Molecular characterization of mitochondrial COI gene sequences in Micraspis allardi from Pakistan.

The Coccinellidae is a highly diversified family of order Coleoptera. Coccinellid ladybirds are well known for their role as biological control agent against varied range of agricultural pests. The samples of coccinellid ladybird collected from Pakistan were identified and characterized as Micraspis allardi (Mulsant, 1866). This is one of the least-studied ladybird species with limited work on its ecological distribution as a biological control agent. The genus Micraspis has vast genetic diversity with a possible presence of unknown number of cryptic species. Sequence information of some species of the genus Micraspis are present in NCBI database. However, least molecular data or sequences describing M. allardi could be available from database. Therefore, morphological and molecular characterization was imperative for this species. Here, the samples collected from sugarcane field of Faisalabad District of Pakistan and were identified by using morphological and molecular protocols. For molecular identification, two different regions of mitochondrial cytochrome c oxidase I (COI) gene (COI-5' and COI- 3') were used as molecular markers for the identification of the species. Morphological appearance, DNA sequence similarity searches and phylogenetic analysis collectively indicated it as M. allardi. To the best of our knowledge, this is the first report providing molecular evidence of M. allardi using mitochondrial DNA barcode region (658bp) as well as mtCOI-3' sequences (817bp). The study will help in understanding population genetics through diversity analysis, ecological role, and phenotypic structures associated with the geographic range of this species.

Mitigation of water scarcity with sustained growth of Rice by plant growth promoting bacteria.

Climate change augments the risk to food security by inducing drought stress and a drastic decline in global rice production. Plant growth-promoting bacteria (PGPB) have been known to improve plant growth under drought stress. Here in the present study, we isolated, identified, and well-characterized eight drought-tolerant bacteria from the rice rhizosphere that are tolerant to 20% PEG-8000. These strains exhibited multiple plant growth-promoting traits, i.e., 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, exopolysaccharide production, phosphate (P)-solubilizing activity (51-356 µg ml-1), indole-3 acetic acid (IAA) production (14.3-46.2 µg ml-1), and production of organic acids (72-178 µg ml-1). Inoculation of bacterial consortium (Bacillus subtilis NM-2, Brucella haematophilum NM-4, and Bacillus cereus NM-6) significantly improved seedling growth and vigor index (1009.2-1100) as compared to non-inoculated stressed plants (630-957). Through rhizoscanning, efficiency of the consortium was validated by improved root parameters such as root length (17%), diameter, and surface area (18%) of all tested genotypes as compared with respective non-inoculated stressed treatments. Furthermore, the response of consortium inoculation on three rice genotypes was positively correlated with improved plant growth and drought stress ameliorating traits by the accumulation of osmoprotectant, i.e., proline (85.8%-122%), relative water content (51%), membrane stability index (64%), and production of antioxidant enzymes to reduce oxidative damage by reactive oxygen species. A decrease in temperature and improved chlorophyll content of inoculated plants were found using infrared thermal imaging and soil plant analyzer development (SPAD), respectively. The key supporting role of inoculation toward stress responses was validated using robust techniques like infrared thermal imaging and an infrared gas analyzer. Furthermore, principal component analysis depicts the contribution of inoculation on stress responses and yield of tested rice genotypes under water stress. The integration of drought-tolerant rice genotype (NIBGE-DT02) and potential bacterial strains, i.e., NM-2, NM-4, and NM-6, can serve as an effective bioinoculant to cope with water scarcity under current alarming issues related to food security in fluctuating climate.

Exploring biocontrol and growth-promoting potential of multifaceted PGPR isolated from natural suppressive soil against the causal agent of chickpea wilt.

Chickpea is an important nutritive food crop both for humans and animals. Chickpea wilt caused by Fusarium oxysporum f.sp. ciceris (Foc) results in huge yield losses every year. Chickpea being a food crop requires the development of an eco-friendly bio-pesticide to effectively control the chickpea wilt disease. In this study, more than 50 bacterial stains isolated from the rhizosphere of healthy plants growing in wilt sick soil were examined for their Foc antagonist activities. Out of these, 17 strains showing > 90% growth inhibition of Foc were then characterized for their plant growth-promoting (PGP) and biocontrol traits. The biocontrol and PGP traits identified include amylase, hydrogen cyanide, protease, cellulase, chitinase activities, p-solubilization, nitrogen-fixing, and indole-3-acetic acid production. Two bacterial strains, IR-27 and IR-57, exhibiting the highest Foc proliferation inhibition and the PGP potential along with a consortium of four different strains (Serratia sp. IN-1, Serratia sp. IS-1, Enterobacter sp. IN-2, Enterobacter sp. IN-6) were used for controlling the chickpea wilt disease and growth promotion of the chickpea plants. Confocal laser scanning microscopy revealed their root colonization ability with partial or complete elimination of broken Foc mycelia and hyphae from roots. The bacterial inoculations particularly the consortium significantly suppressed the disease and improved the overall root morphology traits (root length, root surface area, root volume, forks, tips, and crossings), resulting in enhanced growth of the chickpea plants. Significant changes in growth (107% increase in root length, 23% increase in shoot length, and 54% increase in branches) in Foc-challenged plants were observed when inoculated with the consortium. Further investigations revealed that the chickpea plants inoculated with bacterial strains induced the expression of a number of key defence enzymes, including the phenylalanine ammonia lyase, peroxidase, polyphenol peroxidase, ß-1,3 glucanase, which might have helped the plants to thwart the pathogen attack. These findings indicate the potential of our identified bacterial strains to be used as a natural biopesticide for controlling the chickpea wilt disease.

Integrated Analysis of Osmotic Stress and Infrared Thermal Imaging for the Selection of Resilient Rice Under Water Scarcity.

The climate change scenario has increased the severity and frequency of drought stress, which limits the growth and yield of rice worldwide. There is a dire need to select drought-tolerant rice varieties to sustain crop production under water scarcity. Therefore, the present study effectively combined morpho-physiological and biochemical approaches with the technology of infrared thermal imaging (IRTI) for a reliable selection of drought-tolerant genotypes. Initially, we studied 28 rice genotypes including 26 advance lines and three varieties for water stress tolerance under net house conditions. Three genotypes NIBGE-DT-02, KSK-133, and NIBGE-DT-11 were selected based on the Standard Evaluation System (SES) scoring for drought tolerance. NIBGE-DT-02 showed tolerance to polyethylene glycol (20%) induced osmotic stress indicated by a minimum reduction in seedling length, biomass, chlorophyll content, and increased leaf proline content as compared to susceptible varieties under a hydroponic system. NIBGE-DT-02 was further evaluated for water withholding at varying growth stages, i.e., 30 and 60 days after transplantation (DAT) in pots under net house conditions. NIBGE-DT-02 showed a significantly lower reduction (35.9%) in yield as compared to a susceptible variety (78.06%) under water stress at 60 DAT with concomitant induction of antioxidant enzymes such as peroxidase, catalase, and polyphenol oxidase. A significant increase (45.9%) in proline content, a low increase (7.5%) in plant temperature, along with a low reduction in relative water content (RWC) (5.5%), and membrane stability index (MSI) (9%) were observed under water stress at 60 DAT as compared to the well-watered control. Pearson correlation analysis showed the strong correlation of shoot length with MSI and root length with RWC in rice genotypes at the later growth stage. Furthermore, Regression analysis indicated a negative correlation between plant temperature of NIBGE-DT-02 and proline, RWC, MSI, and peroxidase enzyme under variable water stress conditions. All these responses collectively validated the adaptive response of selected genotypes under water stress during different growth stages. Tolerant genotypes can be used in breeding programs aimed at improving drought tolerance and can expand rice cultivation. Furthermore, this study provides a foundation for future research directed to utilize IRTI as a fast and non-destructive approach for the selection of potent rice genotypes better adapted to water scarcity from wide germplasm collection.

Green synthesis of nickel oxide nanoparticles using Allium cepa peels for degradation of Congo red direct dye: an environmental remedial approach.

Direct dyes are used in different textile operations and processings. The textile industries are disposing of unused direct dyes into the aquatic environment which is posing a serious alarming threat to aquatic lives. The current study deals with the synthesis of nickel oxide nanoparticles using Allium cepa peels aqueous extract. Nickel oxide nanoparticles (NiO-NPs) were characterized by scanning electron microscopy (SEM). Synthesized NiO-NPs were used to remove Congo red direct dye. Various experimental factors like concentration of dye and nanoparticles, pH, and temperature were optimized. Congo red direct dye was decolorized up to 90% at optimized conditions (Congo Red Direct dye concentration 0.02%, catalyst dose 0.003 g·L-1, pH 6, and temperature 50 °C). The real textile industry effluent disclosed 70% decolorization at optimized conditions. The percent reduction in total organic carbon (TOC) and chemical oxygen demand (COD) was found to be 73.24% and 74.56% in the case of Congo red dye catalytic treatment and the percent reduction in TOC and COD was found to be 62.47% and 60.23%, respectively, in the treatment of textile effluent using nickel oxide nanoparticles as a catalyst. Treated and untreated dye samples were exposed to Fourier transform infrared (FTIR) and UV-Visible spectral analyses too. The reaction products were studied by degradation pathway.

Seed inoculation of desert-plant growth-promoting rhizobacteria induce biochemical alterations and develop resistance against water stress in wheat.

Water shortage limits agricultural productivity, so strategies to get higher yields in dry agricultural systems is vital to circumvent the effect of climate change and land-shortage. The plant rhizosphere harbors beneficial bacteria able to confer biotic/abiotic tolerance along with a positive impact on plant growth. Herein, three bacterial strains, Proteus mirabilis R2, Pseudomonas balearica RF-2 and Cronobacter sakazakii RF-4 (accessions: LS975374, LS975373, LS975370, respectively) isolated from native desert-weeds were investigated for their response to improve wheat growth under drought stress. The bacteria showed drought tolerance up to 20% polyethylene glycol (PEG; -0.6 MPa), and salt (65-97 g l-1 ), 1-aminocyclopropane-1-carboxylate (ACC)-deaminase activity, P/Zn/K-solubilization, calcite degradation, IAA, and siderophore production. The plant growth-promoting rhizobacteria (PGPR) were evaluated on wheat under water stress. The P. balearica strain RF-2 primed seeds showed a maximum promptness index and germination index under PEG-stress, that is, 68% and 100%, respectively. Inoculation significantly improved plant growth, leaf area, and biomass under water stress. P. mirabilis R2 inoculated plant leaves showed the highest water contents as compared to the plants inoculated with other strains. C. sakazakii RF-4 inoculated plants showed minimum cell injury, electrolyte leakage, and maximum cell membrane stability at PEG stress. After 13 days exposure to drought, C. sakazakii RF-4 treated plants showed an overall higher expression of cytosolic ascorbate peroxidase (cAPX) and ribulose-bisphosphate carboxylase (rbcL) genes. The activity of stress-induced catalase and polyphenol oxidase was reduced, while that of peroxidase and superoxide dismutase increased after inoculation but the response was temporal. Taken together, this data explains that different PGPR (especially C. sakazakii RF-4) modulate differential responses in wheat that eventually leads towards drought tolerance, hence, it has the potential to enhance crop production in arid regions.

Modern Trends in Plant Genome Editing: An Inclusive Review of the CRISPR/Cas9 Toolbox.

Increasing agricultural productivity via modern breeding strategies is of prime interest to attain global food security. An array of biotic and abiotic stressors affect productivity as well as the quality of crop plants, and it is a primary need to develop crops with improved adaptability, high productivity, and resilience against these biotic/abiotic stressors. Conventional approaches to genetic engineering involve tedious procedures. State-of-the-art OMICS approaches reinforced with next-generation sequencing and the latest developments in genome editing tools have paved the way for targeted mutagenesis, opening new horizons for precise genome engineering. Various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases (MNs) have enabled plant scientists to manipulate desired genes in crop plants. However, these approaches are expensive and laborious involving complex procedures for successful editing. Conversely, CRISPR/Cas9 is an entrancing, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for targeted mutagenesis, including single base substitution, multiplex gene editing, gene knockouts, and regulation of gene transcription in plants. Thus, CRISPR/Cas9-based genome editing has demonstrated great potential for crop improvement but regulation of genome-edited crops is still in its infancy. Here, we extensively reviewed the availability of CRISPR/Cas9 genome editing tools for plant biotechnologists to target desired genes and its vast applications in crop breeding research.

Plant Growth Promotion and Suppression of Bacterial Leaf Blight in Rice by Inoculated Bacteria.

The present study was conducted to evaluate the potential of rice rhizosphere associated antagonistic bacteria for growth promotion and disease suppression of bacterial leaf blight (BLB). A total of 811 rhizospheric bacteria were isolated and screened against 3 prevalent strains of BLB pathogen Xanthomonas oryzae pv. oryzae (Xoo) of which five antagonistic bacteria, i.e., Pseudomonas spp. E227, E233, Rh323, Serratia sp. Rh269 and Bacillus sp. Rh219 showed antagonistic potential (zone of inhibition 1-19 mm). Production of siderophores was found to be the common biocontrol determinant and all the strains solubilized inorganic phosphate (82-116 µg mL-1) and produced indole acetic acid (0.48-1.85 mg L-1) in vitro. All antagonistic bacteria were non-pathogenic to rice, and their co-inoculation significantly improved plant health in terms of reduced diseased leaf area (80%), improved shoot length (31%), root length (41%) and plant dry weight (60%) as compared to infected control plants. Furthermore, under pathogen pressure, bacterial inoculation resulted in increased activity of defense related enzymes including phenylalanine ammonia-lyase and polyphenol oxidase, along with 86% increase in peroxidase and 53% increase in catalase enzyme activities in plants inoculated with Pseudomonas sp. Rh323 as well as co-inoculated plants. Bacterial strains showed good colonization potential in the rice rhizosphere up to 21 days after seed inoculation. Application of bacterial consortia in the field resulted in an increase of 31% in grain yield and 10% in straw yield over non-inoculated plots. Although, yield increase was statistically non-significant but was accomplished with overall saving of 20% chemical fertilizers. The study showed that Pseudomonas sp. Rh323 can be used to develop dual-purpose inoculum which can serve not only to suppress BLB but also to promote plant growth in rice.

G5, a Phage Single-Stranded DNA-Binding Protein, Fused with a Nuclear Localization Signal, Attenuates Symptoms and Reduces Begomovirus-Betasatellite Accumulation in Transgenic Plants.

Cotton leaf curl disease is caused by several monopartite begomoviruses and is the major threat to cotton production in the Indian subcontinent. The disease has been shown to be associated with four distinct species, including Cotton leaf curl Kokhran virus (CLCuKoV), and a specific betasatellite-Cotton leaf curl Multan betasatellite (CLCuMuB). Transgenic Nicotiana benthamiana plants were produced which constitutively express the Escherichia coli phage M13 encoded, sequence nonspecific single-stranded (ss) DNA-binding protein, G5 alone and fused with the maize opaque-2 nuclear localization signal (NLS), to evaluate resistance against CLCuKoV-CLCuMuB. Transgenic plants expressing only G5 performed poorly exhibiting symptoms of infection and high virus DNA levels upon inoculation with CLCuKoV and CLCuKoV with CLCuMuB. In contrast, plants transformed with G5 fused to the NLS developed mild symptoms and showed a reduction in virus and betasatellite DNA levels in comparison to nontransformed plants. The results show that G5 may be useful in developing broad-spectrum resistance against ssDNA viruses.