AzuR From the SmtB/ArsR Family of Transcriptional Repressors Regulates Metallothionein in Anabaena sp. Strain PCC 7120

Metallothioneins (MTs) are cysteine-rich, metal-sequestering cytosolic proteins that play a key role in maintaining metal homeostasis and detoxification. We had previously characterized NmtA, a MT from the heterocystous, nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 and demonstrated its role in providing protection against cadmium toxicity. In this study, we illustrate the regulation of Anabaena NmtA by AzuR (Alr0831) belonging to the SmtB/ArsR family of transcriptional repressors. There is currently no experimental evidence for any functional role of AzuR. It is observed that azuR is located within the znuABC operon but in the opposite orientation and remotely away from the nmtA locus. Sequence analysis of AzuR revealed a high degree of sequence identity with Synechococcus SmtB and a distinct α5 metal binding site similar to that of SmtB. In order to characterize AzuR, we overexpressed it in Escherichia coli and purified it by chitin affinity chromatography. Far-UV circular dichroism spectroscopy indicated that the recombinant AzuR protein possessed a properly folded structure. Glutaraldehyde cross-linking and size-exclusion chromatography revealed that AzuR exists as a dimer of ∼28 kDa in solution. Analysis of its putative promoter region [100 bp upstream of nmtA open reading frame (ORF)] identified the presence of a 12–2–12 imperfect inverted repeat as the cis-acting element important for repressor binding. Electrophoretic mobility shift assays (EMSAs) showed concentration-dependent binding of recombinant dimeric AzuR with the promoter indicating that NmtA is indeed a regulatory target of AzuR. Binding of AzuR to DNA was disrupted in the presence of metal ions like Zn2+, Cd2+, Cu2+, Co2+, Ni2+, Pb2+, and Mn2+. The metal-dependent dissociation of protein–DNA complexes suggested the negative regulation of metal-inducible nmtA expression by AzuR. Overexpression of azuR in its native strain Anabaena 7120 enhanced the susceptibility to cadmium stress significantly. Overall, we propose a negative regulation of Anabaena MT by an α5 SmtB/ArsR metalloregulator AzuR.

VIRULENCE OF ENTOMOPATHOGENIC NEMATODES TO THREE SOFT SCALE INSECT SPECIES

Virulence of commercial strains of entomopathogenic nematode (EPN) Steinernema carpocapsae (Weiser), Steinernema feltiae Filipjev and Heterorhabditis bacteriophora Poinar (Rhabditida: Steinernematidae, Heterorhabditidae), were tested on young females of Pulvinaria hydrangeae Steinweden, Partenolecanium corni (Bouché) and Coccus pseudomagnoliarum (Kuwana) (Hemiptera, Coccidae) and a native strain of H. bacteriophora was tested on citricola scale, C. pseudomagnoliarum. The laboratory tests were carried out in small plastic Petri dishes with infective juveniles (IJ) applied on filter paper covering the bottom. Results showed that all EPN tested were virulent to all three soft scale insect species. Recorded mortality of insects at the highest concentration of approximately 50 IJ per cm2 exceeded 64% in all combinations of nematode and scale species tested. Within the same nematode concentration, scales’ mortality of 90% and above was recorded for P. corni with all nematode species, P. hydrangeae with S. carpocapsae (Nemastar) and C. pseudomagnoliarum with native H. bacteriophora strain. All scale species were good hosts for all nematode species. This is the first report of EPNs to parasitize soft scale insects and one of the few among Coccoidea. The level of parasitism recorded is promising for potential use in management of scale insects.

A DedA Family Membrane Protein in Indium Extrusion in Rhodanobacter sp. B2A1Ga4

Indium (In) is a critical metal widely used in electronic equipment, and the supply of this precious metal is a major challenge for sustainable development. The use of microorganisms for the recovery of this critical high-tech element has been considered an excellent eco-friendly strategy. The Rhodanobacter sp. B2A1Ga4 strain, highly resistant to In, was studied in order to disclose the bacterial mechanisms closely linked to the ability to cope with this metal. The mutation of the gene encoding for a DedA protein homolog, YqaA, affected drastically the In resistance and the cellular metabolic activity of strain Rhodanobacter sp. B2A1Ga4 in presence of this metal. This indicates that this protein plays an important role in its In resistance phenotype. The negative impact of In might be related to the high accumulation of the metal into the mutant cells showing In concentration up to approximately 4-fold higher than the native strain. In addition, the expression of the yqaA gene in this mutant reverted the bacterial phenotype with a significant decrease of In accumulation levels into the cells and an increase of In resistance. Membrane potential measurements showed similar values for native and mutant cells, suggesting that there was no loss of proton-motive force in the mutant cells. The results from this study suggest a potential role of this DedA family protein as a membrane transporter involved in the In efflux process. The mutant strain also has the potential to be used as a biotool in bioaccumulation strategies, for the recovery of In in biomining activities.

Entomopathogenic Potential of Simplicillium lanosoniveum Native Strain in Suppressing Invasive Whitefly, Aleurodicusrugioperculatus Martin (Hemiptera: Aleyrodidae), Infesting Coconut

In 2016, infestation of an exotic polyphagous pest, the rugose spiraling whitefly (RSW), Aleurodicus rugioperculatus Martin (Hemiptera: Aleyrodidae), was documented on coconut for the first time in India. Instantaneously, RSW has garnered wide attention owing to its damage severity and rapid spread across the coconut-growing regions of the country. Hence, an attempt was made to devise a sustainable integrated pest management (IPM) module using biological control agents as a mainstay component. The present study documented the identification and characterization of a potential entomopathogenic fungal isolate for the management of RSW. An entomopathogenic fungus isolated from nymphal cadavers of RSW was identified as Simplicillium lanosoniveum based on morphological and phylogenetic analyses. A gradient of five conidial concentrations (1 × 104, 1 × 105, 1 × 106, 1 × 107 and 1 × 108 conidia/mL) of the S.lanosoniveum were tested against eggs, first instars, second to third instars and pupae of RSW. Results revealed that S.lanosoniveum is highly virulent to all developmental stages of RSW by causing mortality rates of 95.20%, 87.33%, 85.38% and 72.85%, in eggs, initial, middle and later instar nymphs of RSW, respectively, at the highest tested concentration (1 × 108 conidia/mL) at seven days after exposure. The LC50 and LT50 values of S.lanosoniveum were 4.72 × 104, 4.94 × 104, 5.11 × 105, 5.92 × 105 conidia/mL and 4.27, 4.86, 4.56, 5.89 days against eggs, initial, middle and later instar nymphs of RSW, respectively. Further, preliminary field trials with S.lanosoniveum strain at 1 × 108 conidia/mL exhibited a significant reduction in the egg and nymphal population by 57.8% and 56.3%, respectively. This report thus demonstrated that the newly isolated S.lanosoniveum is an effective pathogen at suppressing all the developmental stages of RSW. This is the first record of S.lanosoniveum infecting RSW, and it has a great potential to be developed as a mycoinsecticide.

Plant Growth Ameliorating and Rhizosphere Competent Native Acinetobacter pittii Strain F2 5 from the Rhizosphere of Zea mays L.

Background: Acinetobacter is an aerobic gram negative, non-spore forming, oxidase negative, catalase positive, non-motile encapsulated coccobacilli. They are ubiquitous in nature mostly isolated from soil, water, food, sewage, animal and human skin, marine water and rhizosphere of maize and wheat. The present study was focused on isolation and characterization of a native strain of rhizospheric Acinetobacter for plant growth promoting ability through different microbiological and morphometric parameters under greenhouse conditions. Methods: Rhizosphere soil samples were collected from maize fields and processed as per the standard microbiological procedure. The morphology was reconfirmed through scanning electron microscopy. DNA extraction was performed by using al. Two sets of primers (universal and specific for H. pylori) were used to amplify the 16S ribosomal gene. Sanger sequencing was applied and the resulted sequences were matched with the sequences of the National Center for Biotechnology Information (NCBI) nucleotide database. The evolutionary aspects were analyzed using MEGA7 software. Result: The strain exhibited plant growth promoting attributes of ammonia generation, inorganic and organic phosphate solubilization 1-aminocyclopropane-1-carboxylate deaminase activity. The strain was identified as Acinetobacter pittii through 16S rRNA sequencing and was designated as Acinetobacter pittii strain F2 5 with the accession number KM677194. Scanning electron microscopy was carried out to reconfirm the morphology of the strain. Under laboratory and green house conditions the strain improved the shoot and root length and its biomass of the treated maize seedlings as compared to the uninoculated control underscoring the plant growth potential of these strains in sustainable agricultural practices.

Influence of the Medium Composition and the Culture Conditions on Surfactin Biosynthesis by a Native Bacillus subtilis natto BS19 Strain

An effective microbial synthesis of surfactin depends on the composition of the culture medium, the culture conditions and the genetic potential of the producer strain. The aim of this study was to evaluate the suitability of various medium components for the surfactin producing strain and to determine the impact of the culture conditions on the biosynthesis of surfactin isoforms by the newly isolated native strain Bacillus subtilis natto BS19. The efficiency of surfactin biosynthesis was determined by measuring the surface tension of the medium before and after submerged culture (SmF) and by qualitative and quantitative analysis of the obtained compound by high performance liquid chromatography. The highest efficiency of surfactin biosynthesis was achieved using starch as the carbon source and yeast extract as the nitrogen source at pH 7.0 and 37 °C. Potato peelings were selected as an effective waste substrate. It was shown that the increase in the percentage of peel extract in the culture medium enhanced the biosynthesis of surfactin (mg/L) (2–30.9%; 4–46.0% and 6–58.2%), while reducing surface tension of the medium by about 50%. The obtained results constitute a promising basis for further research on biosynthesis of surfactin using potato peelings as a cheap alternative to synthetic medium components.

Effect of Beauveria bassiana-Seed Treatment on Zea mays L. Response against Spodoptera frugiperda

Spodoptera frugiperda is a widely distributed insect pest that causes major economic losses in various crops, particularly maize. On the other hand, Beauveria bassiana is an entomopathogenic fungus that establishes symbiotic associations with many plants and contributes to tolerance against biotic and abiotic stresses. In the present work, in laboratory experiments, the effects of the B. bassiana strain GHA, in addition to a native strain (PTG4), delivered via seed treatment in maize seedlings, were evaluated on S. frugiperda growth, development, and mortality. We inoculated maize seeds with 1 × 106B. bassiana blastospores; then these seeds were germinated and grown to seedlings under growth chamber conditions. Third-instar S. frugiperda larvae were allowed to feed on B. bassiana-treated and -untreated (negative control) seedlings until reaching the sixth instar and transferred to an artificial diet until reaching adult stage. Results showed that larvae feeding on B. bassiana strain PTG4-treated plants prolonged their larval stage. Furthermore, feeding on plants treated with B. bassiana strains yielded fewer S. frugiperda male moths compared with feeding with the untreated control plants. Under field conditions, 1 × 106 (first trial) and 1 × 108 (second trial) of B. bassiana (GHA strain) blastospores were used for corn seed inoculation. In the first field trial, there were a higher number of larvae in the negative control plants compared to those in the plants treated with B. bassiana. No larvae were found in negative control and B. bassiana-treated plants in the second field trial. In conclusion, seed treatment with B. bassiana in maize reduced S. frugiperda infestation of maize plants in field trials. S. frugiperda development was also affected in laboratory trials.