Characterization of two novel fusariviruses co-infecting a single isolate of phytopathogenic fungus Botrytis cinerea.

A wide diversity of mycoviruses has been reported from Botrytis species, some with the potential to suppress the pathogenic abilities of this fungus. Considering their importance, this study was devised to find potential hypovirulence-associated mycoviruses found in Botrytis cinerea strains isolated from Pakistani strawberry fields. Here we report the complete genome characterization of two fusariviruses co-infecting a single isolate of phytopathogenic fungus B. cinerea (Kst14a). The viral genomes were sequenced by deep sequencing using total RNA fractions of the Kst14a isolate. The identified viruses were tentatively named Botrytis cinerea fusarivirus 9 (BcFV9) and Botrytis cinerea fusarivirus 3a (BcFV3a). Both viruses had a single-segmented (ssRNA) genome having a size of 6424 and 8370 nucleotides encoding two discontinuous open reading frames (ORFs). ORF-1 of both mycoviruses encodes for a polyprotein having a conserved domain of RNA-dependent RNA polymerase (RdRP) and a helicase domain (Hel) which function in RNA replication, while ORF2 encodes a hypothetical protein with an unknown function, respectively. Phylogenetic analysis indicated that BcFV9 made a clade with the genus Alphafusarivirus and BcFV3a fall in the genus Betafusarivirus in the family Fusariviridae. To our knowledge, this is the first report of two fusariviruses identified in isolates of B. cinerea from Pakistan. Both mycoviruses successfully transfected to a compatible strain of B. cinerea (Mst11). A comparison of virus-free (VF) and virus-infected (VI) isogenic lines showed the presence of these viruses was causing hypovirulence in infected strains. Virus-infected strains also had a small lesion size while testing the pathogenicity via apple assay.

Mycorrhizosphere bacteria inhibit greenhouse gas emissions from microplastics contaminated soil by regulating soil enzyme activities and microbial community structure.

Microplastics (MPs) accumulation in terrestrial ecosystems can affect greenhouse gases (GHGs) production by altering microbial and soil structure. Presently, research on the MPs effect on plants is not consistent, and underlying molecular mechanisms associated with GHGs are yet unknown. For the first time, we conducted a microcosm study to explore the impact of MPs addition (Raw vs. aged) and Trichoderma longibrachiatum and Bacillus subtilis inoculation (Sole vs. combination) on GHGs emission, soil community structure, physiochemical properties, and enzyme activities. Our results indicated that the addition of aged MPs considerably enhanced the GHGs emissions (N2O (+16%) and CO2 (+21%), respectively), C and N cycling gene expression, microbial biomass carbon, and soil physiochemical properties than raw MPs. However, the soil microbial community structure and enzyme activities were enhanced in raw MPs added treatments, irrespective of the MPs type added to soil. However, microbial inoculation significantly reduced GHGs emission by altering the expression of C and N cycling genes in both types of MPs added treatments. The soil microbial community structure, enzymes activities, physiochemical properties and microbial biomass carbon were enhanced in the presence of microbial inoculation in both type of MPs. Among sole and combined inoculation of Trichoderma and Bacillus subtilis, the co-applied Trichoderma and Bacillus subtilis considerably reduced the GHGs emission (N2O (-64%) and CO2 (-61%), respectively) by altering the expression of C and N cycling genes regardless of MPs type used. The combined inoculation also enhanced soil enzyme activities, microbial community structure, physiochemical properties and microbial biomass carbon in both types of MPs treatment. Our findings provide evidence that polyethylene MPs likely pose a high risk of GHGs emission while combined application of Trichoderma and Bacillus subtilis significantly reduced GHGs emission by altering C and N cycling gene expression, soil microbial community structure, and enzyme activities under MPs pollution in a terrestrial ecosystem.

Genome mining, antimicrobial and plant growth-promoting potentials of halotolerant Bacillus paralicheniformis ES-1 isolated from salt mine.

Salinity severely affects crop yield by hindering nitrogen uptake and reducing plant growth. Plant growth-promoting bacteria (PGPB) are capable of providing cross-protection against biotic/abiotic stresses and facilitating plant growth. Genome-level knowledge of PGPB is necessary to translate the knowledge into a product as efficient biofertilizers and biocontrol agents. The current study aimed to isolate and characterize indigenous plant growth-promoting strains with the potential to promote plant growth under various stress conditions. In this regard, 72 bacterial strains were isolated from various saline-sodic soil/lakes; 19 exhibited multiple in vitro plant growth-promoting traits, including indole 3 acetic acid production, phosphate solubilization, siderophore synthesis, lytic enzymes production, biofilm formation, and antibacterial activities. To get an in-depth insight into genome composition and diversity, whole-genome sequence and genome mining of one promising Bacillus paralicheniformis strain ES-1 were performed. The strain ES-1 genome carries 12 biosynthetic gene clusters, at least six genomic islands, and four prophage regions. Genome mining identified plant growth-promoting conferring genes such as phosphate solubilization, nitrogen fixation, tryptophan production, siderophore, acetoin, butanediol, chitinase, hydrogen sulfate synthesis, chemotaxis, and motility. Comparative genome analysis indicates the region of genome plasticity which shapes the structure and function of B. paralicheniformis and plays a crucial role in habitat adaptation. The strain ES-1 has a relatively large accessory genome of 649 genes (~ 19%) and 180 unique genes. Overall, these results provide valuable insight into the bioactivity and genomic insight into B. paralicheniformis strain ES-1 with its potential use in sustainable agriculture.

Identification of novel totiviruses from the ascomycetous fungus Geotrichum candidum.

Mycoviruses are widely distributed across the kingdom Fungi, including ascomycetous yeast strains of the class Saccharomycetes. Geotrichum candidum is an important fungal pathogen belonging to Saccharomycetes and has a diverse host range. Here, we report the characterization of four new classical totiviruses from two distinct Geotrichum candidum strains from Pakistan. The four identified viruses were tentatively named "Geotrichum candidum totivirus 1, 2, 3a, and 3b" (GcTV1-3b). The complete dsRNA genomes of the identified totiviruses are 4621, 4592, 4576, and 4576 bp in length, respectively. All totivirus genomes have two open reading frames, encoding a capsid protein (CP) and an RNA-dependent RNA polymerase (RdRP), respectively. The downstream RdRP domain is assumed to be expressed as a CP-RdRP fusion product via -1 frameshifting mediated by a heptameric slippery site. Sequence comparisons and phylogenetic analysis showed that each of the discovered viruses belongs to a new species of the genus Totivirus in the family Totiviridae, with GcTV1 and GcTV3 (a and b strains) clustering in one subgroup and GcTV2 in another subgroup.

A novel victorivirus from the phytopathogenic fungus Neofusicoccum parvum.

Neofusicoccum parvum is an important plant-pathogenic ascomycetous fungus that causes trunk diseases in a variety of plants. A limited number of reports on mycoviruses from this fungus are available. Here, we report the characterization of a novel victorivirus, Neofusicoccum parvum victorivirus 3 (NpVV3). An agarose gel dsRNA profile of a Pakistani strain of N. parvum, NFN, showed a band of ~5 kbp that was not detectable in Japanese strains of N. parvum. Taking a high-throughput and Sanger sequencing approach, the complete genome sequence of NpVV3 was determined to be 5226 bp in length with two open reading frames (ORF1 and ORF2) that encode a capsid protein (CP) and an RNA-dependent RNA polymerase (RdRP). The RdRP appears to be translated by a stop/restart mechanism facilitated by the junction sequence AUGucUGA, as is found in some other victoriviruses. BLASTp searches showed that NpVV3 CP and RdRP share the highest amino acid sequence identity (80.5% and 72.4%, respectively) with the corresponding proteins of NpVV1 isolated from a French strain of N. parvum. However, NpVV3 was found to be different from NpVV1 in its terminal sequences and the stop/restart facilitator sequence. NpVV3 particles ~35 nm in diameter were partially purified and used to infect an antiviral-RNA-silencing-deficient strain (∆dcl2) of an experimental ascomycetous fungal host, Cryphonectria parasitica. NpVV3 showed symptomless infection in the new host strain.

Assessment of mycoviral diversity in Pakistani fungal isolates revealed infection by 11 novel viruses of a single strain of Fusarium mangiferae isolate SP1.

An extensive screening survey was conducted on Pakistani filamentous fungal isolates for the identification of viral infections. A total of 396 fungal samples were screened, of which 36 isolates were found double-stranded (ds) RNA positive with an overall frequency of 9% when analysed by a classical dsRNA isolation method. One of 36 dsRNA-positive strains, strain SP1 of a plant pathogenic fungus Fusarium mangiferae, was subjected to virome analysis. Next-generation sequencing and subsequent completion of the entire genome sequencing by a classical Sanger sequencing method showed the SP1 strain to be co-infected by 11 distinct viruses, at least seven of which should be described as new taxa at the species level according to the ICTV (International Committee on Taxonomy of Viruses) species demarcation criteria. The newly identified F. mangiferae viruses (FmVs) include two partitivirids, one betapartitivirus (FmPV1) and one gammapartitivirus (FmPV2); six mitovirids, three unuamitovirus (FmMV2, FmMV4, FmMV6), one duamitovirus (FmMV5), and two unclassified mitovirids (FmMV1, FmMV3); and three botourmiavirids, two magoulivirus (FmBOV1, FmBOV3) and one scleroulivirus (FmBOV2). The number of coinfecting viruses is among the largest ones of fungal coinfections. Their molecular features are thoroughly described here. This represents the first large virus survey in the Indian sub-continent.

A second capsidless hadakavirus strain with 10 positive-sense single-stranded RNA genomic segments from Fusarium nygamai.

A unique capsidless virus with a positive-sense, single-stranded RNA genome (hadakavirus 1, HadV1), a member of the extended picorna-like supergroup, was isolated previously from the phytopathogenic fungus Fusarium oxysporum. Here, we describe the molecular and biological characterisation of a second hadakavirus strain from Fusarium nygamai, which has not been investigated in detail previously as a virus host. This virus, hadakavirus 1 strain 1NL (HadV1-1NL), has features similar to the first hadakavirus, HadV1-7n, despite having a different number of segments (10 for HadV1-1NL vs. 11 for HadV1-7n). The 10 genomic RNA segments of HadV1-1NL range in size from 0.9 kb to 2.5 kb. All HadV1-1NL segments show 67% to 86% local nucleotide sequence identity to their HadV1-7n counterparts, whereas HadV1-1NL has no homolog of HadV1-7n RNA8, which encodes a zinc-finger motif. Another interesting feature is the possible coding incapability of HadV1-1NL RNA10. HadV1-1NL was predicted to be capsidless based on the RNase A susceptibility of its replicative form dsRNA. Phenotypic comparison of multiple virus-infected and virus-free single-spore isolates indicated asymptomatic infection by HadV1-1NL. Less-efficient vertical transmission via spores was observed as the infected fungal colonies from which the spores were derived became older, as was observed for HadV1-7n. This study shows a second example of a hadakavirus that appears to have unusual features.

Plant uptake and leaching potential upon application of amendments in soils spiked with heavy metals (Cd and Pb).

The aim of this study was to assess the effects of soil amendments (organic and inorganic) on phytoavailability and leaching of cadmium (Cd) and lead (Pb) during enhanced phytoextraction. The vertical column study was carried out to investigate the metal leaching meanwhile studying plant biomass and metals uptake for phytoextraction by Pelargonium hortroum. For this purpose, soil amendments at several concentration levels, such as ethylene diaminetetraacetic acid (EDTA 0, 4, 5 mmol kg-1), ammonium nitrate (0, 8, 10 mmol kg-1), citric acid (0, 8, 10 mmol kg-1), compost (0, 8%, 10%) and titanium dioxide nanoparticles (TNPs, 0, 80, 100 mg kg-1) were used. Results revealed that EDTA efficiently improved Cd and Pb accumulation (mg kg-1) in shoots and roots. Cd accumulation was significantly increased by 270%, 44%, 145%, and 6.4% in shoot and 94%, 19%, 48% and 14% in root upon EDTA, ammonium nitrate, citric acid and TNPs application, respectively compared to the control without amendment (WA). Similarly, Pb accumulation was significantly increased by 71%, 58% and 52% in shoot and 88.8%, 70.6% and 64.6% in root upon exposure to higher levels of EDTA, citric acid and ammonium nitrate, respectively when compared to the WA control. Application of TNPs reduced the Pb-accumulation in shoots and roots by 33% and 28%, respectively. Similarly, the Pb-accumulation in shoots and roots was decreased by 39% and 35%, respectively upon compost addition. Plant biomass was significantly increased upon application of soil amendments, with the exception of EDTA. The maximum Cd and Pb uptake was found in citric acid amended soil. Leachate analysis revealed that the concentrations of Cd and Pb were increased by 109% and 101% in leachates upon EDTA application as compared to other amendments. In comparison with other amendments, citric acid may be recommended as an environmentally friendly alternative for non-biodegradable EDTA for enhanced phytoextraction of Cd and Pb.

Genome-wide analysis of wheat calcium ATPases and potential role of selected ACAs and ECAs in calcium stress.

BACKGROUND: P2- type calcium ATPases (ACAs-auto inhibited calcium ATPases and ECAs-endoplasmic reticulum calcium ATPases) belong to the P- type ATPase family of active membrane transporters and are significantly involved in maintaining accurate levels of Ca2+, Mn2+ and Zn2+ in the cytosol as well as playing a very important role in stress signaling, stomatal opening and closing and pollen tube growth. Here we report the identification and possible role of some of these ATPases from wheat. RESULTS: In this study, ACA and ECA sequences of six species (belonging to Poaceae) were retrieved from different databases and a phylogenetic tree was constructed. A high degree of evolutionary relatedness was observed among P2 sequences characterized in this study. Members of the respective groups from different plant species were observed to fall under the same clade. This pattern highlights the common ancestry of P2- type calcium ATPases. Furthermore, qRT-PCR was used to analyse the expression of selected ACAs and ECAs from Triticum aestivum (wheat) under calcium toxicity and calcium deficiency. The data indicated that expression of ECAs is enhanced under calcium stress, suggesting possible roles of these ATPases in calcium homeostasis in wheat. Similarly, the expression of ACAs was significantly different in plants grown under calcium stress as compared to plants grown under control conditions. This gives clues to the role of ACAs in signal transduction during calcium stress in wheat. CONCLUSION: Here we concluded that wheat genome consists of nine P2B and three P2A -type calcium ATPases. Moreover, gene loss events in wheat ancestors lead to the loss of a particular homoeolog of a gene in wheat. To elaborate the role of these wheat ATPases, qRT-PCR was performed. The results indicated that when plants are exposed to calcium stress, both P2A and P2B gene expression get enhanced. This further gives clues about the possible role of these ATPases in wheat in calcium management. These findings can be useful in future for genetic manipulations as well as in wheat genome annotation process.

The human papillomavirus type 16 L1 protein directly interacts with E2 and enhances E2-dependent replication and transcription activation.

The human papillomavirus (HPV) E2 protein is a multifunctional protein essential for the control of virus gene expression, genome replication and persistence. E2 is expressed throughout the differentiation-dependent virus life cycle and is functionally regulated by association with multiple viral and cellular proteins. Here, we show for the first time to our knowledge that HPV16 E2 directly associates with the major capsid protein L1, independently of other viral or cellular proteins. We have mapped the L1 binding region within E2 and show that the α-2 helices within the E2 DNA-binding domain mediate L1 interaction. Using cell-based assays, we show that co-expression of L1 and E2 results in enhanced transcription and virus origin-dependent DNA replication. Upon co-expression in keratinocytes, L1 reduces nucleolar association of E2 protein, and when co-expressed with E1 and E2, L1 is partially recruited to viral replication factories. Furthermore, co-distribution of E2 and L1 was detected in the nuclei of upper suprabasal cells in stratified epithelia of HPV16 genome-containing primary human keratinocytes. Taken together, our findings suggest that the interaction between E2 and L1 is important for the regulation of E2 function during the late events of the HPV life cycle.

Identification of the odorant binding proteins of Western Flower Thrips (Frankliniella occidentalis), characterization and binding analysis of FoccOBP3 with molecular modelling, molecular dynamics simulations and a confirmatory field trial.

Olfactory systems are indispensable for insects as they, including Western Flower Thrips (Frankliniella occidentalis), use olfactory cues for ovipositing and feeding. F. occidentalis use odorant binding proteins (OBPs) to transport semiochemicals to odorant receptors to induce a behavioural response from the sensillum lymph of the insect's antennae. This study identifies four OBPs of F. occidentalis and analyses their expression at three stages of growth: larvae, adult males and adult females. Further, it investigates the presence of conserved motifs and their phylogenetic relationship to other insect species. Moreover, FoccOBP3 was in silico characterized to analyse its structure along with molecular docking and molecular dynamics simulations to understand its binding with semiochemicals of F. occidentalis. Molecular docking revealed the interactions of methyl isonicotinate, p-anisaldehyde and (S)-(-)-verbenone with FoccOBP3. Moreover, molecular dynamics simulations showed bonding stability of these ligands with FoccOBP3, and field trials validated that Lurem TR (commercial product) and p-anisaldehyde had greater attraction as compared to (S)-(-)-verbenone, given the compound's binding with FoccOBP3. The current study helps in understanding the tertiary structure and interaction of FoccOBP3 with lures using computational and field data and will help in the identification of novel lures of insects in the future, given the importance of binding with OBPs.Communicated by Ramaswamy H. Sarma.

The good, the bad and the cryptic: The multifaceted roles of mycoviruses and their potential applications for a sustainable agriculture.

Mycoviruses are natural inhabitants of fungi and have been identified in almost all fungal taxonomic groups. Mycoviruses that infect phytopathogenic fungi are now becoming a hot research area due to their potential for the biocontrol of important plant pathogens. But, before considering a mycovirus for biocontrol, we should be fully aware of the effects it induces in a fungal host and its interactions with other viruses, fungal strains and even the host plants. Mycoviral infections are generally associated with different effects, ranging from hypovirulence to hypervirulence, but they can often be cryptic (latent infections). The cryptic lifestyle has been associated to many mycoviruses, but thanks to growing knowledge we are now aware that it is often associated to axenic conditions while the real effects can be observed only in nature. Other mycoviruses either promote (hypervirulence) or (hypovirulence) fungal pathogenicity by a strong impact on the fungal physiology or by blocking the production of toxins or effectors. Finally, indirect effects of mycoviral infections can also be provided to the plant that hosts the fungal isolate, highlighting not only their potential as direct biocontrol agents but also as priming agents for plant resilience to biotic and abiotic stresses. This review provides a broad overview of mycoviral interactions both with their hosts and with other mycoviruses, highlighting the most interesting examples. In contrast to what has been observed to date, we believe that the collective availability of these data will not only improve our understanding of mycoviruses, but also increase our confidence in considering them as alternative measures against fungal diseases to improve the sustainable production of food and feed commodities.

Mycovirus-induced hypovirulence in notorious fungi Sclerotinia: a comprehensive review.

Members of the genus Sclerotinia are notorious plant pathogens with a diverse host range that includes many important crops. A huge number of mycoviruses have been identified in this genus; some of these viruses are reported to have a hypovirulent effect on the fitness of their fungal hosts. These mycoviruses are important to researchers from a biocontrol perspective which was first implemented against fungal diseases in 1990. In this review, we have presented the data of all hypovirulent mycoviruses infecting Sclerotinia sclerotiorum isolates. The data of hypovirulent mycoviruses ranges from 1992 to 2023. Currently, mycoviruses belonging to 17 different families, including (+) ssRNA, (-ssRNA), dsRNA, and ssDNA viruses, have been reported from this genus. Advances in studies had shown a changed expression of certain host genes (responsible for cell cycle regulation, DNA replication, repair pathways, ubiquitin proteolysis, gene silencing, methylation, pathogenesis-related, sclerotial development, carbohydrate metabolism, and oxalic acid biosynthesis) during the course of mycoviral infection, which were termed differentially expressed genes (DEGs). Together, research on fungal viruses and hypovirulence in Sclerotinia species can deepen our understanding of the cellular processes that affect how virulence manifests in these phytopathogenic fungi and increase the potential of mycoviruses as a distinct mode of biological control. Furthermore, the gathered data can also be used for in-silico analysis, which includes finding the signature sites [e.g., hypovirus papain-like protease (HPP) domain, "CCHH" motif, specific stem-loop structures, p29 motif as in CHV1, A-rich sequence, CA-rich sequences as in MoV1, GCU motif as in RnMBV1, Core motifs in hypovirus-associated RNA elements (HAREs) as in CHV1] that are possibly responsible for hypovirulence in mycoviruses.

An Overview of Mycoviral Curing Strategies Used in Evaluating Fungal Host Fitness.

The number of novel mycoviruses is increasing at a high pace due to advancements in sequencing technologies. As a result, an uncountable number of mycoviral sequences are available in public sequence repositories. However, only genomic information is not sufficient to understand the impact of mycoviruses on their host biology. Biological characterization is required to determine the nature of mycoviruses (cryptic, hypervirulent, or hypovirulent) and to search for mycoviruses with biocontrol and therapeutic potential. Currently, no particular selective method is used as the gold standard against these mycoviral infections. Given the importance of curing, we present an overview of procedures used in preparation of isogenic lines, along with their benefits and drawbacks. We concluded that a combination of single-spore isolation and hyphal tipping is the best fit for preparation of isogenic lines. Furthermore, recent bioinformatic approaches should be introduced in the field of mycovirology to predict virus-specific antivirals to get robust results.

Genome-wide identification and comparative in-silico characterization of ß-galactosidase (GH-35) in ascomycetes and its role in germ tube development of Aspergillus fumigatus via RNA-seq analysis.

ß-galactosidase (Lactase), an enzyme belonging to the glycoside hydrolase family causing the hydrolysis and trans-glycosylation of ß-D-galactosides, has a vital role in dairy industries. The current investigation emphasizes on in-silico identification and comparative analysis of different fungal lactases present in Aspergillus fumigatus, Aspergillus oryzae, Botrytis cinerea, and Fusarium fujikuroi. Prediction of motifs and domains, chromosomal positioning, gene structure, gene ontology, sub-cellular localization and protein modeling were performed using different bioinformatics tools to have an insight into the structural and functional characteristics of ß-galactosidases. Evolutionary and homology relationships were established by phylogenetic and synteny analyses. A total of 14 ß-gal genes (GH-35) were identified in these species. Identified lactases, having 5 domains, were predicted to be stable, acidic, non-polar and extracellularly localized with roles in polysaccharide catabolic process. Results showed variable exonic/intronic ratios of the gene structures which were randomly positioned on chromosomes. Moreover, synteny blocks and close evolutionary relationships were observed between Aspergillus fumigatus and Aspergillus oryzae. Structural insights allowed the prediction of best protein models based on the higher ERRAT and Q-MEAN values. And RNA-sequencing analysis, performed on A. fumigatus, elucidated the role of ß-gal in germ tube development. This study would pave the way for efficient fungal lactase production as it identified ß-gal genes and predicted their various features and also it would provide a road-way to further the understanding of A. fumigatus pathogenicity via the expression insights of ß-gal in germ tube development.

Biochar alleviated the toxic effects of PVC microplastic in a soil-plant system by upregulating soil enzyme activities and microbial abundance.

Plastics have become an emerging pollutant threatening the sustainability of agroecosystems and global food security. Biochar, a pro-ecosystem/negative carbon emission technology can be exploited as a circular approach for the conservation of plastics contaminated agricultural soils. However, relatively few studies have focused on the effects of biochar on plant growth and soil biochemical properties in a microplastic contaminated soil. This study investigated the effects of a cotton stalk (Gossypium hirsutum L.) biochar on plant growth, soil microbes, and enzyme activity in PVC microplastic (PVC-MPs) contaminated soil. Biochar amendment increased shoot dry matter production in PVC-MPs contaminated soil. However, PVC-MPs alone significantly reduced the soil urease and dehydrogenase activity, soil organic and microbial biomass carbon, bacterial/fungal community percentage, and their abundance (16S rRNA and 18S rRNA genes, respectively). Interestingly, biochar amendment with PVC-MPs significantly alleviated the hazardous effects. Principal component and redundancy analysis of the soil properties, bacterial 16S rRNA genes, and fungal ITS in the biochar-amended PVC-MPs treatments revealed that the observed traits formed an obvious cluster compared to non-biochar treatments. To sum up, this study indicated that PVC-MPs contamination was not benign, while biochar shielded the hazardous effects and sustained soil microbial functionality.

Mycovirus Hunting Revealed the Presence of Diverse Viruses in a Single Isolate of the Phytopathogenic Fungus Diplodia seriata From Pakistan.

Diplodia seriata in the family Botryosphaeriaceae is a cosmopolitan phytopathogenic fungus and is responsible for causing cankers, fruit rot and leaf spots on economically important plants. In this study, we characterized the virome of a single Pakistani strain (L3) of D. seriata. Several viral-like contig sequences were obtained via a previously conducted next-generation sequencing analysis. Multiple infection of the L3 strain by eight RNA mycoviruses was confirmed through RT-PCR using total RNA samples extracted from this strain; the entire genomes were determined via Sanger sequencing of RT-PCR and RACE clones. A BLAST search and phylogenetic analyses indicated that these eight mycoviruses belong to seven different viral families. Four identified mycoviruses belong to double-stranded RNA viral families, including Polymycoviridae, Chrysoviridae, Totiviridae and Partitiviridae, and the remaining four identified mycoviruses belong to single-stranded RNA viral families, i.e., Botourmiaviridae, and two previously proposed families "Ambiguiviridae" and "Splipalmiviridae". Of the eight, five mycoviruses appear to represent new virus species. A morphological comparison of L3 and partially cured strain L3ht1 suggested that one or more of the three viruses belonging to Polymycoviridae, "Splipalmiviridae" and "Ambiguiviridae" are involved in the irregular colony phenotype of L3. To our knowledge, this is the first report of diverse virome characterization from D. seriata.

Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and comparative insights.

Anopheles stephensi is an important vector of malaria in the South Asia, the Middle East, and Eastern Africa. The olfactory system of An. stephensi plays an important role in host-seeking, oviposition, and feeding. Odorant binding proteins (OBPs) are globular proteins that play a pivotal role in insect olfaction by transporting semiochemicals through the sensillum lymph to odorant receptors (ORs). Custom motifs designed from annotated OBPs of Aedes aegypti, Drosophila melanogaster, and Anopheles gambiae were used for the identification of putative OBPs from protein sequences of the An. stephensi Indian strain. Further, BLASTp was also performed to identify missing OBPs and ORs. Subsequently, the presence of domains common to OBPs was confirmed. Identified OBPs were further classified into three sub-classes. Phylogenetic and syntenic analyses were carried out to find homology, and thus the evolutionary relationship between An. stephensi OBPs and ORs with those of An. gambiae, Ae. aegypti and D. melanogaster. Gene structure and physicochemical properties of the OBPs and ORs were also predicted. A total of 44 OBPs and 45 ORs were predicted from the protein sequences of An. stephensi. OBPs were further classified into the classic (27), atypical (10) and plus-C (7) OBP subclasses. The phylogeny revealed close relationship of An. stephensi OBPs and ORs with An. gambiae homologs whereas only five OBPs and two ORs of An. stephensi were related to Ae. aegypti OBPs and ORs, respectively. However, D. melanogaster OBPs and ORs were distantly rooted. Synteny analyses showed the presence of collinear block between the OBPs and ORs of An. stephensi and An. gambiae as well as Ae. aegypti's. No homology was found with D. melanogaster OBPs and ORs. As an important component of the olfactory system, correctly identifying a species' OBPs and ORs provide a valuable resource for downstream translational research that will ultimately aim to better control the malaria vector An. stephensi.

Whole-genome sequence of a putative pathogenic Bacillus sp. strain SD-4 isolated from cattle feed.

OBJECTIVES: The present study describes the draft genome sequence of a novel Bacillus sp. strain SD-4 isolated from animal feed. The study aims to get a deeper insight into antimicrobial resistance and secondary metabolite biosynthetic gene clusters (BGCs) and the association between them. METHODS: The strain SD-4 was preliminarily evaluated for antibacterial activities, motility, biofilm formation, and enterotoxin production using in vitro assays. The genome of strain SD-4 was sequenced using the Illumina HiSeq 2500 platform with paired-end reads. The reads were assembled and annotated using SPAdes and PGAP, respectively. The genome was further analysed using several bioinformatics tools, including TYGS, AntiSMASH, RAST, PlasmidFinder, VFDB, VirulenceFinder, CARD, PathogenFinder, MobileElement finder, IslandViewer, and CRISPRFinder. RESULTS: In vitro assays showed that the strain is motile, synthesises biofilm, and produces an enterotoxin and antibacterial metabolites. The genome analysis revealed that the strain SD-4 carries antimicrobial resistance genes (ARGs), virulence factors, and beneficial secondary metabolite BGCs. Further genome analysis showed interesting genome architectures containing several mobile genetic elements, including two plasmid replicons (repUS22 and rep20), five prophages, and at least four genomic islands (GIs), including one Listeria pathogenicity island LIPI-1. Moreover, the strain SD-4 is identified as a putative human pathogen. CONCLUSION: The genome of strain SD-4 harbours several BGCs coding for biologically active metabolites. It also contains antimicrobial resistance genes and is identified as a potential human pathogen. These results can be used to better comprehend antibiotic resistance in environmental bacteria that are not influenced by human intervention.

High Occurrence of Multidrug-Resistant Escherichia coli Strains in Bovine Fecal Samples from Healthy Cows Serves as Rich Reservoir for AMR Transmission.

OBJECTIVES: Antibiotics are valuable therapeutics. However, the unwarranted and excessive use of these antimicrobials in food animals and the consequent contamination of the environment have been associated with the emergence and spread of antimicrobial resistance. Continuous surveillance and monitoring of antimicrobial resistance among E. coli isolates is recommended, not only for bovine health but also for public health. This study aims to assess the antimicrobial resistance profile, virulence potential, and genetic characterization of fecal E. coli isolates from healthy cows. METHODOLOGY: The in vitro, phenotypic antibiotic resistance of isolates was measured via the Kirby-Bauer disc-diffusion method against twenty-seven antibiotics. The ß-lactamase enzymatic activities of the strains were also investigated. For the assessment of virulence potential, fecal E. coli isolates were subjected to several in vitro pathogenicity assays, including biofilm formation ability, blood hemolysis, complement resistance, and growth in human urine. Phylogroup determination and virulence-associated genes were detected via multiplex PCR. RESULTS: In vitro antibiotic resistance profiling showed that 186/200 (93%) of the isolates were multidrug-resistant (MDR), with the highest resistance against penicillin, tetracycline, fluoroquinolone, and macrolide classes of antibiotics. Of particular concern was the phenotypic resistance to colistin in 52/200 isolates (26%), though 16% of the total isolates harbored mcr1, the genetic determinant of colistin. Despite the scarce use of fluoroquinolone, cephalosporin, and carbapenem in the agricultural sector, resistance to these classes was evident due to the presence of extended-spectrum ß-lactamase (ESBL) in 41% of E. coli isolates. The ß-lactamase genotyping of E. coli isolates showed that 47% of isolates harbored either blaCTX or blaTEM. Approximately 32% of isolates were resistant to serum complement, and their growth in human urine was evident in 18% of isolates, indicating a possible infection of these isolates in high nitrogenous condition. Phylogrouping showed that the most prevalent phylogenetic group among fecal E. coli isolates was phylogroup B1 (57%), followed by phylogroups A (33%), D (6%), and B2 (4%). The most prevalent virulence-associated genes in fecal E. coli were fimH, iss and tatT. Results showed that ten isolates (5%) harbored the stx1 gene, the genetic marker of enterohemorrhagic E. coli. This study provides insights into the antibiotic resistance and virulence profiling of the fecal E. coli isolates from healthy cows. These results emphasize the need for imposing regulations on the proper use of antibiotics and growth promoters in food-producing animals.