Selective control of parasitic nematodes using bioactivated nematicides.

Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amid limited arable land1-4. Most traditional nematicides have been banned owing to poor nematode selectivity, leaving farmers with inadequate means of pest control4-12. Here we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome-p450-mediated bioactivation in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita, a highly destructive plant-parasitic nematode. Tests against numerous phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than most marketed nematicides. Selectivins are first-in-class bioactivated nematode controls that provide efficacy and nematode selectivity.

Harnessing nature's arsenal: Ochrobactrum bacteria metabolites in the battle against root- knot nematode - Insights from in vitro and molecular docking studies.

Agricultural Productivity and plant health are threatened by the root-knot nematode. The use of biocontrol agents reduces the need for chemical nematicides and improves the general health of agricultural ecosystems by offering a more environmentally friendly and sustainable method of managing nematode infestations. Plant-parasitic nematodes can be efficiently managed with the use of entomopathogenic nematodes (EPNs), which are widely used biocontrol agents. This study focused on the nematicidal activity of the secondary metabolites present in the bacteria Ochrobactrum sp. identified in the EPN, Heterorhabditisindica against Root-Knot Nematode (Meloidogyne incognita). Its effect on egg hatching and survival of juveniles of root- knot nematode (RKN) was examined. The ethyl acetate component of the cell-free culture (CFC) filtrate of the Ochrobactrum sp. bacteria was tested at four different concentrations (25 %, 50 %, 75 % and 100 %) along with broth and distilled water as control. The bioactive compounds of Ochrobactrum sp. bacteria showed the highest suppression of M. incognita egg hatching (100 %) and juvenile mortality (100 %) at 100 % concentration within 24 h of incubation. In this study, unique metabolite compounds were identified through the Gas Chromatography- Mass Spectrometry (GC-MS) analysis, which were found to have anti- nematicidal activity. In light of this, molecular docking studies were conducted to determine the impact of biomolecules from Ochrobactrum sp. using significant proteins of M. incognita, such as calreticulin, sterol carrier protein 2, flavin-containing monooxygenase, pectate lyase, candidate secreted effector, oesophageal gland cell secretory protein and venom allergen-like protein. The results also showed that the biomolecules from Ochrobactrum sp. had a significant inhibitory effect on the different protein targets of M. incognita. 3-Epimacronine and Heraclenin were found to inhibit most of the chosen target protein. Among the targets, the docking analysis revealed that Heraclenin exhibited the highest binding affinity of -8.6 Kcal/mol with the target flavin- containing monooxygenase. Further, the in vitro evaluation of 3- Epimacronine confirmed their nematicidal activity against M. incognita at different concentrations. In light of this, the present study has raised awareness of the unique biomolecules of the bacterial symbiont Ochrobactrum sp. isolated from H. indica that have nematicidal properties.

Natural nematicidal metabolites and advances in their biocontrol capacity on plant parasitic nematodes.

Covering: 2010 to 2021Natural nematicidal metabolites are important sources of nematode control. This review covers the isolation and structural determination of nematicidal metabolites from 2010 to 2021. We summarise chemical structures, bioactivity, metabolic regulation and biosynthesis of potential nematocides, and structure-activity relationship and application potentiality of natural metabolites in plant parasitic nematodes' biocontrol. In doing so, we aim to provide a comprehensive overview of the potential roles that natural metabolites can play in anti-nematode strategies.

Characterization of chaetoglobosin producing Chaetomium globosum for the management of Fusarium-Meloidogyne wilt complex in tomato.

AIM: Simultaneous management of FOL and RKN causing wilt complex in tomato by chaetoglobosin-producing Chaetomium globosum. METHODS AND RESULTS: Random survey was carried out to isolate Fusarium and Chaetomium. Twelve Fusarium isolates were characterized, and FOL4 (virulent) was molecularly identified. Wilt complex by FOL, RKN was assessed individually and in combination under greenhouse. RKN (1000 juveniles ml-1) inoculation followed by FOL4 (5 × 105 spores ml-1) accounted for 90% incidence. The chaetoglobosin-producing Chaetomium was isolated, characterized morphologically and molecularly. Among 55 isolates, nine showed >50% inhibition against FOL, and crude culture filtrate showed a significant reduction in RKN egg hatching (15.66%) and juvenile mortality (100%). Chaetomium Cg 40 was confirmed as C. globosum using SCAR marker (OK032373). Among 40 volatile compounds, hexadecanoic acid and 1,2-epoxy-5,9-cyclododecadiene exhibited antifungal and nematicidal properties in GC-MS. High-performance liquid chromatography revealed chaetoglobosin A (0.767 µg µl-1), and the presence of bioactive molecules chaetoglobosin (528.25 m/z), chaetomin (710 m/z), chaetocin (692.8 m/z), chaetoviridin (432.85 m/z), and chaetomugilin (390 m/z) was confirmed by LC/MS/MS. Cg 40 and Cg 6 were able to synthesize the pks1a, b gene responsible for chaetoglobosin, sporulation, and melanin biosynthesis was confirmed by PCR. The application of an aqueous formulation as seed treatment, seedling dip, and soil drenching (application) recorded lowest wilt incidence (11.11%) and gall index (1) with the maximum growth parameter (plant height 51.9 cm), fruit yield (287.5 g), and lycopene content (11.46 mg/100 g). CONCLUSIONS: Cg 40 and Cg 6, containing polyketides, secondary metabolites, antibiotics, chaetoglobosin, and plant growth-promoting ability, showed antifungal and nematicidal properties against the FOL-RKN wilt complex in tomato in vitro and pot culture experiments.

Nematicidal activity of volatile organic compounds produced by Bacillus altitudinis AMCC 1040 against Meloidogyne incognita.

The application of nematicidal microorganisms and their virulence factors provides more opportunities to control root-knot nematodes. Bacillus altitudinis AMCC 1040, previously isolated from suppressive soils, showed significant nematicidal activity, and in this study, nematicidal substances produced by Bacillus altitudinis AMCC 1040 were investigated. The results of the basic properties of active substances showed that these compounds have good thermal stability and passage, are resistant to acidic environment and sensitive to alkaline conditions. Further analysis showed that it is a volatile component. Using HS-SPME-GC/MS, the volatile compounds produced by Bacillus altitudinis AMCC 1040 were identified and grouped into four major categories: ethers, alcohols, ketone, and organic acids, comprising a total of eight molecules. Six of them possess nematicidal activities, including 2,3-butanedione, acetic acid, 2-isopropoxy ethylamine, 3-methylbutyric acid, 2-methylbutyric acid and octanoic acid. Our results further our understanding of the effects of Bacillus altitudinis and its nematicidal metabolites on the management of Meloidogyne incognita and may help in finding less toxic nematicides to control root knot nematodes.

Nematicidal effects of a peptidase (NlpC/P60) from the phytopathogen Pseudomonas syringae MB03 on the model nematode Caenorhabditis elegans and plant-parasitic Meloidogyne incognita.

BACKGROUND: Plant-parasitic nematodes (PPNs) are severe threats to agricultural yields and continue to be challenging to treat in several crops worldwide. Microbial-based control has been suggested as a better alternative to chemical control. In this study, we aimed to identify and characterize nematicidal virulence factors of a common phytopathogenic bacterium, Pseudomonas syringae, mainly focusing on the nematicidal and suppressive activities of an NlpC/P60 family peptidase, namely, Peptidase03, against the model nematode Caenorhabditis elegans and an agriculturally important PPN, Meloidogyne incognita. METHODS AND RESULTS: Genome-wide virulence factor prediction of the P. syringae wild-type strain MB03 revealed numerous nematode pathogenic determinants. We selected 11 predicted nematicidal genes for cloning and induced expression in an Escherichia coli expression system and then performed comparative nematicidal bioassays on the model nematode C. elegans. The recombinant strain expressing Peptidase03 showed the highest level of toxicity against C. elegans, with 75.9% mortality, compared to the other tested strains. Purified Peptidase03 showed significant toxicity against C. elegans and M. incognita, with half lethal concentration (LC50) values of 147.9 µg/mL and 211.50 µg/mL, respectively. We also demonstrated that Peptidase03 could damage the intestinal tissues of C. elegans and exhibit detrimental effects on its growth, brood size, and locomotion. CONCLUSIONS: The Peptidase03 protein from P. syringae MB03 had significant nematicidal and suppressive activities against C. elegans and M. incognita, thereby showing potential for the development of an effective PPN-controlling agent for use in agricultural practice.

Antibiotic and Nematocidal Metabolites from Two Lichen Species Collected on the Island of Lampedusa (Sicily).

The antibiotic and nematocidal activities of extracts from two coastal lichen species collected on Lampedusa Island (Sicily), Ramalina implexa Nyl. and Roccella phycopsis Ach., were tested. Methyl orsellinate, orcinol, (+)-montagnetol, and for the first time 4-chlororcinol were isolated from Roccella phycopsis. (+)-Usnic acid was obtained from Ramalina implexa. The crude organic extract of both lichen species showed strong antibiotic activity against some bacterial species and nematocidal activity. Among all the pure metabolites tested against the infective juveniles (J2) of the root-knot nematode (RKN) Meloydogine incognita, (+)-usnic acid, orcinol, and (+)-montagnetol had significant nematocidal activity, comparable with that of the commercial nematocide Velum® Prime, and thus they showed potential application in agriculture as a biopesticide. On the contrary, methyl orsellinate and 4-chlororcinol had no nematocidal effect. These results suggest that the substituent pattern at ortho-para-position in respect to both hydroxyl groups of resorcine moiety, which is present in all metabolites, seems very important for nematocidal activity. The organic extracts of both lichens were also tested against some Gram-positive and Gram-negative bacteria. Both extracts were active against Gram-positive species. The extract of Ramalina implexa showed, among Gram-negative species, activity against Escherichia coli and Acinetobacter baumannii, while that from Roccella phycopsis was effective towards all test strains, with the exception of Pseudomonas aeruginosa. The antimicrobial activity of (+)-usnic acid, methyl orsellinate, and (+)-montagnetol is already known, so tests were focused on orcinol and 4-chlororcinol. The former showed antibacterial activity against all Gram positive and Gram-negative test strains, with the exception of A. baumannii and K. pneumoniae, while the latter exhibited a potent antibacterial activity against Gram-positive test strains and among Gram-negative strains, was effective against A. baumannii and K. pneumonia. These results suggest, for orcinol and 4-chlororcinol, an interesting antibiotic potential against both Gram-positive and Gram-negative bacterial strains.

Toxicity of Bacillus thuringiensis Strains Derived from the Novel Crystal Protein Cry31Aa with High Nematicidal Activity against Rice Parasitic Nematode Aphelenchoides besseyi.

The plant parasitic nematode, Aphelenchoides besseyi, is a serious pest causing severe damage to various crop plants and vegetables. The Bacillus thuringiensis (Bt) strains, GBAC46 and NMTD81, and the biological strain, FZB42, showed higher nematicidal activity against A. besseyi, by up to 88.80, 82.65, and 75.87%, respectively, in a 96-well plate experiment. We screened the whole genomes of the selected strains by protein-nucleic acid alignment. It was found that the Bt strain GBAC46 showed three novel crystal proteins, namely, Cry31Aa, Cry73Aa, and Cry40ORF, which likely provide for the safe control of nematodes. The Cry31Aa protein was composed of 802 amino acids with a molecular weight of 90.257 kDa and contained a conserved delta-endotoxin insecticidal domain. The Cry31Aa exhibited significant nematicidal activity against A. besseyi with a lethal concentration (LC50) value of 131.80 µg/mL. Furthermore, the results of in vitro experiments (i.e., rhodamine and propidium iodide (PI) experiments) revealed that the Cry31Aa protein was taken up by A. besseyi, which caused damage to the nematode's intestinal cell membrane, indicating that the Cry31Aa produced a pore-formation toxin. In pot experiments, the selected strains GBAC46, NMTD81, and FZB42 significantly reduced the lesions on leaves by up to 33.56%, 45.66, and 30.34% and also enhanced physiological growth parameters such as root length (65.10, 50.65, and 55.60%), shoot length (68.10, 55.60, and 59.45%), and plant fresh weight (60.71, 56.45, and 55.65%), respectively. The number of nematodes obtained from the plants treated with the selected strains (i.e., GBAC46, NMTD81, and FZB42) and A. besseyi was significantly reduced, with 0.56, 0.83., 1.11, and 5.04 seedling mL-1 nematodes were achieved, respectively. Moreover, the qRT-PCR analysis showed that the defense-related genes were upregulated, and the activity of hydrogen peroxide (H2O2) increased while malondialdehyde (MDA) decreased in rice leaves compared to the control. Therefore, it was concluded that the Bt strains GBAC46 and NMTD81 can promote rice growth, induce high expression of rice defense-related genes, and activate systemic resistance in rice. More importantly, the application of the novel Cry31Aa protein has high potential for the efficient and safe prevention and green control of plant parasitic nematodes.

Use of RNAi as a preliminary tool for screening putative receptors of nematicidal toxins from Bacillus thuringiensis.

Bacillus thuringiensis is a potential control agent for plant-parasitic nematodes. Nematode intestinal receptors for Cry21-type toxins are poorly known. Therefore, a strategy was tested as a primary screening tool to find possible Cry toxin receptors, using a nematicidal Bt strain and the RNAi technique on Caenorhabditis elegans. Six genes encoding intestinal membrane proteins were selected (abt-4, bre-1, bre-2, bre-3, asps-1, abl-1) as possible targets for Cry proteins. Fractions of each selected gene were amplified by PCR. Amplicons were cloned into the L4440 vector to transform the E. coli HT155 (DE3) strain. Transformed bacteria were used to silence the selected genes using the RNAi feeding method. Nematodes with silenced genes were tested with the Bt strain LBIT-107, which harbors the nematicidal protein Cry21Aa3, among others. Results indicated that nematodes with the silenced abt-4 gene were 69.5% more resistant to the LBIT-107 strain, in general, and 79% to the Cry21Aa3 toxin, specifically.

Nematicidal Volatiles from Bacillus atrophaeus GBSC56 Promote Growth and Stimulate Induced Systemic Resistance in Tomato against Meloidogyne incognita.

Bacillus volatiles to control plant nematodes is a topic of great interest among researchers due to its safe and environmentally friendly nature. Bacillus strain GBSC56 isolated from the Tibet region of China showed high nematicidal activity against M. incognita, with 90% mortality as compared with control in a partition plate experiment. Pure volatiles produced by GBSC56 were identified through gas chromatography and mass spectrometry (GC-MS). Among 10 volatile organic compounds (VOCs), 3 volatiles, i.e., dimethyl disulfide (DMDS), methyl isovalerate (MIV), and 2-undecanone (2-UD) showed strong nematicidal activity with a mortality rate of 87%, 83%, and 80%, respectively, against M. incognita. The VOCs induced severe oxidative stress in nematodes, which caused rapid death. Moreover, in the presence of volatiles, the activity of antioxidant enzymes, i.e., SOD, CAT, POD, and APX, was observed to be enhanced in M. incognita-infested roots, which might reduce the adverse effect of oxidative stress-induced after infection. Moreover, genes responsible for plant growth promotion SlCKX1, SlIAA1, and Exp18 showed an upsurge in expression, while AC01 was downregulated in infested plants. Furthermore, the defense-related genes (PR1, PR5, and SlLOX1) in infested tomato plants were upregulated after treatment with MIV and 2-UD. These findings suggest that GBSC56 possesses excellent biocontrol potential against M. incognita. Furthermore, the study provides new insight into the mechanism by which GBSC56 nematicidal volatiles regulate antioxidant enzymes, the key genes involved in plant growth promotion, and the defense mechanism M. incognita-infested tomato plants use to efficiently manage root-knot disease.

Heterologous and Engineered Biosynthesis of Nematocidal Polyketide-Nonribosomal Peptide Hybrid Macrolactone from Extreme Thermophilic Fungi.

Fungal polyketide-nonribosomal peptide (PK-NRP) hybrid macrolactones are a growing family of natural products with biomedical and agricultural activities. One of the most important families is the thermolides, which are produced by extreme thermophilic fungi and exhibit strong nematocidal activity. We show here that the genes ThmABCE from Talaromyces thermophilus NRRL 2155 are critical for thermolide synthesis. Two separate single-module hrPKS (ThmA) and NRPS (ThmB) enzymes collaborate to synthesize the core macrolactone backbone (6 or 7), and the NRPS ThmB-CT domain catalyzes the key macrocyclization step in PK-NRP intermediate release via ester bond formation, representing a novel function of fungal NRPS C domains. We also show that heterologous and engineered expression of the Thm genes in the type strains of Aspergillus nidulans and Escherichia coli not only dramatically enhances the yields of thermolides but also affords different esterified analogues, such as butyryl- (thermolides J and K, 15 and 16), hexanoyl-, and octanyl- derivatives or mixed thermolides. Thermolides L and M (18 and 19), discovered via genome mining-based combinatorial biosynthesis, represent the first l-phenylalanine-based thermolides. Our work shows a unique biosynthetic mechanism of PK-NRP hybrid macrolactones from extremophiles, which led to the discovery of novel compounds and furthers our biosynthetic knowledge.

Biology and applications of Clonostachys rosea.

Clonostachys rosea is a promising saprophytic filamentous fungus that belongs to phylum Ascomycota. Clonostachys rosea is widespread around the world and exists in many kinds of habitats, with the highest frequency in soil. As an excellent mycoparasite, C. rosea exhibits strong biological control ability against numerous fungal plant pathogens, nematodes and insects. These behaviours are based on the activation of multiple mechanisms such as secreted cell-wall-degrading enzymes, production of antifungal secondary metabolites and induction of plant defence systems. Besides having significant biocontrol activity, C. rosea also functions in the biodegradation of plastic waste, biotransformation of bioactive compounds, as a bioenergy sources and in fermentation. This mini review summarizes information about the biology and various applications of C. rosea and expands on its possible uses.

Fungi from the extremes of life: an untapped treasure for bioactive compounds.

More than 80% of the Earth surface is consisted of hostile and harsh environments, classified as extreme from an anthropogenic perspective. Microorganisms with acclimatized nature dominate these extreme ecosystems of the biosphere. Survivals in such environments initiate an inductive force leading to the production of noteworthy metabolites having peculiar biochemistry. Recent investigations on extremophilic fungi for unprecedented bioactive compounds emphasize their remarkable potential as sources of new therapeutics. The present review covers the literature published in the last 15 years and highlights the biological activities and structure of compounds isolated from the extremophilic fungi. The compounds are grouped based on their biological functions such as cytotoxicity, lipid-lowering ability, and antimicrobial, antioxidant, nematocidal, anti-inflammatory, anti-malarial, and antifouling activities. A total of 155 compounds isolated from 25 Penicillium species, 16 Aspergillus species, and 23 other species are presented, which include 105 new and 50 known bioactive compounds. Out of these, 77 have known cytotoxic activity and 46 are antimicrobial in nature, while there are 32 other compounds with different activities including nematocidal, anti-allergic, antioxidant, and anti-inflammatory. KEY POINTS: • A broad compilation of bioactive compounds from extremophilic fungi. • Classification of bioactive compounds based on their biological functions. • Production of cytotoxic compounds is common among all kind of extremophilic fungi. • Bioactive compounds have no direct role in adaptation process of extremophiles.

Identification and Nematicidal Characterization of Proteases Secreted by Endophytic Bacteria Bacillus cereus BCM2.

The endophytic bacterium Bacillus cereus BCM2 has shown great potential as a biocontrol organism against Meloidogyne incognita, which causes severe root-knot diseases in crops. In our previous study, the metabolite of BCM2 showed high nematicidal activity against the M. incognita second-stage juveniles. However, the mechanism employed by endophytic bacteria to infect and kill nematodes is still unclear. Here, we investigate both the endophytic bacterial extracellular proteins with nematicidal activity and their mechanism of killing nematodes. The first step was detecting the nematicidal activities of crude proteins. The results show that the nematode mortality rate reached 100% within 72 h, and the crude proteins damaged both the cuticle and eggshell, before finally destroying the targets. This suggests possible proteinaceous pathogeny in BCM2. Throughout the process, the fine-detail changes in the nematode cuticle and the intestinal structure were observed using scanning electron microscopy and transmission electron microscopy. These images show that BCM2 extracellular proteins did not damage the internal organization of the nematode but did severely damage its cuticle, which led to content leakage. From the crude proteins, chitosanase, alkaline serine protease, and neutral protease were purified and identified. The M. incognita-B. cereus BCM2 microenvironment simulation demonstrates that BCM2 adheres to the surface of nematodes and helps the metabolites that were produced by BCM2 to rapidly recognize and kill M. incognita. This relationship between plants, endophytic bacteria, and nematodes offers insight into the biological mechanisms that can be utilized for of nematode management.

Isolation and characterization of soil bacteria able to rapidly degrade the organophosphorus nematicide fosthiazate.

Foshtiazate is an organophosphorus nematicide commonly used in protected crops and potato plantations. It is toxic to mammals, birds and honeybees, it is persistent in certain soils and can be transported to water resources. Recent studies by our group demonstrated, for the first time, the development of enhanced biodegradation of fosthiazate in agricultural soils. However, the micro-organisms driving this process are still unknown. We aimed to isolate soil bacteria responsible for the enhanced biodegradation of fosthiazate and assess their degradation potential against high concentrations of the nematicide. Enrichment cultures led to the isolation of two bacterial cultures actively degrading fosthiazate. Denaturating Gradient Gel Electrophoresis analysis revealed that they were composed of a single phylotype, identified via 16S rRNA cloning and phylogenetic analysis as Variovorax boronicumulans. This strain showed high degradation potential against fosthiazate. It degraded up to 100 mg l-1 in liquid cultures (DT50  = 11·2 days), whereas its degrading capacity was reduced at higher concentration levels (500 mg l-1 , DT50  = 20 days). This is the first report for the isolation of a fosthiazate-degrading bacterium, which showed high potential for use in future biodepuration and bioremediation applications. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reported for the first time the isolation and molecular identification of bacteria able to rapidly degrade the organophosphorus nematicide fosthiazate; one of the few synthetic nematicides still available on the global market. Further tests demonstrated the high capacity of the isolated strain to degrade high concentrations of fosthiazate suggesting its high potential for future bioremediation applications in contaminated environmental sites, considering high acute toxicity and high persistence and mobility of fosthiazate in acidic and low in organic matter content soils.

A Chemosensory GPCR as a Potential Target to Control the Root-Knot Nematode Meloidogyne incognita Parasitism in Plants.

Root-knot nematodes (RKN), from the Meloidogyne genus, have a worldwide distribution and cause severe economic damage to many life-sustaining crops. Because of their lack of specificity and danger to the environment, most chemical nematicides have been banned from use. Thus, there is a great need for new and safe compounds to control RKN. Such research involves identifying beforehand the nematode proteins essential to the invasion. Since G protein-coupled receptors GPCRs are the target of a large number of drugs, we have focused our research on the identification of putative nematode GPCRs such as those capable of controlling the movement of the parasite towards (or within) its host. A datamining procedure applied to the genome of Meloidogyne incognita allowed us to identify a GPCR, belonging to the neuropeptide GPCR family that can serve as a target to carry out a virtual screening campaign. We reconstructed a 3D model of this receptor by homology modeling and validated it through extensive molecular dynamics simulations. This model was used for large scale molecular dockings which produced a filtered limited set of putative antagonists for this GPCR. Preliminary experiments using these selected molecules allowed the identification of an active compound, namely C260-2124, from the ChemDiv provider, which can serve as a starting point for further investigations.

Genetic and Biochemical Characterization of a Gene Operon for trans-Aconitic Acid, a Novel Nematicide from Bacillus thuringiensis.

trans-Aconitic acid (TAA) is an isomer of cis-aconitic acid (CAA), an intermediate of the tricarboxylic acid cycle that is synthesized by aconitase. Although TAA production has been detected in bacteria and plants for many years and is known to be a potent inhibitor of aconitase, its biosynthetic origins and the physiological relevance of its activity have remained unclear. We have serendipitously uncovered key information relevant to both of these questions. Specifically, in a search for novel nematicidal factors from Bacillus thuringiensis, a significant nematode pathogen harboring many protein virulence factors, we discovered a high yielding component that showed activity against the plant-parasitic nematode Meloidogyne incognita and surprisingly identified it as TAA. Comparison with CAA, which displayed a much weaker nematicidal effect, suggested that TAA is specifically synthesized by B. thuringiensis as a virulence factor. Analysis of mutants deficient in plasmids that were anticipated to encode virulence factors allowed us to isolate a TAA biosynthesis-related (tbr) operon consisting of two genes, tbrA and tbrB We expressed the corresponding proteins, TbrA and TbrB, and characterized them as an aconitate isomerase and TAA transporter, respectively. Bioinformatics analysis of the TAA biosynthetic gene cluster revealed the association of the TAA genes with transposable elements relevant for horizontal gene transfer as well as a distribution across B. cereus bacteria and other B. thuringiensis strains, suggesting a general role for TAA in the interactions of B. cereus group bacteria with nematode hosts in the soil environment. This study reveals new bioactivity for TAA and the TAA biosynthetic pathway, improving our understanding of virulence factors employed by B. thuringiensis pathogenesis and providing potential implications for nematode management applications.

Cry6Aa1, a Bacillus thuringiensis nematocidal and insecticidal toxin, forms pores in planar lipid bilayers at extremely low concentrations and without the need of proteolytic processing.

Cry6Aa1 is a Bacillus thuringiensis (Bt) toxin active against nematodes and corn rootworm insects. Its 3D molecular structure, which has been recently elucidated, is unique among those known for other Bt toxins. Typical three-domain Bt toxins permeabilize receptor-free planar lipid bilayers (PLBs) by forming pores at doses in the 1-50 µg/ml range. Solubilization and proteolytic activation are necessary steps for PLB permeabilization. In contrast to other Bt toxins, Cry6Aa1 formed pores in receptor-free bilayers at doses as low as 200 pg/ml in a wide range of pH (5.5-9.5) and without the need of protease treatment. When Cry6Aa1 was preincubated with Western corn rootworm (WCRW) midgut juice or trypsin, 100 fg/ml of the toxin was sufficient to form pores in PLBs. The overall biophysical properties of the pores were similar for all three forms of the toxin (native, midgut juice- and trypsin-treated), with conductances ranging from 28 to 689 pS, except for their ionic selectivity, which was slightly cationic for the native and midgut juice-treated Cry6Aa1, whereas dual selectivity (to cations or anions) was observed for the pores formed by the trypsin-treated toxin. Enrichment of PLBs with WCRW midgut brush-border membrane material resulted in a 2000-fold reduction of the amount of native Cry6Aa1 required to form pores and affected the biophysical properties of both the native and trypsin-treated forms of the toxin. These results indicate that, although Cry6Aa1 forms pores, the molecular determinants of its mode of action are significantly different from those reported for other Bt toxins.

Nematode-specific cadherin CDH-8 acts as a receptor for Cry5B toxin in Caenorhabditis elegans.

Parasitic nematodes of animals and plants cause worldwide devastating impacts on people's lives and agricultural crops. The crystal protein Cry5B produced by Bacillus thuringiensis has efficient and specific activity against a wide range of nematodes. However, the action mode of this toxin has not yet been thoroughly determined. Here, a nematode-specific cadherin CDH-8 was demonstrated to be a receptor for Cry5B toxin by using Caenorhabditis elegans as a model, providing evidence that the cadherin mutant worm cdh-8(RB815) possesses significant resistance to Cry5B, and the CDH-8 fragments bind specifically to Cry5B. Furthermore, CDH-8 was identified to be required for the oligomerization of Cry5B toxin in vivo and contribute to the internalization and pore formation of Cry5B in nematode cells. This study will facilitate a better understanding of the action mode of nematicidal Cry toxins and help the design of Cry toxin-based products for the control of plant or animal parasitic nematodes.

The Nif3-Family Protein YqfO03 from Pseudomonas syringae MB03 Has Multiple Nematicidal Activities against Caenorhabditis elegans and Meloidogyne incognita.

The nematicidal activity of the common plant-pathogenic bacterium Pseudomonas syringae against certain nematodes has been recently identified, but little is known about its virulence factors. In the current study, predictive analysis of nematode-virulent factors in the genome of a P. syringae wild-type strain MB03 revealed a variety of factors with the potential to be pathogenic against nematodes. One of these virulence factors that was predicted with a high score, namely, YqfO03, was a protein with structural domains that are similar to the Nif3 superfamily. This protein was expressed and purified in Escherichia coli, and was investigated for nematicidal properties against the model nematode Caenorhabditis elegans and an agriculturally important pest Meloidogyne incognita. Our results showed that YqfO03 exhibits lethal activity toward C. elegans and M. incognita worms, and it also caused detrimental effects on the growth, brood size, and motility of C. elegans worms. However, C. elegans worms were able to defend themselves against YqfO03 via a physical defense response by avoiding contact with the protein. Discovery of the diverse nematicidal activities of YqfO03 provides new knowledge on the biological function of a bacterial Nif3-family protein and insight into the potential of this protein as a specific means of controlling agricultural nematode pests.