Manipulating the Destiny of Wild Populations Using CRISPR.

Genetic biocontrol aims to suppress or modify populations of species to protect public health, agriculture, and biodiversity. Advancements in genome engineering technologies have fueled a surge in research in this field, with one gene editing technology, CRISPR, leading the charge. This review focuses on the current state of CRISPR technologies for genetic biocontrol of pests and highlights the progress and ongoing challenges of using these approaches.

Evolutionary Ecology of Wolbachia Releases for Disease Control.

Wolbachia is an endosymbiotic Alphaproteobacteria that can suppress insect-borne diseases through decreasing host virus transmission (population replacement) or through decreasing host population density (population suppression). We contrast natural Wolbachia infections in insect populations with Wolbachia transinfections in mosquitoes to gain insights into factors potentially affecting the long-term success of Wolbachia releases. Natural Wolbachia infections can spread rapidly, whereas the slow spread of transinfections is governed by deleterious effects on host fitness and demographic factors. Cytoplasmic incompatibility (CI) generated by Wolbachia is central to both population replacement and suppression programs, but CI in nature can be variable and evolve, as can Wolbachia fitness effects and virus blocking. Wolbachia spread is also influenced by environmental factors that decrease Wolbachia titer and reduce maternal Wolbachia transmission frequency. More information is needed on the interactions between Wolbachia and host nuclear/mitochondrial genomes, the interaction between invasion success and local ecological factors, and the long-term stability of Wolbachia-mediated virus blocking.

Genomic and metabolomic diversity within a familial population of Aspergillus flavus.

Aspergillus flavus is an agriculturally significant micro-fungus having potential to contaminate food and feed crops with toxic secondary metabolites such as aflatoxin (AF) and cyclopiazonic acid (CPA). Research has shown A. flavus strains can overcome heterokaryon incompatibility and undergo meiotic recombination as teleomorphs. Although evidence of recombination in the AF gene cluster has been reported, the impacts of recombination on genotype and metabolomic phenotype in a single generation are lacking. In previous studies, we paired an aflatoxigenic MAT1-1 A. flavus strain with a non-aflatoxigenic MAT1-2 A. flavus strain that had been tagged with green fluorescent protein and then 10 F1 progenies (a mix of fluorescent and non-fluorescent) were randomly selected from single-ascospore colonies and broadly examined for evidence of recombination. In this study, we determined four of those 10 F1 progenies were recombinants because they were not vegetatively compatible with either parent or their siblings, and they exhibited other distinctive traits that could only result from meiotic recombination. The other six progenies examined shared genomic identity with the non-aflatoxigenic, fluorescent, and MAT1-2 parent, but were metabolically distinct. This study highlights phenotypic and genomic changes that may occur in a single generation from the outcrossing of sexually compatible strains of A. flavus.

Bacterial Volatile Compounds: Functions in Communication, Cooperation, and Competition.

Bacteria produce a multitude of volatile compounds. While the biological functions of these deceptively simple molecules are unknown in many cases, for compounds that have been characterized, it is clear that they serve impressively diverse purposes. Here, we highlight recent studies that are uncovering the volatile repertoire of bacteria, and the functional relevance and impact of these molecules. We present work showing the ability of volatile compounds to modulate nutrient availability in the environment; alter the growth, development, and motility of bacteria and fungi; influence protist and arthropod behavior; and impact plant and animal health. We further discuss the benefits associated with using volatile compounds for communication and competition, alongside the challenges of studying these molecules and their functional roles. Finally, we address the opportunities these compounds present from commercial, clinical, and agricultural perspectives.

Mycoviral gene integration converts a plant pathogenic fungus into a biocontrol agent.

Mycovirus-infected fungi can suffer from poor growth, attenuated pigmentation, and virulence. However, the molecular mechanisms of how mycoviruses confer these symptoms remain poorly understood. Here, we report a mycovirus Stemphylium lycopersici alternavirus 1 (SlAV1) isolated from a necrotrophic plant pathogen Stemphylium lycopersici that causes altered colony pigmentation and hypovirulence by specifically interfering host biosynthesis of Altersolanol A, a polyketide phytotoxin. SlAV1 significantly down-regulates a fungal polyketide synthase (PKS1), the core enzyme of Altersolanol A biosynthesis. PKS1 deletion mutants do not accumulate Altersolanol A and lose pathogenicity to tomato and lettuce. Transgenic expression of SlAV1 open-reading frame 3 (ORF3) in S. lycopersici inhibits fungal PKS1 expression and Altersolanol A accumulation, leading to symptoms like SlAV1-infected fungal strains. Multiple plant species sprayed with mycelial suspension of S. lycopersici or S. vesicarium strains integrating and expressing ORF3 display enhanced resistance against virulent strains, converting the pathogenic fungi into biocontrol agents. Hence, our study not only proves inhibiting a key enzyme of host phytotoxin biosynthesis as a molecular mechanism underlying SlAV1-mediated hypovirulence of Stemphylium spp., but also demonstrates the potential of mycovirus-gene integrated fungi as a potential biocontrol agent to protect plants from fungal diseases.

Leveraging a natural murine meiotic drive to suppress invasive populations.

Invasive rodents are a major cause of environmental damage and biodiversity loss, particularly on islands. Unlike insects, genetic biocontrol strategies including population-suppressing gene drives with biased inheritance have not been developed in mice. Here, we demonstrate a gene drive strategy (tCRISPR) that leverages super-Mendelian transmission of the t haplotype to spread inactivating mutations in a haplosufficient female fertility gene (Prl). Using spatially explicit individual-based in silico modeling, we show that tCRISPR can eradicate island populations under a range of realistic field-based parameter values. We also engineer transgenic tCRISPR mice that, crucially, exhibit biased transmission of the modified t haplotype and Prl mutations at levels our modeling predicts would be sufficient for eradication. This is an example of a feasible gene drive system for invasive alien rodent population control.

Insights into the biocontrol and plant growth promotion functions of Bacillus altitudinis strain KRS010 against Verticillium dahliae.

BACKGROUND: Verticillium wilt, caused by the fungus Verticillium dahliae, is a soil-borne vascular fungal disease, which has caused great losses to cotton yield and quality worldwide. The strain KRS010 was isolated from the seed of Verticillium wilt-resistant Gossypium hirsutum cultivar "Zhongzhimian No. 2." RESULTS: The strain KRS010 has a broad-spectrum antifungal activity to various pathogenic fungi as Verticillium dahliae, Botrytis cinerea, Fusarium spp., Colletotrichum spp., and Magnaporthe oryzae, of which the inhibition rate of V. dahliae mycelial growth was 73.97% and 84.39% respectively through confrontation test and volatile organic compounds (VOCs) treatments. The strain was identified as Bacillus altitudinis by phylogenetic analysis based on complete genome sequences, and the strain physio-biochemical characteristics were detected, including growth-promoting ability and active enzymes. Moreover, the control efficiency of KRS010 against Verticillium wilt of cotton was 93.59%. After treatment with KRS010 culture, the biomass of V. dahliae was reduced. The biomass of V. dahliae in the control group (Vd991 alone) was 30.76-folds higher than that in the treatment group (KRS010+Vd991). From a molecular biological aspect, KRS010 could trigger plant immunity by inducing systemic resistance (ISR) activated by salicylic acid (SA) and jasmonic acid (JA) signaling pathways. Its extracellular metabolites and VOCs inhibited the melanin biosynthesis of V. dahliae. In addition, KRS010 had been characterized as the ability to promote plant growth. CONCLUSIONS: This study indicated that B. altitudinis KRS010 is a beneficial microbe with a potential for controlling Verticillium wilt of cotton, as well as promoting plant growth.

In silico biotechnological potential of Bacillus sp. strain MHSD_37 bacterial endophyte.

BACKGROUND: Endophytic bacteria possess a range of unique characteristics that enable them to successfully interact with their host and survive in adverse environments. This study employed in silico analysis to identify genes, from Bacillus sp. strain MHSD_37, with potential biotechnological applications. RESULTS: The strain presented several endophytic lifestyle genes which encode for motility, quorum sensing, stress response, desiccation tolerance and root colonisation. The presence of plant growth promoting genes such as those involved in nitrogen fixation, nitrate assimilation, siderophores synthesis, seed germination and promotion of root nodule symbionts, was detected. Strain MHSD_37 also possessed genes involved in insect virulence and evasion of defence system. The genome analysis also identified the presence of genes involved in heavy metal tolerance, xenobiotic resistance, and the synthesis of siderophores involved in heavy metal tolerance. Furthermore, LC-MS analysis of the excretome identified secondary metabolites with biological activities such as anti-cancer, antimicrobial and applications as surfactants. CONCLUSIONS: Strain MHSD_37 thereby demonstrated potential biotechnological application in bioremediation, biofertilisation and biocontrol. Moreover, the strain presented genes encoding products with potential novel application in bio-nanotechnology and pharmaceuticals.

Unraveling the genomic secrets of Tritonibacter mobilis AK171: a plant growth-promoting bacterium isolated from Avicennia marina.

The scarcity of freshwater resources resulting in a significant yield loss presents a pressing challenge in agriculture. To address this issue, utilizing abundantly available saline water could offer a smart solution. In this study, we demonstrate that the genome sequence rhizosphere bacterium Tritonibacter mobilis AK171, a halophilic marine bacterium recognized for its ability to thrive in saline and waterlogged environments, isolated from mangroves, has the remarkable ability to enable plant growth using saline irrigation. AK171 is characterized as rod-shaped cells, displays agile movement in free-living conditions, and adopts a rosette arrangement in static media. Moreover, The qualitative evaluation of PGP traits showed that AK171 could produce siderophores and IAA but could not solubilize phosphate nor produce hydrolytic enzymes it exhibits a remarkable tolerance to high temperatures and salinity. In this study, we conducted a comprehensive genome sequence analysis of T. mobilis AK171 to unravel the genetic mechanisms underlying its plant growth-promoting abilities in such challenging conditions. Our analysis revealed diverse genes and pathways involved in the bacterium's adaptation to salinity and waterlogging stress. Notably, T. mobilis AK171 exhibited a high level of tolerance to salinity and waterlogging through the activation of stress-responsive genes and the production of specific enzymes and metabolites. Additionally, we identified genes associated with biofilm formation, indicating its potential role in establishing symbiotic relationships with host plants. Furthermore, our analysis unveiled the presence of genes responsible for synthesizing antimicrobial compounds, including tropodithietic acid (TDA), which can effectively control phytopathogens. This genomic insight into T. mobilis AK171 provides valuable information for understanding the molecular basis of plant-microbial interactions in saline and waterlogged environments. It offers potential applications for sustainable agriculture in challenging conditions.

Immune system modulation & virus transmission during parasitism identified by multi-species transcriptomics of a declining insect biocontrol system.

BACKGROUND: The Argentine stem weevil (ASW, Listronotus bonariensis) is a significant pasture pest in Aotearoa New Zealand, primarily controlled by the parasitoid biocontrol agent Microctonus hyperodae. Despite providing effective control of ASW soon after release, M. hyperodae parasitism rates have since declined significantly, with ASW hypothesised to have evolved resistance to its biocontrol agent. While the parasitism arsenal of M. hyperodae has previously been investigated, revealing many venom components and an exogenous novel DNA virus Microctonus hyperodae filamentous virus (MhFV), the effects of said arsenal on gene expression in ASW during parasitism have not been examined. In this study, we performed a multi-species transcriptomic analysis to investigate the biology of ASW parasitism by M. hyperodae, as well as the decline in efficacy of this biocontrol system. RESULTS: The transcriptomic response of ASW to parasitism by M. hyperodae involves modulation of the weevil's innate immune system, flight muscle components, and lipid and glucose metabolism. The multispecies approach also revealed continued expression of venom components in parasitised ASW, as well as the transmission of MhFV to weevils during parasitism and some interrupted parasitism attempts. Transcriptomics did not detect a clear indication of parasitoid avoidance or other mechanisms to explain biocontrol decline. CONCLUSIONS: This study has expanded our understanding of interactions between M. hyperodae and ASW in a biocontrol system of critical importance to Aotearoa-New Zealand's agricultural economy. Transmission of MhFV to ASW during successful and interrupted parasitism attempts may link to a premature mortality phenomenon in ASW, hypothesised to be a result of a toxin-antitoxin system. Further research into MhFV and its potential role in ASW premature mortality is required to explore whether manipulation of this viral infection has the potential to increase biocontrol efficacy in future.

Molecular Basis of Yeasts Antimicrobial Activity-Developing Innovative Strategies for Biomedicine and Biocontrol.

In the context of the growing concern regarding the appearance and spread of emerging pathogens with high resistance to chemically synthetized biocides, the development of new agents for crops and human protection has become an emergency. In this context, the yeasts present a huge potential as eco-friendly agents due to their widespread nature in various habitats and to their wide range of antagonistic mechanisms. The present review focuses on some of the major yeast antimicrobial mechanisms, their molecular basis and practical applications in biocontrol and biomedicine. The synthesis of killer toxins, encoded by dsRNA virus-like particles, dsDNA plasmids or chromosomal genes, is encountered in a wide range of yeast species from nature and industry and can affect the development of phytopathogenic fungi and other yeast strains, as well as human pathogenic bacteria. The group of the "red yeasts" is gaining more interest over the last years, not only as natural producers of carotenoids and rhodotorulic acid with active role in cell protection against the oxidative stress, but also due to their ability to inhibit the growth of pathogenic yeasts, fungi and bacteria using these compounds and the mechanism of competition for nutritive substrate. Finally, the biosurfactants produced by yeasts characterized by high stability, specificity and biodegrability have proven abilities to inhibit phytopathogenic fungi growth and mycelia formation and to act as efficient antibacterial and antibiofilm formation agents for biomedicine. In conclusion, the antimicrobial activity of yeasts represents a direction of research with numerous possibilities of bioeconomic valorization as innovative strategies to combat pathogenic microorganisms.

Comprehensive genomic analysis of Burkholderia arboris PN-1 reveals its biocontrol potential against Fusarium solani-induced root rot in Panax notoginseng.

Panax notoginseng (Burkill) F.H. Chen, a valuable traditional Chinese medicine, faces significant yield and quality challenges stemming from root rot primarily caused by Fusarium solani. Burkholderia arboris PN-1, isolated from the rhizosphere soil of P. notoginseng, demonstrated a remarkable ability to inhibit the growth of F. solani. This study integrates phenotypic, phylogenetic, and genomic analyses to enhance our understanding of the biocontrol mechanisms employed by B. arboris PN-1. Phenotype analysis reveals that B. arboris PN-1 effectively suppresses P. notoginseng root rot both in vitro and in vivo. The genome of B. arboris PN-1 comprises three circular chromosomes (contig 1: 3,651,544 bp, contig 2: 1,355,460 bp, and contig 3: 3,471,056 bp), with a 66.81% GC content, housing 7,550 protein-coding genes. Notably, no plasmids were detected. Phylogenetic analysis places PN-1 in close relation to B. arboris AU14372, B. arboris LMG24066, and B. arboris MEC_B345. Average nucleotide identity (ANI) values confirm the PN-1 classification as B. arboris. Comparative analysis with seven other B. arboris strains identified 4,628 core genes in B. arboris PN-1. The pan-genome of B. arboris appears open but may approach closure. Whole-genome sequencing revealed 265 carbohydrate-active enzymes and identified 9 gene clusters encoding secondary metabolites. This comprehensive investigation enhances our understanding of B. arboris genomes, paving the way for their potential as effective biocontrol agents against fungal plant pathogens in the future.

Trichoderma cf. asperellum and plant-based titanium dioxide nanoparticles initiate morphological and biochemical modifications in Hordeum vulgare L. against Bipolaris sorokiniana.

BACKGROUND: Spot blotch is a serious foliar disease of barley (Hordeum vulgare L.) plants caused by Bipolaris sorokiniana, which is a hemibiotrophic ascomycete that has a global impact on productivity. Some Trichoderma spp. is a promising candidate as a biocontrol agent as well as a plant growth stimulant. Also, the application of nanomaterials in agriculture limits the use of harmful agrochemicals and helps improve the yield of different crops. The current study was carried out to evaluate the effectiveness of Trichoderma. cf. asperellum and the biosynthesized titanium dioxide nanoparticles (TiO2 NPs) to manage the spot blotch disease of barley caused by B. sorokiniana and to assess the plant's innate defense response. RESULTS: Aloe vera L. aqueous leaf extract was used to biosynthesize TiO2 NPs by reducing TiCl4 salt into TiO2 NPs and the biosynthesized NPs were detected using SEM and TEM. It was confirmed that the NPs are anatase-crystalline phases and exist in sizes ranging from 10 to 25 nm. The T. cf. asperellum fungus was detected using morphological traits and rDNA ITS analysis. This fungus showed strong antagonistic activity against B. sorokiniana (57.07%). Additionally, T. cf. asperellum cultures that were 5 days old demonstrated the best antagonistic activity against the pathogen in cell-free culture filtrate. Also, B. sorokiniana was unable to grow on PDA supplemented with 25 and 50 mg/L of TiO2 NPs, and the diameter of the inhibitory zone increased with increasing TiO2 NPs concentration. In an in vivo assay, barley plants treated with T. cf. asperellum or TiO2 NPs were used to evaluate their biocontrol efficiency against B. sorokiniana, in which T. cf. asperellum and TiO2 NPs enhanced the growth of the plant without displaying disease symptoms. Furthermore, the physiological and biochemical parameters of barley plants treated with T. cf. asperellum or TiO2 NPs in response to B. sorokiniana treatment were quantitively estimated. Hence, T. cf. asperellum and TiO2 NPs improve the plant's tolerance and reduce the growth inhibitory effect of B. sorokiniana. CONCLUSION: Subsequently, T. cf. asperellum and TiO2 NPs were able to protect barley plants against B. sorokiniana via enhancement of chlorophyll content, improvement of plant health, and induction of the barley innate defense system. The present work emphasizes the major contribution of T. cf. asperellum and the biosynthesized TiO2 NPs to the management of spot blotch disease in barley plants, and ultimately to the enhancement of barley plant quality and productivity.

Creating large chromosomal segment deletions in Aspergillus flavus by a dual CRISPR/Cas9 system: Deletion of gene clusters for production of aflatoxin, cyclopiazonic acid, and ustiloxin B.

Aspergillus flavus produces hepatocarcinogenic aflatoxin that adversely impacts human and animal health and international trade. A promising means to manage preharvest aflatoxin contamination of crops is biological control, which employs non-aflatoxigenic A. flavus isolates possessing defective aflatoxin gene clusters to outcompete field toxigenic populations. However, these isolates often produce other toxic metabolites. The CRISPR/Cas9 technology has greatly advanced genome editing and gene functional studies. Its use in deleting large chromosomal segments of filamentous fungi is rarely reported. A system of dual CRISPR/Cas9 combined with a 60-nucleotide donor DNA that allowed removal of A. flavus gene clusters involved in production of harmful specialized metabolites was established. It efficiently deleted a 102-kb segment containing both aflatoxin and cyclopiazonic acid gene clusters from toxigenic A. flavus morphotypes, L-type and S-type. It further deleted the 27-kb ustiloxin B gene cluster of a resulting L-type mutant. Overall efficiencies of deletion ranged from 66.6 % to 85.6 % and efficiencies of deletions repaired by a single copy of donor DNA ranged from 50.5 % to 72.7 %. To determine the capacity of this technique, a pigment-screening setup based on absence of aspergillic acid gene cluster was devised. Chromosomal segments of 201 kb and 301 kb were deleted with efficiencies of 57.7 % to 69.2 %, respectively. This system used natural A. flavus isolates as recipients, eliminated a forced-recycling step to produce recipients for next round deletion, and generated maker-free deletants with sequences predefined by donor DNA. The research provides a method for creating genuine atoxigenic biocontrol strains friendly for field trial release.

Predators control pests and increase yield across crop types and climates: a meta-analysis.

Pesticides have well-documented negative consequences to control crop pests, and natural predators are alternatives and can provide an ecosystem service as biological control agents. However, there remains considerable uncertainty regarding whether such biological control can be a widely applicable solution, especially given ongoing climatic variation and climate change. Here, we performed a meta-analysis focused on field studies with natural predators to explore broadly whether and how predators might control pests and in turn increase yield. We also contrasted across studies pest suppression by a single and multiple predators and how climate influence biological control. Predators reduced pest populations by 73% on average, and increased crop yield by 25% on average. Surprisingly, the impact of predators did not depend on whether there were many or a single predator species. Precipitation seasonality was a key climatic influence on biological control: as seasonality increased, the impact of predators on pest populations increased. Taken together, the positive contribution of predators in controlling pests and increasing yield, and the consistency of such responses in the face of precipitation variability, suggest that biocontrol has the potential to be an important part of pest management and increasing food supplies as the planet precipitation patterns become increasingly variable.

Unraveling the enigma of root-knot nematodes: from origins to advanced management strategies in agriculture.

MAIN CONCLUSION: Integrated management strategies, including novel nematicides and resilient cultivars, offer sustainable solutions to combat root-knot nematodes, crucial for safeguarding global agriculture against persistent threats. Root-knot nematodes (RKN) pose a significant threat to a diverse range of host plants, with their obligatory endoparasitic nature leading to substantial agricultural losses. RKN spend much of their lives inside or in contact by secreting plant cell wall-modifying enzymes resulting in the giant cell development for establishing host-parasite relationships. Additionally, inflicting physical harm to host plants, RKN also contributes to disease complexes creation with fungi and bacteria. This review comprehensively explores the origin, history, distribution, and physiological races of RKN, emphasizing their economic impact on plants through gall formation. Management strategies, ranging from cultural and physical to biological and chemical controls, along with resistance mechanisms and marker-assisted selection, are explored. While recognizing the limitations of traditional nematicides, recent breakthroughs in non-fumigant alternatives like fluensulfone, spirotetramat, and fluopyram offer promising avenues for sustainable RKN management. Despite the success of resistance mechanisms like the Mi gene, challenges persist, prompting the need for integrative approaches to tackle Mi-virulent isolates. In conclusion, the review stresses the importance of innovative and resilient control measures for sustainable agriculture, emphasizing ongoing research to address evolving challenges posed by RKN. The integration of botanicals, resistant cultivars, and biological controls, alongside advancements in non-fumigant nematicides, contributes novel insights to the field, laying the ground work for future research directions to ensure the long-term sustainability of agriculture in the face of persistent RKN threats.

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.

Antibacterial synergy between a phage endolysin and citric acid against the Gram-negative kiwifruit pathogen Pseudomonas syringae pv. actinidiae.

Horticultural diseases caused by bacterial pathogens provide an obstacle to crop production globally. Management of the infection of kiwifruit by the Gram-negative phytopathogen Pseudomonas syringae pv. actinidiae (Psa) currently includes copper and antibiotics. However, the emergence of bacterial resistance and a changing regulatory landscape are providing the impetus to develop environmentally sustainable antimicrobials. One potential strategy is the use of bacteriophage endolysins, which degrade peptidoglycan during normal phage replication, causing cell lysis and the release of new viral progeny. Exogenous use of endolysins as antimicrobials is impaired by the outer membrane of Gram-negative bacteria that provides an impermeable barrier and prevents endolysins from accessing their target peptidoglycan. Here, we describe the synergy between citric acid and a phage endolysin, which results in a reduction of viable Psa below detection. We show that citric acid drives the destabilization of the outer membrane via acidification and sequestration of divalent cations from the lipopolysaccharide, which is followed by the degradation of the peptidoglycan by the endolysin. Scanning electron microscopy revealed clear morphological differences, indicating cell lysis following the endolysin-citric acid treatment. These results show the potential for citric acid-endolysin combinations as a possible antimicrobial approach in agricultural applications. IMPORTANCE: The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) causes major impacts to kiwifruit horticulture, and the current control strategies are heavily reliant on copper and antibiotics. The environmental impact and increasing resistance to these agrichemicals are driving interest in alternative antimicrobials including bacteriophage-derived therapies. In this study, we characterize the endolysin from the Otagovirus Psa374 which infects Psa. When combined with citric acid, this endolysin displays an impressive antibacterial synergy to reduce viable Psa below the limit of detection. The use of citric acid as a synergistic agent with endolysins has not been extensively studied and has never been evaluated against a plant pathogen. We determined that the synergy involved a combination of the chelation activity of citric acid, acidic pH, and the specific activity of the ΦPsa374 endolysin. Our study highlights an exciting opportunity for alternative antimicrobials in agriculture.

Biocontrol potential and growth-promoting effect of endophytic fungus Talaromyces muroii SD1-4 against potato leaf spot disease caused by Alternaria alternata.

BACKGROUND: Alternaria alternata is the primary pathogen of potato leaf spot disease, resulting in significant potato yield losses globally. Endophytic microorganism-based biological control, especially using microorganisms from host plants, has emerged as a promising and eco-friendly approach for managing plant diseases. Therefore, this study aimed to isolate, identify and characterize the endophytic fungi from healthy potato leaves which had great antifungal activity to the potato leaf spot pathogen of A. alternata in vitro and in vivo. RESULTS: An endophytic fungal strain SD1-4 was isolated from healthy potato leaves and was identified as Talaromyces muroii through morphological and sequencing analysis. The strain SD1-4 exhibited potent antifungal activity against the potato leaf spot pathogen A. alternata Lill, with a hyphal inhibition rate of 69.19%. Microscopic and scanning electron microscope observations revealed that the strain SD1-4 grew parallel to, coiled around, shrunk and deformed the mycelia of A. alternata Lill. Additionally, the enzyme activities of chitinase and ß-1, 3-glucanase significantly increased in the hyphae of A. alternata Lill when co-cultured with the strain SD1-4, indicating severe impairment of the cell wall function of A. alternata Lill. Furthermore, the mycelial growth and conidial germination of A. alternata Lill were significantly suppressed by the aseptic filtrate of the strain SD1-4, with inhibition rates of 79.00% and 80.67%, respectively. Decrease of leaf spot disease index from 78.36 to 37.03 was also observed in potato plants treated with the strain SD1-4, along with the significantly increased plant growth characters including plant height, root length, fresh weight, dry weight, chlorophyll content and photosynthetic rate of potato seedlings. CONCLUSION: The endophyte fungus of T. muroii SD1-4 isolated from healthy potato leaves in the present study showed high biocontrol potential against potato leaf spot disease caused by A. alternata via direct parasitism or antifungal metabolites, and had positive roles in promoting potato plant growth.

A potent endophytic fungus Purpureocillium lilacinum YZ1 protects against Fusarium infection in field-grown wheat.

Fusarium diseases pose a severe global threat to major cereal crops, particularly wheat. Existing biocontrol strains against Fusarium diseases are believed to primarily rely on antagonistic mechanisms, but not widely used under field conditions. Here, we report an endophytic fungus, Purpureocillium lilacinum YZ1, that shows promise in combating wheat Fusarium diseases. Under glasshouse conditions, YZ1 inoculation increased the survival rate of Fusarium graminearum (Fg)-infected wheat seedlings from 0% to > 60% at the seedling stage, and reduced spikelet infections by 70.8% during anthesis. In field trials, the application of YZ1 resulted in an impressive 89.0% reduction in Fg-susceptible spikelets. While a slight antagonistic effect of YZ1 against Fg was observed on plates, the induction of wheat systemic resistance by YZ1, which is distantly effective, non-specific, and long-lasting, appeared to be a key contributor to YZ1's biocontrol capabilities. Utilizing three imaging methods, we confirmed YZ1 as a potent endophyte capable of rapid colonization of wheat roots, and systematically spreading to the stem and leaves. Integrating dual RNA-Seq, photosynthesis measurements and cell wall visualization supported the link between YZ1's growth-promoting abilities and the activation of wheat systemic resistance. In conclusion, endophytes such as YZ1, which exhibits non-antagonistic mechanisms, hold significant potential for industrial-scale biocontrol applications.