Nano-formulated pesticides are increasingly desired to control the insect pest and plant disease with superior efficacy for guaranteeing the high yield and quality in crop production. However, the impact of nanocarrier morphology on pesticide resistance against rainwash, photolysis, and overall pesticide bioactivity remains unknown. In this work, a series of well-defined and morphology-controllable polymer nanocarriers for pesticide are fabricated through polymerization-induced self-assembly. All these generated soft nanocarriers with hydrophobic regions exhibit excellent pesticide loading capacity over 70%. After foliar spraying, the generated one-dimensional worm-like nanopesticides exhibit an extremely high retention of 80% on leaves after 10 mm rainfall (only 10% for naked pesticide) and a good resistance to photodegradation under UV irradiation (less than 50% for worm-like micelle vs 70% for naked pesticide degradation after 20 h irradiation under 365 nm). Therefore, worm-like nanocarriers show higher bioactivity than that of spherical nanocarriers. In general, the comprehensive performance order of pesticide-loaded polymer nanocarriers is worm-like micelle > spherical vesicle > spherical micelle. Moreover, the facilely resultant soft nanocarriers of pesticides possess a remarkable low cytotoxicity and excellent biocompatibility in human cells. This nanoplatform possesses simple fabrication, structure controllability, excellent performances, and environmental friendliness, enabling the nanocarriers promising for effective pesticide delivery.

Entomopathogenic fungi offer an ecologically sustainable and highly effective alternative to chemical pesticides for managing plant pests. However, the efficacy of mycoinsecticides in pest control suffers from environmental abiotic stresses, such as solar UV radiation and temperature fluctuations, which seriously hinder their practical application in the field. Herein, we discovered that the synthetic amphiphilic thermal-responsive polymers are able to significantly enhance the resistance of Metarhizium robertsii conidia against thermal and UV irradiation stresses. The thermosensitive polymers with extremely low cytotoxicity and good biocompatibility can be engineered onto the M. robertsii conidia surface by anchoring hydrophobic alkyl chains. Further investigations revealed that polymer supplementation remarkably augmented the capacity for penetration and the virulence of M. robertsii under heat and UV stresses. Notably, broad-spectrum entomopathogenic fungi can be protected by the polymers. The molecular mechanism was elucidated through exploring RNA sequencing and in vivo/vitro enzyme activity assays. This work provides a novel avenue for fortifying the resilience of entomopathogenic fungi, potentially advancing their practical application as biopesticides.

Glycopolymer-supported silver nanoparticles (AgNPs) have demonstrated a promising alternative to antibiotics for the treatment of multidrug-resistant bacteria-infected diseases. In this contribution, we report a class of biohybrid glycopolymersome-supported AgNPs, which are capable of effectively killing multidrug-resistant bacteria and disrupting related biofilms. First of all, glycopolymersomes with controllable structures were massively fabricated through reversible addition-fragmentation chain transfer (RAFT) polymerization-induced self-assembly (PISA) in an aqueous solution driven by complementary hydrogen bonding interaction between the pyridine and amide groups of N-(2-methylpyridine)-acrylamide (MPA) monomers. Subsequently, Ag+ captured by glycopolymersomes through the coordination between pyridine-N and Ag+ was reduced into AgNPs stabilized by glycopolymersomes upon addition of the NaBH4 reducing agent, leading to the formation of the glycopolymersome@AgNPs biohybrid. As a result, they showed a wide-spectrum and enhanced removal of multidrug-resistant bacteria and biofilms compared to naked AgNPs due to the easier adhesion onto the bacterial surface and diffusion into biofilms through the specific protein-carbohydrate recognition. Moreover, the in vivo results revealed that the obtained biohybrid glycopolymersomes not only demonstrated an effective treatment for inhibiting the cariogenic bacteria but also were able to repair the demineralization of caries via accumulating Ca2+ through the recognition between carbohydrates and Ca2+. Furthermore, glycopolymersomes@AgNPs showed quite low in vitro hemolysis and cytotoxicity and almost negligible acute toxicity in vivo. Overall, this type of biohybrid glycopolymersome@AgNPs nanomaterial provides a new avenue for enhanced antibacterial and antibiofilm activities and the effective treatment of oral microbial-infected diseases.

Transgenic RNA interference (RNAi) represents a burgeoning and promising alternative avenue to manage plant diseases and insect pests in plants. Nonviral nanostructured dsRNA carriers have been demonstrated to possess great potential to facilitate the application of RNAi. However, it remains a critical challenge to achieve the targeted and effective release of dsRNA into the pest cells, limiting the efficiency of the biological control of pests and diseases in practical applications. In this study, we designed and constructed a new type of core-shell polymeric nanostructure (CSPN) with controllable structure, eco-friendliness, and good biocompatibility, on which dsRNA can be efficiently loaded. Once loaded into CSPNs, the dsRNA can be effectively prevented from nonsense degradation by enzymes before entering cells, and it shows targeted and image-guided release triggered by intracellular ATP, which significantly increases the efficiency of gene transfection. Significantly, the in vivo study of the typical lepidoptera silkworm after oral feeding demonstrates the potential of dsCHT10 in CSPNs for a much better knockdown efficiency than that of naked dsCHT10. This innovation enables the nanotechnology developed for the disease microenvironment-triggered release of therapeutic genes for application in sustainable crop protection.

Polymer-based nanomaterials have exhibited promising alternative avenues to combat the globe challenge of multidrug-resistant bacterial infection. However, most of the reported polymeric nanomaterials have facially linear amphiphilic structures with positive net charges, which may lead to nonspecific binding, high hemolysis, and uncontrollable self-organization, limiting their practical applications. In this contribution, we report a one-dimensional glyconanorod (GNR) through self-assembly of well-defined β-cyclodextrin-based glycoconjugates (RMan) featuring hydrophobic carbon-based chains and amide rhodamines with an adenosine triphosphate (ATP)-recognition site and targeted and hydrophilic mannoses and positively net-charged ethylene amine groups. The GNRs show superior targeting sensing and killing for Gram-negative Escherichia coli (E. coli) dominantly through the multivalent recognition between mannoses on the nanorod and the lectin on the surface of E. coli. Moreover, red fluorescence was light on due to the hydrogen bonding between amide rhodamine and ATP. Benefiting from the designs, the GNRs are capable of possessing a higher therapeutic index and of encapsulating other antibiotics. They exhibit an enhanced effect against E. coli strains. Intriguingly, the GNRs displayed a more reduced hemolysis effect and lower cytotoxicity compared to that of ethylene glyco-modified nanorods. These results reveal that the glyconanomaterials not only feature superior and targeted bacterial sensing and antibacterial activity, but also better biocompatibility compared with the widely used PEG-covered nanomaterials. Furthermore, the in vivo studies demonstrate that the targeted and ATP-responsive GNRs complexed with antibiotics showed better treatment using a mouse model of abdominal sepsis following intraperitoneal E. coli infection. The present work describes a targeted and effective sensing and antibacterial platform based on glycoconjugates that have potential applications for the treatment of infections caused by pathogenic microorganisms.

A library of 14 dynamic glycopeptide amphiphilic dendrimers composed of 14 hydrophilic and bioactive saccharides (seven kinds) as dendrons and 7 hydrophobic peptides (di- and tetrapeptides) as arms with β-cyclodextrin (CD) as a core were facially designed and synthesized in several steps. Fourteen saccharides were first conjugated to the C-2 and C-3 positions of CD, forming glycodendrons. Subsequently, seven oligopeptide arms were introduced at the C-6 positions of a CD moiety by an acylhydrazone dynamic covalent bond, resulting in unique Janus amphiphilic glycopeptide dendrimers with precise and varied molecular structures. The kinds of hydrophilic parts of saccharides and hydrophobic parts of peptides were easily varied to prepare a series of amphiphilic Janus glycopeptide dendrimers. Intriguingly, these obtained amphiphilic glycopeptide dendrimers showcased very different self-assembly behaviors from the traditional amphiphilic linear block-copolymers and self-assembled into different glyco-nanostructures with controllable morphologies including glycospheres, worm-like micelles, and fibers depending upon the repeat unit ratio of saccharides and phenylalanine. Both glycodendrons and glycopeptide assemblies displayed strong and specific recognitions with C-type mannose-specific lectin. Moreover, these glycopeptide nanomaterials can encapsulate exemplary hydrophobic molecules such as Nile red (NR). The dye-loaded glycopeptide nanostructures showed a pH-controllable release behavior around the physiological and acidic tumor environment. Furthermore, cell experiments demonstrated that such glyco-nanostructures can further facilitate the functions of a model drug of the pyridone agent to reduce the expression of monocyte chemotactic protein-1 (MCP-1) and interleukin -1beta (IL-1β) in the primary peritoneal macrophages via encapsulating drugs. Considering all the abovementioned advantages including unique and precise structures, bioactivity, targeting, and controllable cargo release, we believe that these findings can not only enrich the library of glycopeptides but also provide a new avenue to the fabrication of smart and structure-controllable glyco-nanomaterials which hold great potential biological applications such as targeted delivery and release of therapeutic and bioactive molecules.

A diversity of pyridine-containing polymeric nanomaterials with controllable structures and multiple responses were developed through complementary hydrogen-bonding directed polymerization-induced self-assembly in aqueous solution.

Summary The slow lethality of fungal biopesticides to insects restrains their widespread application as a strategy of pest control. In this study, unary, binary and ternary transgenic Metarhizium robertsii were created by integrating genes that encode the scorpion neurotoxin Bjα IT, the cuticle‐degrading protease Pr1A, and a double‐stranded RNA (dsRNA) that targets host gnbp3 , individually or collectively under a constitutive promoter to enhance virulence. Compared with the parental wild type, all unary transgenic strains had increased virulence against four insect species, Tenebrio molitor , Locusta migratoria , Plutella xylostella and Galleria mellonella , whereas the binary transgenic strain expressing both pr1A and Bjα IT had increased virulence to T. molitor and L. migratoria , with no change in virulence against P. xylostella and G. mellonella . Importantly, all ternary transgenic strains simultaneously expressing pr1A , Bjα IT, and the dsRNA specific to host gnbp3 exhibited the highest increase in insect‐specific virulence. This finding highlights a novel strategy for genetic engineering of dsRNAs that target genes associated with the host immune response alongside virulence genes to maximize fungal virulence and lethality against insect pests.

G-protein-coupled receptor K (GPRK), which is a class VI fungal G-protein-coupled receptor (GPCR), plays a critical role in plant immunity against pathogens by mediating the endocytic pathway, influencing metabolism in response to environmental signals, and regulating asexual reproduction and pathogenic development. However, the function of these proteins in entomopathogenic fungi has rarely been investigated. Accordingly, we characterized MrGPRK, a GPCR in the entomopathogenic fungus Metarhizium robertsii containing a C-terminal seven-transmembrane and a conserved regulator of G protein signaling domain, and found that it localized to endosomes. Mutant phenotype assays showed that a ΔMrGprk strain displayed increased defects in radial growth (~28%) and decreased conidial production (~80%) compared with a wild-type strain. Decreased conidiation rates coincided well with the repression of conidiation-related regulatory genes, including three key conidial transcription factors: brlA, abaA, and wetA. MrGprk deficiency impaired full virulence (both topical and injectable inoculations). Further analysis demonstrated that deleting fungal MrGprk decreased the rates of appressorium formation and suppressed the transcription of several genes contributing to appressorial turgor pressure, cuticle penetration, and pH regulation. Additionally, the ΔMrGprk strain showed higher cyclic (cAMP) levels, suggesting that this GPCR is critical for cAMP signal transduction. In summary, MrGPRK was found to contribute to vegetative growth, conidial production, and full virulence of M. robertsii. These findings are conducive to a better understanding of the roles of GPCRs in the development and pathogenicity of entomopathogenic fungi.

Ketol-acid reductoisomerase (ILVC) is the second enzyme in the branched-chain amino acid (BCAA) biosynthesis, which regulates many physiological activities in a variety of organisms from bacteria to fungi and plants. In this work, function mechanisms of ILVC in Metarhizium robertsii Metchnikoff (Hypocreales: Clavicipitaceae) were explored with site-directed mutagenesis, reductase activity assays and transcriptomics analysis. The reductase activity assays showed that ILVC from phytopathogenic fungi exhibited significantly higher activities than those from entomopathogenic fungi but lower than those from yeast. Site-directed mutagenesis and enzymatic activities of MrILVC with different active-site mutants (Arg-113, Ser-118, Asp-152, Asp-260, and Glu-264) confirmed that active sites of MrILVC are conserved with plant and bacterial ILVCs. Deleting MrilvC causes the complete failures of vegetative growth and conidial germination, feeding with branched-chain amino acids (BCAAs) recovers the fungal growth but not conidial germination, while both characteristics are restored when supplemented with yeast extract. Compared to ΔMrilvC cultured in czapek agar (CZA), plenty of genes involved in the biosynthesis of antibiotics and amino acids were up- or down-regulated in the wild type or ΔMrilvC feeding with either BCAAs or yeast extract. Further analysis showed some genes, such as catalase A, participate in mycelial growth and conidial germination was down-regulated in ΔMrilvC from CZA, revealing that MrILVC might control the fungal development by gene regulation and BCAAs or yeast extract could play partial roles of MrILVC. This study will advance our understanding of ILVC function mechanisms in fungi.

Conidiobolus spp. are important saprophytic basal fungi. However, to date, no genomic-level data for decaying plant materials in the genus Conidiobolus has been reported. Here, we report that the 33.4-Mb genome of Conidiobolus heterosporus encodes 10,857 predicted genes. Conidiobolus heterosporus harbors 394 CAZyme-encoding genes belonging to 4 major modules but does not encode a polysaccharide lyase (PL). Many carbohydrate esterases (CEs) belonging to the family CE12 play crucial roles as pectin acetylesterases, and 14 genes were upregulated in the IM (fungus grown on inducing medium) among 17 expressed CE12 family genes. In addition, most of the genes in the GH132 CAZyme family showed a greater than 5-fold increase in expression in the IM compared with that in the wild type. Furthermore, 122 P450-encoding genes grouped into 11 families were detected in the fungal genome, most of which belonged to the CYP547 family (36 genes) followed by CYP548 (27 genes) and CYP5856 (25 genes). Interestingly, members of the families CYP5014 and CYP5136 were identified, the first time such enzymes have been described in a fungus. Our findings provide new insights into the genomics and genomic features of the saprophytic basal fungus C. heterosporus.

The genus Conidiobolus is an important group in entomophthoroid fungi and is considered to be polyphyletic in recent molecular phylogenies. To re-evaluate and delimit this genus, multi-locus phylogenetic analyses were performed using the large and small subunits of nuclear ribosomal DNA (nucLSU and nucSSU), the small subunit of the mitochondrial ribosomal DNA (mtSSU) and the translation elongation factor 1-alpha (EF-1α). The results indicated that the Conidiobolus is not monophyletic, being grouped into a paraphyletic grade with four clades. Consequently, the well-known Conidiobolus is revised and three new genera Capillidium, Microconidiobolus and Neoconidiobolus are proposed along with one new record and 22 new combinations. In addition, the genus Basidiobolus is found to be basal to the other entomophthoroid taxa and the genus Batkoa locates in the Entomophthoraceae clade.

The genus Conidiobolus Bref. is widely distributed and the Conidiobolus sensu lato contained three other genera, Capillidium , Microconidiobolus and Neoconidiobolus . A molecular phylogeny based on the nuclear large subunit of rDNA (nucLSU), the mitochondrial small subunit of rDNA (mtSSU) and the translation elongation factor 1-alpha gene (TEF1) revealed three novel species within the clade of Conidiobolus s.s., i.e. C. bifurcatus sp. nov. , C. taihushanensis sp. nov. and C. variabilis sp. nov. These three species were isolated from plant debris in eastern China. Morphologically, C. bifurcatus sp. nov. is characterised by its secondary conidiophores often branched at the tip to form two short stipes each bearing a secondary conidium. C. taihushanensis sp. nov. is different from the others in its straight apical mycelia and the production of 2–5 conidia. C. variabilis sp. nov. is distinctive because of its various shapes of primary conidia. All these three new taxa are illustrated herein with an update key to the species of the genus Conidiobolus s.s.

Dnm1 was found to be involved in fungal development and virulence, mediated peroxisomal fission, and normal endocytosis. This finding provides new insights into the cellular processes and pathogenicity in entomopathogenic fungi.

In this study, we report a novel double-stranded RNA (dsRNA) virus, Beauveria bassiana partitivirus 3 (BbPV-3), derived from the entomogenous fungus Beauveria bassiana isolate RCEF5853 from China. The genome of BbPV-3, whose sequence was determined by metagenomic sequencing, RT-PCR, and RACE cloning, comprises two dsRNA genome segments that are 1,856 and 1,719 bp long. The first segment contains a single ORF (ORF-1) encoding a 584-amino-acid-long protein (66.05 kDa) with a conserved RNA-dependent RNA polymerase (RdRp) motif. The second segment also has a single ORF (ORF-2) encoding a 500-amino-acid-long coat protein (CP) (55.9 kDa). The CP and RdRp sequences showed highest identity of 43.4% and 60.2%, respectively, to those of Colletotrichum eremochloae partitivirus 1. Phylogenetic analysis of the RdRp domain of the polyprotein revealed that BbPV-3 grouped together with the members of the genus Epsilonpartitivirus. Hence, we proposed that Beauveria bassiana partitivirus 3 is a novel member of the proposed genus Epsilonpartitivirus.

Metarhizium robertsii, a widely distributed insect pathogen, is presently used as a natural alternative to chemical insecticides. Unfortunately, its worldwide commercial use has been restricted by a short shelf life and inconsistencies in virulence. In our previous study, a gene (GenBank accession number EFZ01626) was found to be significantly upregulated in heat-treated conidia. In the present study, this gene was characterized via gene disruption and complementation strategies. The gene (amplified by rapid amplification of cDNA ends PCR) was 1219 bp long and contained an open reading frame (ORF) of 777 bp. It encoded a protein of 234 amino acid residues with a 26-residue signal peptide. Bioinformatics analyses did not identify conserved functional domains; therefore, it was assumed to be a secreted virulence-associated protein according to its signal peptide and bioassay results. We found that the conidial germination rate of the ΔMrSVP mutant fungi dramatically decreased after heat shock treatment in a thermotolerance test. In addition, transcription levels of all tested heat shock–related genes were significantly lower in the mutant than in the wild type. We also demonstrated that the mean lethal time to death (LT50) of ΔMrSVP significantly increased relative to the wild type in insect bioassays (both topical inoculation and injection) involving Galleria mellonella. Moreover, similar rates of appressorium formation between ΔMrSVP and the wild type—and the significantly different expression of virulence-related genes such as acid trehalase and sucrose nonfermenting protein kinase in the haemocoel after injection—revealed that MrSVP is required for virulence in the insect haemocoel. Overall, our data suggest that the Mrsvp gene contributes to thermotolerance and virulence of M. robertsii. Furthermore, this gene is deeply involved in the mycosis of insect cadavers and in immune escape rather than insect cuticle penetration during infection.

Two new species of Conidiobolus were isolated from Anhui Province, China. A polyphasic taxonomic approach comprising morphological characteristics and molecular data (the nuclear large subunit of ribosomal DNA and the translation elongation factor 1 alpha gene sequences) was applied to determine their novel taxonomic status in the genus Conidiobolus . The new species C. mirabilis is a sister group to C . thromoides , but morphologically differs by smaller primary conidia and a unique formation of zygospores among two to four adjacent hyphal segments. The other new species C . pachyzygosporus characterized with thick-walled zygospores is phylogenetically closely related to C. antarcticus , C. couchii , and C. osmodes .