Bioremediation of heavy metals polluted environment and decolourization of black liquor using microbial biofilms.

BACKGROUND: With increased urbanization and industrialization, modern life has led to an anthropogenic impact on the biosphere. Heavy metals pollution and pollutants from black liquor (BL) have caused severe effects on environment and living organisms. Bacterial biofilm has potential to remediate heavy metals and remove BL from the environment. Hence, this study was planned to investigate the potential of microbial biofilms for the bioremediation of heavy metals and BL polluted environments. METHODS AND RESULTS: Eleven biofilm forming bacterial strains (SB1, SB2, SC1, AF1, 5A, BC-1, BC-2, BC-3, BC-4, BC-5 and BC-6) were isolated and identified upto species level via 16S rRNA gene sequencing. Biofilm strains belonging to Bacillus and Lysinibacillus sphaericus were used to remediate heavy metals (Pb, Ni, Mn, Zn, Cu, and Co). Atomic absorption spectroscopy showed significantly high (P ≤ 0.05) bioremediation potential by L. sphaericus biofilm (1462.0 ± 0.67 µgmL-1) against zinc (Zn). Similarly, Pseudomonas putida biofilm significantly (P ≤ 0.05) decolourized (65.1%) BL. Fourier transform infrared (FTIR) analysis of treated heavy metals showed the shifting of major peaks (1637 & 1629-1647, 1633 & 1635-1643, and 1638-1633 cm-1) corresponding to specific amide groups due to C = O stretching. CONCLUSION: The study suggested that biofilm of the microbial flora from tanneries and pulp paper effluents possesses a strong potential for heavy metals bioremediation and BL decolourization. To our knowledge, this is the first report showing promising biofilm remediation potential of bacterial flora of tanneries and pulp-paper effluent from Kasur and Sheikhupura, Punjab, Pakistan, against heavy metals and BL.

Role of modified diet and gut microbiota in metabolic endotoxemia in mice.

This study was aimed at investigating the effect of cultured gut microbiota (GM) from obese humans coupled HFD in inducing metabolic endotoxemia in humanized mice. In total, 30 strains were isolated from 10 stool samples of obese patients. Following morphological and biochemical characterization, 16S rRNA gene sequencing of six abundant isolates identified these Klebsiella aerogenes, Levilactobacillus brevis, Escherichia coli, Staphylococcus aureus, Bacillus cereus and Bacillus subtilis (MZ052089-MZ052094). In vivo trial using above isolates, known as human gut microbiota (HGM), was performed for six months. Sixteen mice were distributed into four groups, i.e., G1 (control) mice fed with chow diet, group 2 (G2) with HFD, group 3 (G3) with HFD + HGM and group 4 (G4) with chow diet + HGM. Body mass index (BMI) and plasma endotoxins were measured pre- and post-experiment. In vivo study revealed that HFD + HGM caused significant increase (3.9 g/cm at 20 weeks) in the body weight and BMI (0.4 g/cm post-experiment) of G3 mice compared to the other groups. One-way ANOVA showed significantly higher level of endotoxins (2.41, 4.08 and 3.7 mmol/L) in mice groups G2, G3 and G4, respectively, indicating onset of metabolic endotoxemia. Cecal contents of experimental mice groups showed a shift in microbial diversity as observed by all isolates belonging to either Firmicutes or Bacteroidetes phyla, respectively. In conclusion, current study reported that minor alteration in GM composition through HFD feeding and cultured GM transfer has significant impact in development of metabolic endotoxemia, possibly via modified intestinal permeability.

Journey of begomovirus betasatellite molecules: from satellites to indispensable partners.

Betasatellites are a group of circular, single-stranded DNA molecules that are frequently found to be associated with monopartite begomoviruses of the family Geminiviridae. Betasatellites require their helper viruses for replication, movement, and encapsidation and they are often essential for induction of typical disease symptoms. The ßC1 protein encoded by betasatellites is multifunctional that participates in diverse cellular events. It interferes with several cellular processes like normal development, chloroplasts, and innate immune system of plants. Recent research has indicated ßC1 protein interaction with cellular proteins and its involvement in modulation of the host's cell cycle and symptom determination. This article focuses on the functional mechanisms of ßC1 and its interactions with other viral and host proteins.

Structure activity relationship (SAR) and quantitative structure activity relationship (QSAR) studies showed plant flavonoids as potential inhibitors of dengue NS2B-NS3 protease.

BACKGROUND: Due to dengue virus disease, half of the world population is at severe health risk. Viral encoded NS2B-NS3 protease complex causes cleavage in the nonstructural region of the viral polyprotein. The cleavage is essentially required for fully functional viral protein. It has already been reported that if function of NS2B-NS3 complex is disrupted, viral replication is inhibited. Therefore, the NS2B-NS3 is a well-characterized target for designing antiviral drug. RESULTS: In this study docking analysis was performed with active site of dengue NS2B-NS3 protein with selected plant flavonoids. More than 100 flavonoids were used for docking analysis. On the basis of docking results 10 flavonoids might be considered as the best inhibitors of NS2B-NS3 protein. The interaction studies showed resilient interactions between ligand and receptor atoms. Furthermore, QSAR and SAR studies were conducted on the basis of NS2B-NS3 protease complex docking results. The value of correlation coefficient (r) 0.95 shows that there was a good correlation between flavonoid structures and selected properties. CONCLUSION: We hereby suggest that plant flavonoids could be used as potent inhibitors of dengue NS2B-NS3 protein and can be used as antiviral agents against dengue virus. Out of more than hundred plant flavonoids, ten flavonoid structures are presented in this study. On the basis of best docking results, QSAR and SAR studies were performed. These flavonoids can directly work as anti-dengue drug or with little modifications in their structures.

Characterization, phylogeny and recombination analysis of Pedilanthus leaf curl virus-Petunia isolate and its associated betasatellite.

BACKGROUND: Geminiviruses cause major losses to several economically important crops. Pedilanthus leaf curl virus (PeLCV) is a pathogenic geminivirus that appeared in the last decade and is continuously increasing its host range in Pakistan and India. This study reports the identification and characterization of PeLCV-Petunia from ornamental plants in Pakistan, as well as geographical, phylogenetic, and recombination analysis. METHODS: Viral genomes and associated satellites were amplified, cloned, and sequenced from Petunia atkinsiana plants showing typical geminivirus infection symptoms. Virus-satellite complex was analyzed for phylogenetic and recombination pattern. Infectious clones of isolated virus and satellite molecules were constructed using a partial dimer strategy. Infectivity analysis of PeLCV alone and in combination with Digera yellow vein betasatellite (DiYVB) was performed by Agrobacterium infiltration of Nicotiana benthamiana and Petunia atkinsiana plants with infectious clones. RESULTS: PeLCV, in association with DiYVB, was identified as the cause of leaf curl disease on P. atkinsiana plants. Sequence analysis showed that the isolated PeLCV is 96-98% identical to PeLCV from soybean, and DiYVB has 91% identity to a betasatellite identified from rose. Infectivity analysis of PeLCV alone and in combination with DiYVB, performed by Agrobacterium infiltration of infectious clones in N. benthamiana and P. atkinsiana plants, resulted in mild and severe disease symptoms 14 days after infiltration, respectively, demonstrating that these viruses are natural disease-causing agents. Southern blot hybridization indicated successful replication of the virus-betasatellite complex in the infected plants. Phylogenetic analysis suggests that PeLCV originated from Pakistan and later spread to India. Recombination analysis predicted that PeLCV is a donor parent for recombination and evolution of two important begomoviruses, Papaya leaf curl virus (PaLCuV) and Radish leaf curl virus (RaLCuV). The molecular phylogeny of genes encoding coat protein (CP) and replication associated protein (Rep) depict a complex evolutionary pattern of the viruses, with wide diversity in both of the genes. CONCLUSIONS: This study presents PeLCV and DiYVB as a new natural combination resulting in leaf curl disease on P. atkinsiana plants. Phylogenetic analysis, in addition to recent agricultural reports, identify PeLCV as an emerging broad host range Begomovirus that is resident in Pakistan and, more recently, has also spread to India. Recombination analysis showed that PeLCV was involved in a natural recombinational event leading to the evolution of two recombinant begomoviruses, RaLCuV and PaLCuV.

Molecular analysis of partial VP-2 gene amplified from rectal swab samples of diarrheic dogs in Pakistan confirms the circulation of canine parvovirus genetic variant CPV-2a and detects sequences of feline panleukopenia virus (FPV).

BACKGROUND: The infection in dogs due to canine parvovirus (CPV), is a highly contagious one with high mortality rate. The present study was undertaken for a detailed genetic analysis of partial VP2 gene i.e., 630 bp isolated from rectal swab samples of infected domestic and stray dogs from all areas of district Faisalabad. Monitoring of viruses is important, as continuous prevalence of viral infection might be associated with emergence of new virulent strains. METHODS: In the present study, 40 rectal swab samples were collected from diarrheic dogs from different areas of district Faisalabad, Pakistan, in 2014-15 and screened for the presence of CPV by immunochromatography. Most of these dogs were stray dogs showing symptoms of diarrhea. Viral DNA was isolated and partial VP2 gene was amplified using gene specific primer pair Hfor/Hrev through PCR. Amplified fragments were cloned in pTZ57R/T (Fermentas) and completely sequenced. Sequences were analyzed and assembled by the Lasergene DNA analysis package (v8; DNAStar Inc., Madison, WI, USA). RESULTS: The results with immunochromatography showed that 33/40 (82%) of dogs were positive for CPV. We were able to amplify a fragment of 630 bp from 25 samples. In 25 samples the sequences of CPV-2a were detected showing the amino acid substitution Ser297Ala and presence of amino acid (426-Asn) in partial VP2 protein. Interestingly the BLAST analysis showed the of feline panleukopenia virus (FPV) sequences in 3 samples which were already positive for new CPV-2a, with 99% sequence homology to other FPV sequences present in GenBank. CONCLUSIONS: Phylogenetic analysis showed clustering of partial CPV-VP-2 gene with viruses from China, India, Japan and Uruguay identifying a new variant, whereas the 3 FPV sequences showed immediate ancestral relationship with viruses from Portugal, South Africa and USA. Interesting observation was that CPV are clustering away from the commercial vaccine strains. In this work we provide a better understanding of CPV prevailing in Pakistan at molecular level. The detection of FPV could be a case of real co-infection or a case of dual presence, due to ingestion of contaminated food.

Labeling phospholipid membranes with lipid mimetic luminescent metal complexes.

Lipid-mimetic metallosurfactant based luminophores are promising candidates for labeling phospholipid membranes without altering their biophysical characteristics. The metallosurfactants studied exhibit high structural and physicochemical similarity to phospholipid molecules, designed to incorporate into the membrane structure without the need for covalent attachment to a lipid molecule. In this work, two lipid-mimetic phosphorescent metal complexes are described: [Ru(bpy)2(dn-bpy)](2+) and [Ir(ppy)2(dn-bpy)](+) where bpy is 2,2'-bipyridine, dn-bpy is 4,4'-dinonyl-2,2'-bipyridine and ppy is 2-phenylpyridine. Apart from being lipid-mimetic in size, shape and physical properties, both complexes exhibit intense photoluminescence and enhanced photostability compared with conventional organic fluorophores, allowing for prolonged observation. Moreover, the large Stokes shift and long luminescence lifetime associated with these complexes make them more suitable for spectroscopic studies. The complexes are easily incorporated into dimyristoil-phosphatidyl-choline (DMPC) liposomes by mixing in the organic solvent phase. DLS reveals the labeled membranes form liposomes of similar size to that of neat DMPC membrane. Synchrotron Small-Angle X-ray Scattering (SAXS) measurements confirmed that up to 5% of either complex could be incorporated into DMPC membranes without producing any structural changes in the membrane. Fluorescence microscopy reveals that 0.5% label content is sufficient for imaging. Atomic Force Microscopic imaging confirms that liposomes of the labeled bilayers on a mica surface can fuse into a flat lamellar membrane that is morphologically identical to neat lipid membranes. These results demonstrate the potential of such lipid-mimetic luminescent metal complexes as a new class of labels for imaging lipid membranes.

Effect of high temperature on grain filling period, yield, amylose content and activity of starch biosynthesis enzymes in endosperm of basmati rice.

BACKGROUND: High temperature during grain filling affects yield, starch amylose content and activity of starch biosynthesis enzymes in basmati rice. To investigate the physiological mechanisms underpinning the effects of high temperature on rice grain, basmati rice was grown under two temperature conditions - 32 and 22 °C - during grain filling. RESULTS: High temperature decreased the grain filling period from 32 to 26 days, reducing yield by 6%, and caused a reduction in total starch (3.1%) and amylose content (22%). Measurable activities of key enzymes involved in sucrose to starch conversion, sucrose synthase, ADP-glucose pyrophosphorylase, starch phosphorylase and soluble starch synthase in endosperms developed at 32 °C were lower than those at 22 °C compared with similar ripening stage on an endosperm basis. In particular, granule-bound starch synthase (GBSS) activity was significantly lower than corresponding activity in endosperms developing at 22 °C during all developmental stages analyzed. CONCLUSION: Results suggest changes in amylose/amylopectin ratio observed in plants grown at 32 °C was attributable to a reduction in activity of GBSS, the sole enzyme responsible for amylose biosynthesis.

A betasatellite-dependent begomovirus infects ornamental rose: characterization of begomovirus infecting rose in Pakistan.

The Begomovirus genus of the family Geminiviridae comprises the largest group of geminiviruses. The list of begomoviruses is continuously increasing as a result of improvement in the methods for identification. Ornamental rose plants (Rosa chinensis) with highly stunted growth and leaf curling were found in Faisalabad, Pakistan. Plants were analyzed for begomovirus infection, through rolling circle amplification and PCR methods. Based on complete genome sequence homologies with other begomoviruses, a new begomovirus species infecting the rose plants was discovered. In this paper, we propose a new species name, Rose leaf curl virus (RoLCuV), for the virus. RoLCuV showed close identity (83 %) with Tomato leaf curl Pakistan virus, while associated betasatellite showed 96 % identity with Digera arvensis yellow vein betasatellite (DiAYVB), justifying a new isolate for the betasatellite. Recombination analysis of newly identified begomovirus revealed it as a recombinant of tomato leaf curl Pakistan virus from its coat protein region. The infectious molecules for virus/satellite were prepared and inoculated through Agrobacterium tumefaciens to N. benthamiana plants. RoLCuV alone was unable to induce any level of symptoms on N. benthamiana plants, but co-inoculation with cognate betasatellite produced infection symptoms. Further investigation to understand the trans-replication ability of betasatellites revealed their flexibility to interact with Rose leaf curl virus.

Halotolerant bacterial biofilms for desalination and water treatment: a pilot study.

Salinity has a significant impact on the water quality and crop yield. Physical desalination techniques were once thought to be expensive and time-consuming. Among biological techniques, halotolerant bacteria were thought to be the fastest and most effective way to reduce the salt content in brackish saltwater water. In the current study, halotolerant bacterial biofilms were used to desalinate saline water on abiotic substrates (such as sand, pebbles, glass beads, and plastic beads), and studied subsequently for the effects on Zea mays germination. Briefly, salt samples (SLT7 and SLT8) from the Khewra site in Punjab, Pakistan, as well as seawater and sea sand samples (USW1, USW3, USW6, DSW1, DSW4, SS1, and SS3) from Karachi, Sindh, Pakistan's Arabian Sea, were collected. Halotolerant bacteria were isolated and characterized. Crystal violet ring assays and capsule staining were used to estimate extracellular polymeric substance (EPS) and biofilm development, respectively. All halotolerant bacterial strains were spore formers and produced EPS and formed biofilms well. 16S rRNA gene sequencing of the best halotolerant bacteria, USW6, showed the closest (100%) similarity to Bacillus aerius strain G-07 (a novel species) (accession number ON202984). A pilot-scale experiment for desalinating the artificial water (supplemented with 1 M NaCl) using biofilm adhered abiotic beads showed declined level of NaCl from 1 M to 0.00003 M after 15 days in treated water. Also, Zea mays germination was observed in the plants using treated water compared to no growth in the non-treated saline water. Estimations of chlorophyll, total soluble sugar, and protein revealed that plants cultivated using elute collected from a desalinated pilot scale setup contained less chlorophyll (i.e., 5.994 and 116.76). Likewise, plants grown with elute had a total soluble protein and sugar content of 1.45 mg/ml and 1.3 mg/ml, respectively. Overall, in treated water plants, a minor drop in chlorophyll content, a slight increase in total soluble sugar content, and a slight increase in protein content were noted. The study concluded that biofilm-treated desalt water has the potential to significantly reduce the effects of droughts, soil salinization, and economic and environmental issues associated with agricultural drainage. The results specified the application of halotolerant bacteria biofilms (Bacillus aerius, a novel species, USW6) for water desalination to overcome the problem of water scarcity caused by global warming and the increased salinity.

REPercussions: how geminiviruses recruit host factors for replication.

Circular single-stranded DNA viruses of the family Geminiviridae encode replication-associated protein (Rep), which is a multifunctional protein involved in virus DNA replication, transcription of virus genes, and suppression of host defense responses. Geminivirus genomes are replicated through the interaction between virus Rep and several host proteins. The Rep also interacts with itself and the virus replication enhancer protein (REn), which is another essential component of the geminivirus replicase complex that interacts with host DNA polymerases α and δ. Recent studies revealed the structural and functional complexities of geminivirus Rep, which is believed to have evolved from plasmids containing a signature domain (HUH) for single-stranded DNA binding with nuclease activity. The Rep coding sequence encompasses the entire coding sequence for AC4, which is intricately embedded within it, and performs several overlapping functions like Rep, supporting virus infection. This review investigated the structural and functional diversity of the geminivirus Rep.

Investigation of NLR Genes Reveals Divergent Evolution on NLRome in Diploid and Polyploid Species in Genus Trifolium.

Crop wild relatives contain a greater variety of phenotypic and genotypic diversity compared to their domesticated counterparts. Trifolium crop species have limited genetic diversity to cope with biotic and abiotic stresses due to artificial selection for consumer preferences. Here, we investigated the distribution and evolution of nucleotide-binding site leucine-rich repeat receptor (NLR) genes in the genus of Trifolium with the objective to identify reference NLR genes. We identified 412, 350, 306, 389 and 241 NLR genes were identified from Trifolium. subterraneum, T. pratense, T. occidentale, subgenome-A of T. repens and subgenome-B of T. repens, respectively. Phylogenetic and clustering analysis reveals seven sub-groups in genus Trifolium. Specific subgroups such as G4-CNL, CCG10-CNL and TIR-CNL show distinct duplication patterns in specific species, which suggests subgroup duplications that are the hallmarks of their divergent evolution. Furthermore, our results strongly suggest the overall expansion of NLR repertoire in T. subterraneum is due to gene duplication events and birth of gene families after speciation. Moreover, the NLRome of the allopolyploid species T. repens has evolved asymmetrically, with the subgenome -A showing expansion, while the subgenome-B underwent contraction. These findings provide crucial background data for comprehending NLR evolution in the Fabaceae family and offer a more comprehensive analysis of NLR genes as disease resistance genes.

Evolution of NLR genes in genus Arachis reveals asymmetric expansion of NLRome in wild and domesticated tetraploid species.

Arachis hypogaea is an allotetraploid crop widely grown in the world. Wild relatives of genus Arachis are the rich source of genetic diversity and high levels of resistance to combat pathogens and climate change. The accurate identification and characterization of plant resistance gene, nucleotide binding site leucine rich repeat receptor (NLRs) substantially contribute to the repertoire of resistances and improve production. In the current study, we have studied the evolution of NLR genes in genus Arachis and performed their comparative genomics among four diploids (A. duranensis, A. ipaensis, A. cardenasii, A. stenosperma) and two tetraploid (wild: A. monticola and domesticated: A. hypogaea) species. In total 521, 354, 284, 794, 654, 290 NLR genes were identified from A. cardenasii, A. stenosperma and A. duranensis, A. hypogaea, A. monticola and A. ipaensis respectively. Phylogenetic analysis and classification of NLRs revealed that they belong to 7 subgroups and specific subgroups have expanded in each genome leading towards divergent evolution. Gene gain and loss, duplication assay reveals that wild and domesticated tetraploids species have shown asymmetric expansion of NLRome in both sub-genome (AA and BB). A-subgenome of A. monticola exhibited significant contraction of NLRome while B-subgenome shows expansion and vice versa in case of A. hypogaea probably due to distinct natural and artificial selection pressure. In addition, diploid species A. cardenasii revealed the largest repertoire of NLR genes due to higher frequency of gene duplication and selection pressure. A. cardenasii and A. monticola can be regarded as putative resistance resources for peanut breeding program for introgression of novel resistance genes. Findings of this study also emphasize the application neo-diploids and polyploids due to higher quantitative expression of NLR genes. To the best of our knowledge, this is the first study that studied the effect of domestication and polyploidy on the evolution of NLR genes in genus Arachis to identify genomic resources for improving resistance of polyploid crop with global importance on economy and food security.

Letter to the Editor: Mastrevirus sequences in a begomovirus-infected plant.

Recombination is a major driver of diversification of geminiviruses and is believed to be, for a large part, responsible for the present taxonomic structure of the family Geminiviridae. Examples of recent intergeneric recombination between viruses of the genera Begomovirus and Mastrevirus remain to be identified. Here, we show that one of the prerequisites for begomovirus-mastrevirus intergeneric recombination, co-infection of a single plant, does occur. The lack of reported recombination between viruses of these two genera may suggest that there are constraints to viable intergeneric recombinant viruses being produced, possibly due to the extreme genetic distances between extant begomo- and mastreviruses.

Population dynamics of pelagic rotifers in Marala Headworks (Pakistan).

Research work was designed to investigate the density and diversity of pelagic rotifers in a Lake near Marala Headworks. The physico-chemical parameters of water such as pH, dissolved oxygen, temperature, electrical conductivity, transparency and turbidity were evaluated. Correlation between rotifers and these parameters was also studied. Plankton sampling was done on monthly basis in order to check the population density of rotifers. In total, 18 species of rotifers were identified which belonged to 11 genera. The highest number of rotifers and their diversity was shown by genera namely Brachionus, Keratella, and Filinia. The Brachionus calyciflorus was dominant species in all the samples with mean population density (41%). Analysis of variance of physico-chemical parameters presented that the air and water temperature, electrical conductivity, transparency, dissolved oxygen and oxygen saturation were statistically significant in all the months. While pH was statistically non-significant (p≥0.05. Pearson correlation showed that oxygen and transparency were negatively correlated with rotifers density and diversity. Air and water temperature, concentration of hydrogen ions (pH), electrical conductivity and salinity showed positive relationship with density and diversity of rotifers.

Advances in Metabolomics-Driven Diagnostic Breeding and Crop Improvement.

Climate change continues to threaten global crop output by reducing annual productivity. As a result, global food security is now considered as one of the most important challenges facing humanity. To address this challenge, modern crop breeding approaches are required to create plants that can cope with increased abiotic/biotic stress. Metabolomics is rapidly gaining traction in plant breeding by predicting the metabolic marker for plant performance under a stressful environment and has emerged as a powerful tool for guiding crop improvement. The advent of more sensitive, automated, and high-throughput analytical tools combined with advanced bioinformatics and other omics techniques has laid the foundation to broadly characterize the genetic traits for crop improvement. Progress in metabolomics allows scientists to rapidly map specific metabolites to the genes that encode their metabolic pathways and offer plant scientists an excellent opportunity to fully explore and rationally harness the wealth of metabolites that plants biosynthesize. Here, we outline the current application of advanced metabolomics tools integrated with other OMICS techniques that can be used to: dissect the details of plant genotype-metabolite-phenotype interactions facilitating metabolomics-assisted plant breeding for probing the stress-responsive metabolic markers, explore the hidden metabolic networks associated with abiotic/biotic stress resistance, facilitate screening and selection of climate-smart crops at the metabolite level, and enable accurate risk-assessment and characterization of gene edited/transgenic plants to assist the regulatory process. The basic concept behind metabolic editing is to identify specific genes that govern the crucial metabolic pathways followed by the editing of one or more genes associated with those pathways. Thus, metabolomics provides a superb platform for not only rapid assessment and commercialization of future genome-edited crops, but also for accelerated metabolomics-assisted plant breeding. Furthermore, metabolomics can be a useful tool to expedite the crop research if integrated with speed breeding in future.

Diverse Begomoviruses Evolutionarily Hijack Plant Terpenoid-Based Defense to Promote Whitefly Performance.

Arthropod-borne pathogens and parasites are major threats to human health and global agriculture. They may directly or indirectly manipulate behaviors of arthropod vector for rapid transmission between hosts. The largest genus of plant viruses, Begomovirus, is transmitted exclusively by whitefly (Bemisia tabaci), a complex of at least 34 morphologically indistinguishable species. We have previously shown that plants infected with the tomato yellowleaf curl China virus (TYLCCNV) and its associated betasatellite (TYLCCNB) attract their whitefly vectors by subverting plant MYC2-regulated terpenoid biosynthesis, therefore forming an indirect mutualism between virus and vector via plant. However, the evolutionary mechanism of interactions between begomoviruses and their whitefly vectors is still poorly understood. Here we present evidence to suggest that indirect mutualism may happen over a millennium ago and at present extensively prevails. Detailed bioinformatics and functional analysis identified the serine-33 as an evolutionary conserved phosphorylation site in 105 of 119 Betasatellite species-encoded ßC1 proteins, which are responsible for suppressing plant terpenoid-based defense by interfering with MYC2 dimerization and are essential to promote whitefly performance. The substitution of serine-33 of ßC1 proteins with either aspartate (phosphorylation mimic mutants) or cysteine, the amino acid in the non-functional sßC1 encoded by Siegesbeckia yellow vein betasatellite SiYVB) impaired the ability of ßC1 functions on suppression of MYC2 dimerization, whitefly attraction and fitness. Moreover the gain of function mutation of cysteine-31 to serine in sßC1 protein of SiYVB restored these functions of ßC1 protein. Thus, the dynamic phosphorylation of serine-33 in ßC1 proteins helps the virus to evade host defense against insect vectors with an evolutionarily conserved manner. Our data provide a mechanistic explanation of how arboviruses evolutionarily modulate host defenses for rapid transmission.

DNA Barcoding: Molecular Identification and Phylogenetic Analysis of Pheretimoid Earthworm (Metaphire sp. and Amynthas sp.) Based on Mitochondrial Partial COI Gene from Sialkot, Pakistan.

The extremely difficult and challenging process is identifying pheretimoid species, genus Metaphire and Amynthas involving increased homoplasy in various morphological characteristics. The molecular identification, phylogenetic relationships, and evolutionary divergence time of earthworms belonging to the pheretimoid complex were investigated in this study using partial mitochondrial COI (cytochrome C oxidase subunit I) gene sequences ranging from 550-680 bp. Results revealed that 86 pheretimoid earthworms were morphologically different from a total of 342 mature worms. Moreover, 11 pheretimoid species were molecularly identified, including Metaphire posthuma (02), M. anomala (01), M. houlleti (02), M. californica (01), M. birmanica (02), Amynthas minimus (01), A. morrisi (01), and M. bununa (01). A phylogenetic tree was constructed with bootstrap values of 95%, which supported a monophyletic lineage of two well-supported clades formed by 12 partial COI sequences and 48 GenBank sequences using Hirudo medicinalis as an outgroup. The monophyly of these obtained genera indicated overall similarity at species level. Today, species like Amynthas, Metaphire and Pheretima have worm diversity in the form of pheretimoid earthworms, which dates to the Late Miocene (11.2-5.3 Mya) and the Pliocene (5.3-2.4 Mya). Compared to all relevant pheretimoid species, genetic p-distance values ranged from 0.0% to 0.57% (less than 1%). These low range values demonstrated that both genera Metaphire and Amynthas, supported the theory, which states that there are shared similarities among the species, despite different morphology. The current study is the first attempt in Pakistan to identify earthworms through DNA barcoding thus providing a genomic stamp. The work explored the significance of COI gene sequences to construct molecular tools that will be useful to overcome the different obstacles in morphologically similar earthworm identification and their phylogenetic study.

Antimicrobial Efficacy of Biogenic Cobalt and Copper Nanoparticles against Pathogenic Isolates.

Biogenic synthesis of cobalt (Co) and copper (Cu) nanoparticles (NPs) was performed using the bacterial strains Escherichia coli and Bacillus subtilis. Prepared NPs were confirmed by a color change to maroon for CoNPs and green for CuNPs. The NPs characterization using FTIR showed the presence of functional groups, i.e., phenols, acids, protein, and aromatics present in the Co and CuNPs. UV-vis spectroscopy of E. coli and B. subtilis CuNPs showed peaks at 550 and 625 nm, respectively. For E. coli and B. subtilis CoNPs, peaks were observed at 300 nm and 350 nm, respectively. Antibacterial and antifungal activity of B. subtilis and E. coli Co and CuNPs was determined at 100 mg/mL concentration against two bacterial strains at 5, 2.5, and 1.5 mg/mL against fungal two strains F. oxysporum and T. viridi, respectively. B. subtilis CuNPs showed significantly higher inhibition zones (ZOI=25.7-29.7 mm) against E. coli and B. subtilis compared to other biogenic NPs. Likewise, B. Subtilis CuNPs showed lower MIC (4.3 ± 6.3) and MBC (5.3 mg/mL) values against both tested isolates. Antifungal activity of B. subtilis and E. coli CuNPs and CoNPs showed a concentration-dependent decrease in ZOI. Among all biogenic NPs, B. subtilis CoNPs showed the highest ZOI (25-30 mm) against F. oxysporum followed by E. coli CuNPs with maximum ZOI (20-27 mm) against T. viridi. Again, B. subtilis CoNPs and E. coli CuNPs showed lowest MIC and MFC values against both fungal isolates. In conclusion, the current study showed that biogenically synthesized B. subtilis Cu or CoNPs can be used as effective antimicrobial agents due to their potential antibacterial and antifungal potential.

Patterns of Genetic Diversity among Alphasatellites Infecting Gossypium Species.

Alphasatellites are small single-stranded circular DNA molecules associated with geminiviruses and nanoviruses. In this study, a meta-analysis of known alphasatellites isolated from the genus Gossypium (cotton) over the last two decades was performed. The phylogenetic and pairwise sequence identity analysis suggested that cotton-infecting begomoviruses were associated with at least 12 different alphasatellites globally. Three out of twelve alphasatellite were associated with cotton leaf curl geminiviruses but were not isolated from cotton plants. The cotton leaf curl Multan alphasatellite, which was initially isolated from cotton, has now been reported in several plant species, including monocot plants such as sugarcane. Our recombination analysis suggested that four alphasatellites, namely cotton leaf curl Lucknow alphasatellites, cotton leaf curl Multan alphasatellites, Ageratum yellow vein Indian alphasatellites and Ageratum enation alphasatellites, evolved through recombination. Additionally, high genetic variability was detected among the cotton-infecting alphasatellites at the genome level. The nucleotide substitution rate for the replication protein of alphasatellites (alpha-Rep) was estimated to be relatively high (~1.56 × 10-3). However, unlike other begomoviruses and satellites, the first codon position of alpha-Rep rapidly changed compared to the second and third codon positions. This study highlights the biodiversity and recombination of alphasatellites associated with the leaf curl diseases of cotton crops.