Anil Prakash

Bacillus as PGPR in Crop Ecosystem

Gram-positive bacteria, in particular, members of group Bacillus, are among the best-studied experimental systems in bacteriology. Research, in Bacillus subtilis is remarkably diverse, including genetics, biochemistry, cell biology, and ecology, thus has an enormous impact on both basic and applied biology. Multiple species of Bacillus and Paenibacillus occur in the agricultural fields that can promote the crop health in different ways. Some of these species directly stimulate plant growth either through enhancement in acquisition of nutrients or through stimulation of host plant’s defense mechanisms prior to infection; other species can inhibit or suppress the populations of pathogenic microorganisms and/or pests. Although the distribution, diversity, and population dynamics of these two genera have been widely studied using a variety of techniques, much remains to be learned if we are to improve both basic studies of plant–microbe interactions and bacterial ecology, as well as the efforts to improve agricultural technologies. Biological control, using microorganisms to suppress plant disease, offers a powerful alternative to the use of synthetic chemicals. Many species of Bacillus and related forms are potential biological control agents against various pathogenic microbes. Their spore forming ability makes them an ideal candidate for developing efficient biopesticide products from technological point of view. Many isolates of Bacillus spp. have been developed as biocontrol agents (BCAs) of plant pests and pathogens. However, a greater understanding of their ecology including diversity, distribution, and physiology of this Gram-positive genus will be helpful for identification of new strains to be used as successful BCAs. Development of molecular and genomic tools offers new possibilities for improving the selection, characterization, and management of biological control including modifications of wild-type strains to improve their ability for controlling soilborne diseases.

Induced systemic resistance (ISR) in plants: mechanism of action

Plants possess a range of active defense apparatuses that can be actively expressed in response to biotic stresses (pathogens and parasites) of various scales (ranging from microscopic viruses to phytophagous insect). The timing of this defense response is critical and reflects on the difference between coping and succumbing to such biotic challenge of necrotizing pathogens/parasites. If defense mechanisms are triggered by a stimulus prior to infection by a plant pathogen, disease can be reduced. Induced resistance is a state of enhanced defensive capacity developed by a plant when appropriately stimulated. Systemic acquired resistance (SAR) and induced systemic resistance (ISR) are two forms of induced resistance wherein plant defenses are preconditioned by prior infection or treatment that results in resistance against subsequent challenge by a pathogen or parasite. Selected strains of plant growth-promoting rhizobacteria (PGPR) suppress diseases by antagonism between the bacteria and soil-borne pathogens as well as by inducing a systemic resistance in plant against both root and foliar pathogens. Rhizobacteria mediated ISR resembles that of pathogen induced SAR in that both types of induced resistance render uninfected plant parts more resistant towards a broad spectrum of plant pathogens. Several rhizobacteria trigger the salicylic acid (SA)-dependent SAR pathway by producing SA at the root surface whereas other rhizobacteria trigger different signaling pathway independent of SA. The existence of SA-independent ISR pathway has been studied in Arabidopsis thaliana, which is dependent on jasmonic acid (JA) and ethylene signaling. Specific Pseudomonas strains induce systemic resistance in viz., carnation, cucumber, radish, tobacco, and Arabidopsis, as evidenced by an enhanced defensive capacity upon challenge inoculation. Combination of ISR and SAR can increase protection against pathogens that are resisted through both pathways besides extended protection to a broader spectrum of pathogens than ISR/SAR alone. Beside Pseudomonas strains, ISR is conducted by Bacillus spp. wherein published results show that several specific strains of species B. amyloliquifaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B.sphaericus elicit significant reduction in the incidence or severity of various diseases on a diversity of hosts.

Effect of different organic manures/composts on the herbage and essential oil yield of Cymbopogon winterianus and their influence on the native AM population in a marginal alfisol

Four organic amendments: leaf compost (LC), vegetable compost (VC), poultry manure (PM) and sewage sludge (SSL) applied at four doses (40, 80, 100 and 120 tha(-1)) were evaluated for their effect on the herbage yield, essential oil content and inoculum potential (IP) of native arbuscular mycorrhizal fungi (AMF) on three varieties of Java citronella, Cymbopogon winterianus Jowitt (Manjusha, Mandakini, and Bio-13). PM applied at 100 t ha(-1) followed by SSL increased the herbage, essential oil content and dry matter yield significantly. Bio-13 performed better and produced the highest herbage, essential oil and dry matter yield. The type and dose of the various organic amendments also significantly influenced the indigenous AMF infectious propagules in soil. Highest number of AMF propagules were recorded in the LC amended plots in all the three varieties. Amongst the varieties, highest native mycorrhizal inoculum was recorded in the Bio-13. Least number of AM infectious propagules were recorded in the Mandakini plants grown in 40 t ha(-1) SSL.

Application of inorganic carrier-based formulations of fluorescent pseudomonads and Piriformospora indica on tomato plants and evaluation of their efficacy

Aims:  Fluorescent pseudomonads are widely used as bioinoculants for improving plant growth and controlling phytopathogenic fungi. Piriformospora indica (Pi), a symbiotic root endophyte, also has beneficial effects on a number of plants. The present study focuses on the improvement of growth yields of tomato plants and control of Fusarium wilt using inorganic carrier‐based formulations of two fluorescent pseudomonad strains (R62 and R81) and Pi.

Characterization of an antifungal compound produced by Bacillus sp. strain A5F that inhibits Sclerotinia sclerotiorum

A potential antagonist, Bacillus sp. strain A5F was isolated from soybean rhizosphere following in vitro dual plate screening. The bacterium displayed strong inhibitory activity in vitro against soybean stem rot pathogen, Sclerotinia sclerotiorum. The culture supernatant of strain A5F completely suppressed the mycelial growth of the pathogen, indicating that suppression was due to the presence of antifungal compounds in the culture filtrate. The culture filtrate also suppressed other phytopathogenic fungi including Fusarium oxysporum and Macrophomina phaseolina, in vitro suggesting a broad spectrum antagonistic activity against fungal pathogens. Chemical extraction followed by chromatographic analysis resulted in two antifungal fractions. The high resolution‐electron spin ionization‐mass spectrometry (HR‐ESI‐MS) and Nuclear Magnetic Resonance (1D and 2D1H) spectra of these antifungal fractions revealed the presence of antifungal compounds, one of which showed similarity to bacillomycin D. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Shelf-life enhancement of bio-inoculant formulation by optimizing the trace metals ions in the culture medium for production of DAPG using fluorescent pseudomonad R62

Statistical experimental design was used to optimize the concentration of trace elements for production of antifungal compound, 2,4-diacetylphloroglucinol (DAPG), from fluorescent pseudomonad R62 in shake-flask cultivation. The selection of the trace metal ions, influencing DAPG production, was done using Plackett-Burman design (PBD). Only Zn(2+), Mn(2+) and MoO(4)(2-) were the most significant components (p<0.05). A quadratic model was used to fit the response. Application of response surface methodology (RSM) revealed that the optimum values of the salts of the trace elements Zn(2+) (ZnSO(4)·7H(2)O), Mn(2+) (MnCl(2)·4H(2)O), and MoO(4)(2-) (Na(2)MoO(4)·2H(2)O) were 83, 42 and 135μM, respectively, to achieve 125 mg/L of DAPG, which was nearly 13-fold more compared to its production in basal synthetic medium in shake flask. The studies in 14L bioreactor resulted in 135 mg/L of DAPG at the end of 36 h of cultivation. The culture broth containing 125 mg/L of DAPG was found to be sufficient for keeping the bio-inoculant viable in non-sterile talcum powder-based formulations (which contained 25μg DAPG/g carrier) when stored at 28°C for 6 months. The structure of the purified DAPG was confirmed using (1)H NMR and mass spectrometry.

Screening of Resistant Varieties and Antagonistic Fusarium oxysporum forBiocontrol of Fusarium Wilt of Chilli

Chilli is an important vegetable/spice, and its socio-cultural role is remarkable worldwide. The enormous popularity and demand for chilli is providing a boost to the chilli industry, but its production is increasingly constrained by diseases. Chilli producers have reported Fusarium wilt, as the frequently encountered disease. The present investigation focuses on biological control, which is found effective to manage this disease. A survey was conducted at Uttarakhand and Uttar Pradesh states in India, for collection of soil and plant samples. A total of eighty isolates of Fusarium were isolated from these samples. Among these, forty eight isolates of Fusarium oxysporum were identified on the basis of morphological and molecular characteristics, using speciesspecific primers. Pathogenicity test on chilli was conducted. One isolate of F. oxysporum was found most virulent pathogen, while eleven isolates were non- pathogenic isolates. Isolate no. 65 was found most antagonistic towards F. oxysporum, under in-vitro dual culture assay. Thirty chilli varieties were screened for evaluation of resistance. Among these, two varieties were found resistant against the Fusarium wilt. The present investigation focused on recovery of antagonistic Fusarium and resistant varieties of chilli, for controlling and resisting wilt and improving the soil health.

siRNAs targeting PB2 and NP genes potentially inhibit replication of Highly Pathogenic H5N1 Avian Influenza Virus

Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is a threat to animal and public health worldwide. Till date, the H5N1 virus has claimed 402 human lives, with a mortality rate of 58% and has caused the death or culling of millions of poultry since 2003. In this study, we have designed three siRNAs (PB2-2235, PB2-479 and NP-865) targeting PB2 and NP genes of avian influenza virus and evaluated their potential, measured by hemagglutination (HA), plaque reduction and Real time RT-PCR assay, in inhibiting H5N1 virus (A/chicken/Navapur/7972/2006) replication in MDCK cells. The siRNAs caused 8- to 16-fold reduction in virus HA titers at 24 h after challenged with 100TCID50 of virus. Among these siRNAs, PB2-2235 offered the highest inhibition of virus replication with 16-fold reduction in virus HA titer, 80% reduction in viral plaque counts and 94% inhibition in expression of specific RNA at 24 h. The other two siRNAs had 68–73% and 87–88% reduction in viral plaque counts and RNA copy number, respectively. The effect of siRNA on H5N1 virus replication continued till 48h (maximum observation period). These findings suggest that PB2-2235 could efficiently inhibit HPAI H5N1 virus replication.

In silico modelling and validation of differential expressed proteins in lung cancer

Abstract Objective The present study aims predict the three dimensional structure of three major proteins responsible for causing Lung cancer. Methods These are the differentially expressed proteins in lung cancer dataset. Initially, the structural template for these proteins is identified from structural database using homology search and perform homology modelling approach to predict its native 3D structure. Three-dimensional model obtained was validated using Ramachandran plot analysis to find the reliability of the model. Results Four proteins were differentially expressed and were significant proteins in causing lung cancer. Among the four proteins, Matrixmetallo proteinase (P39900) had a known 3D structure and hence was not considered for modelling. The remaining proteins Polo like kinase I Q58A51, Trophinin B1AKF1, Thrombomodulin P07204 were modelled and validated. Conclusions The three dimensional structure of proteins provides insights about the functional aspect and regulatory aspect of the protein. Thus, this study will be a breakthrough for further lung cancer related studies.

Evidence of a humoral immune response against the prokaryotic expressed N-terminal autoprotease (Npro) protein of bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle and sheep belonging to the genus Pestivirus of the family Flaviviridae. Although the BVDV non-structural N-terminal protease (Npro) acts as an interferon antagonist and subverts the host innate immunity, little is known about its immunogenicity. Hence, we expressed a recombinant BVDV Npro-His fusion protein (28 kDa) in E. coli and determined the humoral immune response generated by it in rabbits. The antigenicity of the Npro protein was confirmed by western blot using anti-BVDV hyperimmune cattle, sheep and goat serum, and anti-Npro rabbit serum. When rabbits were immunized with the Npro protein, a humoral immune response was evident by 4 weeks and persisted till 10 weeks post immunization as detected by ELISA and western blot. Despite Npro-specific antibodies remaining undetectable in 80 serum samples from BVDV-infected sheep and goats, BVDV hyperimmune sera along with some of the field cattle, sheep and goat sera with high BVDV neutralizing antibody titres were found positive for Npro antibodies. Our results provide evidence that despite the low immunogenicity of the BVDV Npro protein, a humoral immune response is induced in cattle, sheep and goats only with repeated BVDV exposure.