Neg1 overexpression in Trichoderma harzianum T4 enhanced its ability to produce spores and antagonistic activity against phytopathogenic fungi.

Trichoderma harzianum T4 is a soil fungus that plays an important role in the biological control of plant diseases. The aim of this study was to functionally characterize the ß-1,6-glucanase gene Neg1 in T. harzianum T4 and to investigate the effect of its overexpression on biocontrol traits, especially antagonism against pathogenic fungi. We found that overexpression of Neg1 did not affect growth of T. harzianum but enhanced sporulation of T. harzianum T4 cultures. Generally, spores are closely related to the defense ability of defense fungi and can assist their proliferation and improve their colonization ability. Secondly, overexpression of Neg1 also increased the secretion level of various hydrolytic enzymes and enhanced the antagonistic ability against phytopathogenic fungi of Fusarium spp. The results suggest that Neg1 is a key gene for improving the biocontrol effect of T. harzianum T4, which contributes to a better understanding of the mechanism of action of T. harzianum T4 as a fungal biocontrol agent.

Actinobacteria as Promising Biocontrol Agents for In Vitro and In Planta Degradation and Detoxification of Zearalenone.

Zearalenone (ZEN) is a prevalent mycotoxin found in grains and grain-derived products, inducing adverse health effects in both animals and humans. The in-field application of microorganisms to degrade and detoxify ZEN is a promising strategy to enhance the safety of food and feed. In this study, we investigated the potential of three actinobacterial strains to degrade and detoxify ZEN in vitro and in planta on wheat ears. The residual ZEN concentration and toxicity in the samples were analysed with UHPLC-MS/MS and a bioluminescence BLYES assay, respectively. Streptomyces rimosus subsp. rimosus LMG19352 could completely degrade and detoxify 5 mg/L ZEN in LB broth within 24 h, along with significant reductions in ZEN concentration both in a minimal medium (MM) and on wheat ears. Additionally, it was the only strain that showed a significant colonisation of these ears. Rhodococcus sp. R25614 exhibited partial but significant degradation in LB broth and MM, whereas Streptomyces sp. LMG16995 degraded and detoxified ZEN in LB broth after 72 h by 39% and 33%, respectively. Although all three actinobacterial strains demonstrated the metabolic capability to degrade and detoxify ZEN in vitro, only S. rimosus subsp. rimosus LMG19352 showed promising potential to mitigate ZEN in planta. This distinction underscores the importance of incorporating in planta screening assays for assessing the potential of mycotoxin-biotransforming microorganisms as biocontrol agents.

Assessment of Fungal Lytic Enzymatic Extracts Produced Under Submerged Fermentation as Enhancers of Entomopathogens' Biological Activity.

The application of enzymes in agricultural fields has been little explored. One potential application of fungal lytic enzymes (chitinases, lipases, and proteases) is as an additive to current biopesticides to increase their efficacy and reduce the time of mortality. For this, a screening of lytic overproducer fungi under submerged fermentation with a chemical-defined medium was performed. Then, the enzymatic crude extract (ECE) was concentrated and partially characterized. This characterization consisted of measuring the enzymatic activity (lipase, protease and, chitinase) and determining the enzyme stability after storage at temperatures of - 80, - 20 and, 4 °C. And lastly, the application of these concentrated enzymatic crude extracts (C-ECE) as an enhancer of spores-based fungal biopesticide was proven. Beauveria were not as good producers of lytic enzymes as the strains from Trichoderma and Metarhizium. The isolate M. robertsii Mt015 was selected for the co-production of chitinases and proteases; and the isolate T. harzianum Th180 for co-production of chitinases, lipases, and proteases. The C-ECE of Mt015 had a protease activity of 18.6 ± 1.1 U ml-1, chitinase activity of 0.28 ± 0.01 U ml-1, and no lipase activity. Meanwhile, the C-ECE of Th180 reached a chitinase activity of 0.75 U ml-1, lipase activity of 0.32 U ml-1, and protease activity of 0.24 U ml-1. Finally, an enhancing effect of the enzymatic extracts of M. robertsii (66.7%) and T. harzianum (43.5%) on the efficacy of B. bassiana Bv064 against Diatraea saccharalis larvae was observed. This work demonstrates the non-species-specific enhancing effect of enzymatic extracts on the insecticidal activity of conidial-based biopesticides, which constitutes a contribution to the improvement of biological control agents' performance.

Entomopathogenic fungi in crops protection with an emphasis on bioactive metabolites and biological activities.

Plant pathogens with their abundance are harmful and cause huge damage to different agricultural crops and economy of a country as well as lead towards the shortage of food for humans. For their management, the utilization of entomopathogenic fungi is an eco-friendly technique, sustainable to the environment, safe for humans and has promising effect over chemical-based pesticides. This process requires a biochemical mechanism, including the production of enzymes, toxins, and other metabolites that facilitate host infection and invasion. Essential enzymes such as chitinase, proteinase, and lipase play a direct role in breaking down the host cuticle, the primary barrier to EPF (Entomopathogenic Fungi) infection. Additionally, secondary metabolites such as destruxins in Metarhizium, beauvericin in Beauveria, hirsutellides in Hirsutella, isarolides in Isaria, cordyols in Cordyceps, and vertihemipterins in Verticillium, among others, act both directly and indirectly to disable the defense mechanisms of insect hosts, thereby accelerating the EPF infection process. The chemical composition of these secondary metabolites varies, ranging from simple non-peptide pigments such as oosporine to highly complex piperazine derivatives such as vertihemiptellides. The biocontrol efficacy of EPF is extensively studied, with numerous fungal strains commercially available on a large scale for managing arthropod pests. This review emphasizes the role of proteins and enzymes against crop pathogens, detailing their mode of action, and describing the metabolites from entomopathogenic fungi and their biological activities. In doing so, these findings contribute to establishing a symbiotic equilibrium between agricultural productivity and environmental conservation.

A systematic review on products derived from nematophagous fungi in the biological control of parasitic helminths of animals.

Nematophagous fungi have been widely evaluated in the biological control of parasitic helminths in animals, both through their direct use and the use of their derived products. Fungal bioproducts can include extracellular enzymes, silver nanoparticles (AgNPs), as well as secondary metabolites. The aim of this study was to conduct a systematic review covering the evaluation of products derived from nematophagous fungi in the biological control of parasitic helminths in animals. In total, 33 studies met the inclusion criteria and were included in this review. The majority of the studies were conducted in Brazil (72.7%, 24/33), and bioproducts derived from the fungus Duddingtonia flagrans were the most commonly evaluated (36.3%, 12/33). The studies involved the production of extracellular enzymes (48.4%, 16/33), followed by crude enzymatic extract (27.2%, 9/33), secondary metabolites (15.1%, 5/33) and biosynthesis of AgNPs (9.1%, 3/33). The most researched extracellular enzymes were serine proteases (37.5%, 6/16), with efficacies ranging from 23.9 to 85%; proteases (31.2%, 5/16), with efficacies from 41.4 to 95.4%; proteases + chitinases (18.7%, 3/16), with efficacies from 20.5 to 43.4%; and chitinases (12.5%, 2/16), with efficacies ranging from 12 to 100%. In conclusion, extracellular enzymes are the most investigated derivatives of nematophagous fungi, with proteases being promising strategies in the biological control of animal helminths. Further studies under in vivo and field conditions are needed to explore the applicability of these bioproducts as tools for biological control.

Actinomycetes are a natural resource for sustainable pest control and safeguarding agriculture.

Actinomycetes, a diverse group of bacteria with filamentous growth characteristics, have long captivated researchers and biochemists for their prolific production of secondary metabolites. Among the myriad roles played by actinomycete secondary metabolites, their historical significance in the field of biocontrol stands out prominently. The fascinating journey begins with the discovery of antibiotics, where renowned compounds like streptomycin, tetracycline, and erythromycin revolutionized medicine and agriculture. The history of biocontrol traces its roots back to the early twentieth century, when scientists recognized the potential of naturally occurring agents to combat pests and diseases. The emergence of synthetic pesticides in the mid-twentieth century temporarily overshadowed interest in biocontrol. However, with growing environmental concerns and the realization of the negative ecological impacts of chemical pesticides, the pendulum swung back towards exploring sustainable alternatives. Beyond their historical role as antibiotics, actinomycete-produced secondary metabolites encompass a rich repertoire with biopesticide potential. The classification of these compounds based on chemical structure and mode of action is highlighted, demonstrating their versatility against both plant pathogens and insect pests. Additionally, this review provides in-depth insights into how endophytic actinomycete strains play a pivotal role in biocontrol strategies. Case studies elucidate their effectiveness in inhibiting Spodoptera spp. and nematodes through the production of bioactive compounds. By unraveling the multifunctional roles of endophytic actinomycetes, this review contributes compelling narrative knowledge to the field of sustainable agriculture, emphasizing the potential of these microbial allies in crafting effective, environmentally friendly biocontrol strategies for combating agricultural pests.

Novel Environmentally Friendly RNAi Biopesticides: Targeting V-ATPase in Holotrichia parallela Larvae Using Layered Double Hydroxide Nanocomplexes.

RNA interference (RNAi)-based biopesticides offer an attractive avenue for pest control. Previous studies revealed high RNAi sensitivity in Holotrichia parallela larvae, showcasing its potential for grub control. In this study, we aimed to develop an environmentally friendly RNAi method for H. parallela larvae. The double-stranded RNA (dsRNA) of the V-ATPase-a gene (HpVAA) was loaded onto layered double hydroxide (LDH). The dsRNA/LDH nanocomplex exhibited increased environmental stability, and we investigated the absorption rate and permeability of dsRNA-nanoparticle complexes and explored the RNAi controlling effect. Silencing the HpVAA gene was found to darken the epidermis of H. parallela larvae, with growth cessation or death or mortality, disrupting the epidermis and midgut structure. Quantitative reverse transcription-polymerase chain reaction and confocal microscopy confirmed the effective absorption of the dsRNA/LDH nanocomplex by peanut plants, with distribution in roots, stems, and leaves. Nanomaterial-mediated RNAi silenced the target genes, leading to the death of pests. Therefore, these findings indicate the successful application of the nanomaterial-mediated RNAi system for underground pests, thus establishing a theoretical foundation for developing a green, safe, and efficient pest control strategy.

The novel bacteriocin BacYB1 produced by Leuconostoc mesenteroides YB1: From recent analytical characterization to biocontrol Verticillium dahliae and Agrobacterium tumefaciens.

Biocontrol of phytopathogens involving the use of bioactive compounds produced by lactic acid bacteria (LAB), is a promising approach to manage many diseases in agriculture. In this study, a lactic acid bacterium designated YB1 was isolated from fermented olives and selected for its antagonistic activity against Verticillium dahliae (V. dahliae) and Agrobacterium tumefaciens (A. tumefaciens). Based on the 16S rRNA gene nucleotide sequence analysis (1565 pb, accession number: OR714267), the new isolate YB1 bacterium was assigned as Leuconostoc mesenteroides YB1 (OR714267) strain. This bacterium produces an active peptide "bacteriocin" called BacYB1, which was purified in four steps. Matrix-assisted lasers desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) based approach was performed to identify and characterize BacYB1. The exact mass was 5470.75 Da, and the analysis of the N-terminal sequence (VTRASGASTPPGTASPFKTL) of BacYB1 revealed no significant similarity to currently available antimicrobial peptides. The BacYB1 displayed a bactericidal mode of action against A. tumefaciens. The potentiel role of BacYB1 to supress the growth of A. tumefaciens was confirmed by live-dead cells viability assay. In pot experiments, the biocontrol efficacy of BacYB1 against V. dahliae wilt on young olive trees was studied. The percentage of dead plants (PDP) and the final mean symptomes severity (FMS) of plants articifialy infected by V. dahliae and treated with the pre-purified peptide BacYB1 (preventive and curative treatments) were significantly inferior to untreated plants. Biochemical analysis of leaves of the plants has shown that polyophenols contents were highly detected in plants infected by V. dahliae and the highest contents of chlorophyl a, b and total chlorophyll were recorded in plants treated with the combination of BacYB1 with the biofertilisant Humivital. BacYB1 presents a promising alternative for the control of Verticillium wilt and crown gall diseases.

Exploring chitin: novel pathways and structures as promising targets for biopesticides.

Chitin, the most prevalent polymer in nature, a significant structural polysaccharide that comes in second only to cellulose. Chitin is a crucial component of fungal cell walls and also present in many other creatures, such as viruses, plants, animals, insect exoskeletons, and crustacean shells. Chitin presents itself as a promising target for the development of biopesticides. It focuses on unraveling the unique structures and biochemical pathways associated with chitin, aiming to identify vulnerabilities that can be strategically leveraged for effective and environmentally sustainable pest control. It involves a comprehensive analysis of chitinase enzymes, chitin biosynthesis, and chitin-related processes across diverse organisms. By elucidating the molecular intricacies involved in chitin metabolism, this review seeks to unveil potential points of intervention that can disrupt essential biological processes in target pests without harming non-target species. This holistic approach to understanding chitin-related pathways aims to inform the design and optimization of biopesticides with enhanced specificity and reduced ecological impact. The outcomes of this study hold great promise for advancing innovative and eco-friendly pest management strategies. By targeting chitin structures and pathways, biopesticides developed based on these findings may offer a sustainable and selective alternative to conventional chemical pesticides, contributing to the ongoing efforts towards more environmentally conscious and effective pest control solutions.

Strong antagonism of an endophyte of Fraxinus excelsior towards the ash dieback pathogen, Hymenoscyphus fraxineus, is mediated by the antifungal secondary metabolite PF1140.

Ash dieback, caused by the fungal pathogen Hymenoscyphus fraxineus (Helotiales, Ascomycota), is threatening the existence of the European ash, Fraxineus excelsior. During our search for biological control agents for this devastating disease, endophytic fungi were isolated from healthy plant tissues and co-cultivated with H. fraxineus to assess their antagonistic potential. Among the strains screened, Penicillium cf. manginii DSM 104493 most strongly inhibited the pathogen. Initially, DSM 104493 showed promise in planta as a biocontrol agent. Inoculation of DSM 104493 into axenically cultured ash seedlings greatly decreased the development of disease symptoms in seedlings infected with H. fraxineus. The fungus was thus cultivated on a larger scale in order to obtain sufficient material to identify active metabolites that accounted for the antibiosis observed in dual culture. We isolated PF1140 (1) and identified it as the main active compound in the course of a bioassay-guided isolation strategy. Furthermore, its derivative 2, the mycotoxin citreoviridin (3), three tetramic acids of the vancouverone type (4-6), and penidiamide (7) were isolated by preparative chromatography. The structures were elucidated mainly by NMR spectroscopy and high-resolution mass spectrometry (HRMS), of which compounds 2 and 6 represent novel natural products. Of the compounds tested, not only PF1140 (1) strongly inhibited H. fraxineus in an agar diffusion assay but also showed phytotoxic effects in a leaf puncture assay. Unfortunately, both the latent virulent attributes of DSM 104493 observed subsequent to these experiments in planta and the production of mycotoxins exclude strain Penicillium cf. manginii DSM 104493 from further development as a safe biocontrol agent.IMPORTANCEEnvironmentally friendly measures are urgently needed to control the causative agent of ash dieback, Hymenoscyphus fraxineus. Herein, we show that the endophyte DSM 104493 exhibits protective effects in vitro and in planta. We traced the activity of DSM 104493 to the antifungal natural product PF1140, which unfortunately also showed phytotoxic effects. Our results have important implications for understanding plant-fungal interactions mediated by secondary metabolites, not only in the context of ash dieback but also generally in plant-microbial interactions.

Unraveling the metabolic potential of biocontrol fungi through omics data: a key to enhancing large-scaleapplication strategies.

Biological control of pests and pathogens has attracted much attention due to its green, safe and effective characteristics. However, it faces the dilemma of insignificant effects in large-scale applications. Therefore, an in-depth exploration of the metabolic potential of biocontrol fungi based on big omics data is crucial for a comprehensive and systematic understanding of the specific modes of action operated by various biocontrol fungi. This article analyzes the preferences for extracellular carbon and nitrogen source degradation, secondary metabolites (nonribosomal peptides, polyketide synthases) and their product characteristics and the conversion relationship between extracellular primary metabolism and intracellular secondary metabolism for eight different filamentous fungi with characteristics appropriate for the biological control of bacterial pathogens and phytopathogenic nematodes. Further clarification is provided that Paecilomyces lilacinus, encoding a large number of hydrolase enzymes capable of degrading pathogen protection barrier, can be directly applied in the field as a predatory biocontrol fungus, whereas Trichoderma, as an antibiosis-active biocontrol control fungus, can form dominant strains on preferred substrates and produce a large number of secondary metabolites to achieve antibacterial effects. By clarifying the levels of biological control achievable by different biocontrol fungi, we provide a theoretical foundation for their application to cropping habitats.

Use of Chemicals and Biological Control Agents as Activators of Plant Defense Against Endoparasitic Sedentary Nematodes.

Full compatible interactions between crop plants and endoparasitic sedentary nematodes (ESNs) lead to severe infestation of the roots and plant growth impairing, as well as to the increase of nematode population in the soil that is a threat for the next planting crop. In the absence of activators, basic plant defense is overcome by nematode secretion of effectors that suppress defense gene expression, inhibit ROS generation and the oxidative burst used by plants to hamper nematode feeding site settlement and limit its development and reproduction. Activators can be exogenously added as a preventive measure to prime plants and strengthen their defense against ESNs. Activators can be an array of antioxidant compounds or biocontrol agents, such as mutualist microorganisms living in the rhizosphere (biocontrol fungi (BCF), arbuscular mycorrhizal fungi (AMF), plant growth-promoting bacteria (PGPB), etc.). In this chapter, methods are described for usage of both salicylic acid (SA) and its methylated form (Met-SA), and BCF/AMF as elicitors of resistance of vegetable crops against root-knot nematodes (RKNs). The rhizosphere-living BCF/AMF were recovered from commercial formulates pre-incubated in suitable growth media and provided exclusively as soil drench of potted plants. The plant hormones SA and Met-SA were provided to plants as soil drench, foliar spray, and root dip. It is indicated that activators' dosages and plant age are crucial factors in determining the success of a pre-treatment to reduce nematode infection. Therefore, dosages should be expressed as amounts of activators per g of plant weight at treatment. Thresholds exist above which dosages start to work; overdoses were found to be toxic to plants and useless as activators.

Whole genome analysis of Bacillus velezensis 160, biological control agent of corn head smut.

Corn head smut is a disease caused by the fungus Sporisorium reilianum. This phytosanitary problem has existed for several decades in the Mezquital Valley, an important corn-producing area in central Mexico. To combat the problem, a strain identified as Bacillus subtilis 160 was applied in the field, where it decreased disease incidence and increased crop productivity. In this study, the sequencing and analysis of the whole genome sequence of this strain were carried out to identify its genetic determinants for the production of antimicrobials. The B. subtilis 160 strain was found to be Bacillus velezensis. Its genome has a size of 4,297,348 bp, a GC content of 45.8%, and 4,174 coding sequences. Comparative analysis with the genomes of four other B. velezensis strains showed that they share 2,804 genes and clusters for the production of difficidin, bacillibactin, bacilysin, macrolantin, bacillaene, fengycin, butirosin A, locillomycin, and surfactin. For the latter metabolite, unlike the other strains that have only one cluster, B. velezensis 160 has three. A cluster for synthesizing laterocidine, an antimicrobial reported only in Brevibacillus laterosporus, was also identified. IMPORTANCE: In this study, we performed sequencing and analysis of the complete genome of the strain initially identified as Bacillus subtilis 160 as part of its characterization. This bacterium has shown its ability to control corn head smut in the field, a disease caused by the basidiomycete fungus Sporisorium reilianum. Analyzing the complete genome sequence not only provides a more precise taxonomic identification but also sheds light on the genetic potential of this bacterium, especially regarding mechanisms that allow it to exert biological control. Employing molecular and bioinformatics tools in studying the genomes of agriculturally significant microorganisms offers insights into the development of biofungicides and bioinoculants. These innovations aim to enhance plant growth and pave the way for strategies that boost crop productivity.

Streptomyces Strains and Their Metabolites for Biocontrol of Phytopathogens in Agriculture.

Sustainable agriculture is increasingly linked to biological pesticides as alternatives to agro-chemicals. Streptomyces species suppress plant diseases through their unique traits and numerous metabolites. Although many Streptomyces strains have been developed into commercial products, their roles in the biocontrol of phytopathogens and mechanisms of functional metabolite synthesis remain poorly understood. In this review, biocontrol of plant diseases by Streptomyces is summarized on the basis of classification of fungal and bacterial diseases and secondary metabolites produced by Streptomyces that act on phytopathogenic microorganisms are discussed. The associated non-ribosomal peptide synthetases and polyketide synthetases responsible for biosynthesis of these secondary metabolites are also investigated, and advances in fermentation of Streptomyces are described. Finally, the need to develop precise and effective biocontrol methods for plant diseases is highlighted.

Screening and characterization of Bacillus thuringiensis isolates for high production of Vip3A and Cry proteins and high thermostability to control Spodoptera spp.

Bacillus thuringiensis (Bt) is an entomopathogenic bacterium that produces crystalline (Cry and Cyt) and soluble (vegetative insecticidal proteins or Vips) proteins during the sporulation and vegetative growth phases, respectively. Combining Cry and Vip proteins could delay insect resistance development and exhibit synergistic activity against various insect pests. This study aims to screen Bt isolates collected from Thailand for high Vip3A and Cry protein production levels and high thermostability to control Spodoptera spp. Among the selected Bt isolates with high target protein synthesis, Bt isolate 506 was found to be safe for further biopesticide formulation due to the absence of non-specific metabolite, as determined by the detection of thermo-stable ß-exotoxin I based on biological assays and PCR analysis. Bt isolate 506 showed the presence of Cry1A, Cry2A, and Vip3A-type proteins identified as Cry1Aa45, Cry2Aa22, and Vip3A87, respectively. The insecticidal activity of whole culture extracts containing Vip3A and Cry mixtures and culture supernatants containing secreted Vip3A protein was evaluated against the second-instar larvae of S. exigua and S. frugiperda. The Bt isolate 506 showed high toxicity against both insects, and the insecticidal proteins produced by this isolate retained their activity after heating at 50 °C. This Bt isolate is a promising candidate for further development as a biopesticide against lepidopteran pests.

Eco-friendly biopesticides derived from CO2-Fixing cyanobacteria.

There is currently an escalating global demand for the utilization of plant and natural extracts as pesticides due to their minimal health risks. Cyanobacteria are highly valuable organisms with significant potential in agriculture and are of great interest for the development of agrochemical agents as biopesticides. The flexibility and adaptability of Cyanobacteria to various environmental conditions are facilitated by the presence of specialized enzymes involved in the production of biologically active diverse secondary metabolites, including alkaloids, lipopolysaccharides, non-protein amino acids, non-ribosomal peptides, polyketides, terpenoids, and others. This review focuses on the metabolites synthesized from cyanobacteria that have demonstrated effectiveness as antibacterial, antiviral, antifungal agents, insecticides, herbicides, and more. The potential role of cyanobacteria as an alternative to chemical pesticides for environmental conservation is discussed.

Discovery of a Natural Hybrid Polyketide Produced by Endophytic Cladosporium sphaerospermum for Biocontrol of Phytopathogenic Fungus Botrytis cinerea.

The endophytic fungus Cladosporium sphaerospermum WBS017 exhibits broad-spectrum activity against plant pathogens, with particular effectiveness against Botrytis cinerea. Subsequently, a compound is isolated from strain WBS017 as the main active ingredient against B. cinerea using activity-guided separation and identified as hybrid polyketide (namely cladodionen, CLD) using UV, MS, NMR, etc. In vitro and in vivo antifungal activity tests demonstrate that CLD effectively inhibits the mycelial growth and spore germination, with an IC50 value of 1.13 and 0.095 mM, respectively, and exerts antifungal and fresh-keeping effects on both strawberry and tomato. Microscopy analysis reveals that the inhibitory effects of CLD on hyphae and spore germination are attributed to a decrease in structural stability of mycelia cells as well as the accumulation of reactive oxygen species (ROS). Furthermore, transcriptome analysis further indicates that spore germination is inhibited by suppressing the transcription levels of membrane or membrane-related genes, disturbing the balance of ROS metabolism, altering the primary metabolic pathways, genetic information processing, and cellular processes. Importantly, CLD demonstrates no significant toxicity on zebrafish embryos even at a concentration of 0.226 mM, indicating its potential as a safe biological-control agent. In summary, CLD would be a novel potential biological-control agent and can be considered as a promising fungicide to control B. cinerea.

Enhancement of herbicolin A production by integrated fermentation optimization and strain engineering in Pantoea agglomerans ZJU23.

BACKGROUND: The lipopeptide herbicolin A (HA) secreted by the biocontrol agent Pantoea agglomerans ZJU23 is a promising antifungal drug to combat fungal pathogens by targeting lipid rafts, both in agricultural and clinical settings. Improvement of HA production would be of great significance in promoting its commercialization. This study aims to enhance the HA production in ZJU23 by combining fermentation optimization and strain engineering. RESULTS: Based on the results in the single-factor experiments, corn steep liquor, temperature and initial pH were identified as the significant affecting factors by the Plackett-Burman design. The fermentation medium and conditions were further optimized using the Box-Behnken response surface method, and the HA production of the wild type strain ZJU23 was improved from ~ 87 mg/mL in King's B medium to ~ 211 mg/mL in HA induction (HAI) medium. A transposon library was constructed in ZJU23 to screen for mutants with higher HA production, and two transcriptional repressors for HA biosynthesis, LrhA and PurR, were identified. Disruption of the LrhA gene led to increased mRNA expression of HA biosynthetic genes, and subsequently improved about twofold HA production. Finally, the HA production reached ~ 471 mg/mL in the ΔLrhA mutant under optimized fermentation conditions, which is about 5.4 times higher than before (~ 87 mg/mL). The bacterial suspension of the ΔLrhA mutant fermented in HAI medium significantly enhanced its biocontrol efficacy against gray mold disease and Fusarium crown rot of wheat, showing equivalent control efficacies as the chemical fungicides used in this study. Furthermore, HA was effective against fungicide resistant Botrytis cinerea. Increased HA production substantially improved the control efficacy against gray mold disease caused by a pyrimethanil resistant strain. CONCLUSIONS: This study reveals that the transcriptional repressor LrhA negatively regulates HA biosynthesis and the defined HAI medium is suitable for HA production. These findings provide an extended basis for large-scale production of HA and promote biofungicide development based on ZJU23 and HA in the future.

A novel cyanolytic bacterium, Pseudomonas fluorescens BG-E as a potential biological control agent for freshwater bloom-forming cyanobacteria Pseudanabaena spp.

The majority of bacterial antagonists identified to date are active against Microcystis. Therefore, this study aimed to isolate and characterize novel cyanolytic bacterial strains antagonistic against bloom-forming filamentous cyanobacteria. The bacterial strain BG-E isolated from the Bandagiriya Wewa in Sri Lanka was identified as Pseudomonas fluorescens (MZ007859) based on the 16S rRNA gene sequencing. BG-E showed 82% and 73% cyanolytic activity (CA) against Pseudanabaena sp. LW2 (MW288948) and Pseudanabaena lonchoides LW1 (MW288940), respectively, after 10 days of inoculation. The light microscopic images affirmed the complete disintegration in the filamentous structures of the tested Pseudanabaena species. The bacterial cell density of 15% v/v showed the CA with 95% and 89% cell lysis, respectively, in P. lonchoides and Pseudanabaena sp. LW2. Moreover, the results showed that >50% CA could be achieved by 0.100 and 1.00 (OD730 ) cell densities for these same species. The highest CA of the cell-free supernatant of BG-E against P. lonchoides and bacterial culture against Pseudanabaena sp. LW2 illustrated the species-specific mode of action of BG-E. Although BG-E efficiently lysed the tested cyanobacterial species, the results of the MC-biodegradation assay confirmed its inability to degrade MC-LR cyanotoxin. Further, the BG-E strain lacks the mlrABCD gene cluster which is known to be responsible for the enzymatic degradation of MCs. The overall findings highlighted the applicability of P. fluorescens BG-E as a biological controlling agent to terminate blooms of freshwater filamentous cyanobacteria genus Pseudanabaena. The incorporation of cyanotoxin-degrading heterotrophic bacteria is recommended as a means of controlling toxic Pseudanabaena blooms.

Are biopesticides safe for the environment? Effects of pyrethrum extract on the non-target species Daphnia magna.

Biopesticides are natural compounds considered more safe and sustainable for the environment. However, it is also important to evaluate the potential risk in non-target organisms. Pyrethrum extract (PE) is a biopesticide, widely used for agriculture, veterinary, and aquaculture. This work aimed to evaluate acute (0.6 - 40.0 µg/L; 96 h; E(L)C50 toxicity) and sub-chronic (0.7 - 1.1 µg/L; 10 d; life-history parameters) effects of PE on Daphnia magna. Moreover, a biomarkers approach using antioxidant and biotransformation capacity, lipid peroxidation (LPO), neurotoxicity, and energy reserves content were evaluated. Acute effects (mortality, changes in swimming behavior, oxidative stress, lipid peroxidation, neurotoxicity) were recorded with the increase in PE concentration. Sub-chronic assay showed an increase in energy reserves content, antioxidant parameters, and LPO demonstrating that PE unbalances oxidative metabolism. This study can conclude that PE potentiates toxic effects in D. magna and demonstrates the vulnerability of a non-target organism to PE that is considered environmentally safe.