination, seedling vigour and field emergence, yield, thousand seed weight and fumonisin production. It was found that the pure culture of T. harzianum was more effective in reducing the F. verticillioides and fumonisin incidence followed by Talc formulation than the carbendizim treated and untreated control. Formulations of T. harzianum were effective at reducing the F. verticillioides and Fumonisin infection and also increasing the seed germination, vigour index, field emergence, yield, and thousand seed weight in comparison with the control.

A novel PCR–DNA probe for the detection of fumonisin-producing Fusarium species from major food crops grown in southern India

Fumonisins are a group of 20 chemically related toxic fungal metabolites mainly produced by Fusarium verticillioides and Fusarium proliferatum. In this study, attempt has been made to develop rapid detection of fumonisin-producing Fusarium species from freshly harvested rice and finger millet by novel and sensitive polymerase chain reaction–deoxyribonucleic acid (PCR–DNA) probe. One set of oligonucleotide primers was designed targeting FUM13 gene involved in fumonisin biosynthesis and PCR–DNA probe was developed for dot-blot hybridization assay. The sensitivity of developed PCR–DNA probe was determined; it could detect 10 pg genomic DNA and 1 pg of purified PCR product. To determine the practical usefulness, the developed PCR–DNA probe was evaluated onto the rice grains that were artificially inoculated with toxigenic fungal spores. Sensitivity of the developed probe was found to be 1 × 106 CFU per gram of the rice sample using dot-blot hybridization assay. The specificity of the DNA probe was validated by testing against an array of Fusarium, including Gibberella zeae clade and other fungal strains. All PCR–DNA probe results were further cross-checked with conventional high performance thin layer chromatography (HPTLC) method. PCR–DNA probe results were equivocally matched with the HPTLC method. The novel PCR–DNA probe developed in this study may find application in rapid detection of fumonisin-producing Fusarium isolates from contaminated cereal grains.

Cerebroside mediated elicitation of defense response in chilli (Capsicum annuum L.) against Colletotrichum capsici infection

Abstract Enhancing the host resistance by using naturally occurring elicitors derived from pathogenic organisms is emerging as an ecofriendly approach in plant disease management. Cerebrosides, categorized as glycosphingolipids, were isolated and partially purified from the wilt causing fungus (Fusarium oxysporum f. sp. lycopersici). Cerebroside treatment significantly reduced the anthracnose disease incidence under greenhouse conditions. Cerebroside elicitors were found to stimulate the early H2O2 accumulation followed by the production of plant defense-related enzymes such as Phenylalanine ammonia lyase (PAL), Peroxidase (POX), Polyphenol oxidase (PPO), and Lipoxygenase (LOX) when applied to chilli (Capsicum annuum L.) plants by spray treatment and also induced the accumulation of capsidiol. Defense-related enzyme activities were increased by the elicitor treatment and an high level in activity was maintained during the experimental period. Under greenhouse conditions, the cerebroside elicitors effectively protected chilli plants against infection by anthracnose causing organism, Colletotrichum capsici.

Hepatoprotective action of Orthosiphon diffusus (Benth.) methanol active fraction through antioxidant mechanisms: An in vivo and in vitro evaluation

ETHNOPHARMACOLOGICAL RELEVANCE Preparations of Orthosiphon diffusus (Benth.) have been used by folk medicinal practitioners in the Western Ghats of India for treating inflammation, hepatitis and jaundice for many years and their effectiveness is widely acclaimed among the tribal communities. AIM OF THE STUDY To evaluate the mechanisms behind the antioxidant and hepatoprotective potential of Orthosiphon diffusus methanol active fraction (MAF) using in vivo (rat) and in vitro (cell culture) models. MATERIALS AND METHODS Neutralization of CCl4-induced hepatotoxicity by MAF was evaluated in rats. Towards this, serum levels of hepatic injury markers (lactate dehydrogenase and alkaline phosphatase), antioxidant enzymes in the liver homogenates, and histological examination were performed. In in vitro studies, mechanisms of neutralization of H2O2-induced toxicity by MAF using MTT, Comet assay and up-regulation of antioxidant enzymes at genetic level (RT-PCR) was performed in HepG2 cells. RESULTS Rats pre-treated with Orthosiphon diffusus MAF demonstrated significantly reduced levels of serum LDH (1.3-fold, p<0.05) and ALP (1.6-fold, p<0.05). Similarly, multiple dose MAF administration demonstrated significantly enhanced levels (p<0.05) of antioxidant enzymes in the liver homogenates. Histological analysis revealed complete neutralization of CCl4-induced liver injury by the extract. The in vitro studies demonstrated that, pre-treatment of MAF effectively prevented H2O2-induced oxidative stress, genotoxicity and significantly enhanced (~6-fold, p<0.01) expression of genes for antioxidant enzymes. CONCLUSIONS Orthosiphon diffusus MAF demonstrated significant hepatoprotection against CCl4-induced hepatotoxicity by antioxidant mechanisms comparable to silymarin. H2O2-induced oxidative stress was completely neutralized by MAF through enhanced expression of genes for antioxidant enzymes. Therefore, this study validates the use of Orthosiphon diffusus by folk medicinal practitioners in India. Further, MAF of Orthosiphon diffusus can serve as a strong candidate for the development of herbal hepatoprotective agents.

Chemical and Molecular Methods for Detection of Toxigenic Fungi and Their Mycotoxins from Major Food Crops

Mycotoxins are the secondary metabolites produced by certain molds on a wide range of agricultural commodities and are closely related to human and animal food chains. Mycotoxins are capable of causing disease in humans and other animals, and their detection is largely dependent on the sample matrix and the type of fungus causing their contamination. The strict regulations on trade of contaminated grains and seeds and other produce in industrial countries lead to economic burdens on farmers. In developing countries, the situation is aggravated where regulations may be nonexistent or not enforced and where consumption of home-grown cereals leads to a wide exposure to toxins. Important mycotoxins that occur quite often in food are deoxynivalenol/nivalenol, trichothecenes, zearalenone, ochratoxin A fumonisins, and aflatoxins. High concentrations of mycotoxins such as aflatoxins are consumed by humans in areas of the world with higher-than-average levels of liver cancer, childhood malnutrition, and disease. This chapter introduces rapid, robust, and user-friendly protocols currently applied in the identification of toxigenic fungi and important mycotoxins.

Analysis of genetic and aflatoxin diversity among Aspergillus flavus isolates collected from sorghum seeds.

Thirty‐four Aspergillus flavus isolates were recovered from sorghum seeds sampled across five states in India. Our study included (1) species confirmation through PCR assay, (2) quantification of total aflatoxin concentrations by the indirect competitive‐ELISA (ic‐ELISA) method, and (3) analysis of molecular diversity among the A. flavus isolates using β‐tubulin, ITS, and ISSR markers. Among the isolates studied, 28 were found to be positive for the production of aflatoxins. ITS and β‐tubulin phylogenetic analysis segregated the A. flavus sample population into two major groups or clades with little to no subdivision based on geography. In contrast, ISSR analysis also separated the A. flavus isolates into two main clusters, showing a distance of 0.0–0.5, with one cluster exhibiting a high level of diversity though no geographic or chemotype subdivision could be observed. The majority of sampled A. flavus isolates were highly toxigenic, and also highly diversified in terms of toxin‐producing potential in‐vitro. Genetic diversity among the sorghum isolates of A. flavus further warrants the development of appropriate farming management practices as well as improved aflatoxin detection measures in India.

Draft genome sequence of Sclerospora graminicola, the pearl millet downy mildew pathogen

Highlights • We sequenced the downy mildew pathogen, which is one of the most important production constraints for pearl millet.• In a maiden attempt, the whole-genome of Sclerospora graminicola pathotype 1 from India was sequenced and annotated.• The overall genome coverage achieved was 40×.• Estimate genome size of S. graminicola was 299.9 Mb.• Out of 65,404 genes that were predicted, a total of 38,120 genes were annotated.