Nematicidal Agents Research Articles

The diverse nematicidal properties and biocontrol efficacy of Bacillus thuringiensis Cry6A against the root-knot nematode Meloidogyne hapla

Cry6A toxin from Bacillus thuringiensis is a representative nematicidal crystal protein with a variety of nematicidal properties to free-living nematode Caenorhabditis elegans. Cry6A shares very low homology and different structure with Cry5B, another representative nematicidal crystal protein, and probably acts in a distinct pathway. All these strongly indicate that Cry6A toxin is likely a potent candidate for nematicide. The present study dealt with global investigation to determine the detrimental impacts of Cry6Aa2 toxin on Meloidogyne hapla, a root-knot nematode, and evaluated its biocontrol efficacy in pot experiment. Obtained results indicated that Cry6Aa2 toxin exhibits obvious toxicity to second-stage juvenile of M. hapla, and significantly inhibits egg hatch, motility, and penetration to host plant. Pot experiment suggested that soil drenching with spore-crystal mixture of Cry6Aa2 can clearly lighten the disease of root-knot nematode, including reduction of galling index and egg masses on host plant root, decreasing final population of nematode in soil. Moreover, application of Cry6Aa2 can obviously promote plant growth. These results demonstrated that Cry6Aa2 toxin is a promising nematicidal agent, and possesses great potential in plant-parasitic nematode management and construction of transgenic crop with constant resistance to nematode.

Global DNA hypomethylation: A potential mechanism in King pigeon nerve tissue damage induced by avermectin

As an effective insecticidal and nematicidal agent, avermectin (AVM) has been widely used in agricultural production and stock farming areas. Subsequently, the residues of AVM or its active metabolites in animal manure pose a toxic threat to non-target organisms in the environment. As the most characteristic epigenetic phenomena, DNA methylation status is a useful biological signal for the toxicity assessment of environmental chemical toxicants. In this study, analyses of the overall level of genomic DNA methylation were performed, and the expression levels of DNA methyltransferases (DNMTs), as well as demethylase methyl-CpG-binding domain protein 2 (MBD2), in pigeon brain tissues after subchronic exposure (with a AVM concentration of 20mg/kg, 40mg/kg and 60mg/kg, respectively) to AVM for 30, 60 and 90days were investigated. Global DNA hypomethylation and down-regulation of DNMT mRNA expression occurred in a dose–time-dependent manner in pigeon brains. The expression level of MBD2, which functions as a demethylase, was significantly enhanced in a dose-dependent but not time-dependent manner. In addition, the elevated expression level of MBD2 had a more robust effect on genomic DNA hypomethylation compared to changes in DNMT expression. Taken together, these results suggested that subchronic dose exposures of AVM could affect the global DNA methylation status, and this mechanism is closely related to changes in the expression levels of DNMTs and MBD2.

English

This study discusses and developed methods for obtaining plant extracts/pure compounds and its usages as a nematicidal agent. Freshly hatched second-stage juveniles of two nematode species, Meloidogyne incognita and Cephalobus litoralis were used. A bioassay guided isolation of the extract, fractions and pure compounds were done for their nematicidal activity at different concentrations in comparison with Azadirachta indica, while distilled water was taken as control. The crude extract showed 90% and ethyl acetate fraction 97% mortality rate after 48 h at 1% concentration against M. incognita species and 81 and 50% against C. litoralis specie at the same concentration. Among the pure compounds, 4 and 9 showed maximum mortality of 90 and 91% and compounds 8, 3, 6, 2, 5, 7 and 1 showed 89, 88, 88, 82, 80, 80 and 69% mortality, respectively after 48 h in M. incognita sp. In C. litoralis, compounds 8 and 9 showed 72 and 75%, significant mortality, while 7, 4, 3, 5, 6, 2 and 1 showed 70, 70, 70, 68, 62, 60 and 58% mortality, respectively after 48 h. The plant is of economic importance with nematicidal value. Key words: Meloidogyne incognita, Cephalobus litoralis, compound, mortality, crude extract.

Nematicidal isochromane glycoside from Kigelia pinnata leaves

Synthetic nematicides such as oxamyl and carbofuran play significant roles in the management of plant-parasitic nematodes. However, their negative environmental impacts have it imperative to search for safer alternatives. As part of our contribution in the search for bio-nematicides, compounds from plant extract were screened for possible potent nematicidal agent. A new isochromane carboxylic acid glycoside, isolated from the leaves of Kigelia pinnata (Lam.) Benth (Bignoniaceae) was evaluated for its nematicidal activity. The structure of the proposed compound was characterized by various spectroscopic methods, which included UV, FTIR, 1D-, and 2D-NMR, FAB-MS, TOF-ESI-MS and TOF-ESI-MS/MS (TANDEM). The in vitro experiment conducted on the glycoside against Meloidogyne incognita juveniles and eggs indicated an induced mortality. Its activity can be compared favourably with oxamyl, when tested at 0.1 mg/mL concentration. At four hours of observation, no significant difference (P < 0.05) between oxamyl and the glycoside was observed. The present data sustains that natural glycoside is a promising oxamyl alternate for controlling nematode-induced plant root knots and may contribute to integrated pest management.

Nematicidal Bacteria Associated to Pinewood Nematode Produce Extracellular Proteases

Bacteria associated with the nematode Bursaphelenchus xylophilus, a pathogen of trees and the causal agent of pine wilt disease (PWD) may play a role in the disease. In order to evaluate their role (positive or negative to the tree), strains isolated from the track of nematodes from infected Pinus pinaster trees were screened, in vitro, for their nematicidal potential. The bacterial products, from strains more active in killing nematodes, were screened in order to identify and characterize the nematicidal agent. Forty-seven strains were tested and, of these, 21 strains showed capacity to produce extracellular products with nematicidal activity. All Burkholderia strains were non-toxic. In contrast, all Serratia strains except one exhibited high toxicity. Nematodes incubated with Serratia strains showed, by SEM observation, deposits of bacteria on the nematode cuticle. The most nematicidal strain, Serratia sp. A88copa13, produced proteases in the supernatant. The use of selective inhibitors revealed that a serine protease with 70 kDa was majorly responsible for the toxicity of the supernatant. This extracellular serine protease is different phylogenetically, in size and biochemically from previously described proteases. Nematicidal assays revealed differences in nematicidal activity of the proteases to different species of Bursaphelenchus, suggesting its usefulness in a primary screen of the nematodes. This study offers the basis for further investigation of PWD and brings new insights on the role bacteria play in the defense of pine trees against B. xylophilus. Understanding all the factors involved is important in order to develop strategies to control B. xylophilus dispersion.

The effects of Bacillus thuringiensis Cry6A on the survival, growth, reproduction, locomotion, and behavioral response of Caenorhabditis elegans

Several families of crystal proteins from Bacillus thuringiensis exhibit nematicidal activity. Cry5B protein, a pore-forming toxin, has been intensively studied yielding many insights into the mode of action of crystal protein at molecular level and pathogenesis of pore-forming toxins. However, little attention was paid to Cry6A, another representative nematicidal crystal protein. Cry6A shares very low homology with Cry5B at amino acid sequence and probably acts in a distinct pathway from Cry5B and even the other main commercial crystal proteins. In the current study, we comprehensively investigated the nematicidal properties of Cry6Aa2 against the free-living soil nematode Caenorhabditis elegans and examined the physical response of C. elegans to Cry6Aa2 attack. Our results indicate that Cry6Aa2 exhibits high lethal activity to C. elegans and could cause detrimental effects on C. elegans, including obviously suppressed growth, decreased brood size, and even abnormal motility. Meanwhile, our study additionally shows that C. elegans could defend against the Cry6Aa2 toxin harmful threat through behavioral defense responses, such as reduced oral uptake and physical avoidance. In general, this study suggests that Cry6Aa2 possesses diverse nematicidal properties, which strongly indicates that Cry6Aa2 is a promising potential candidate of nematicidal agent. Moreover, this study highlights the importance of behavioral responses in defense of C. elegans for survival and demonstrates the key role of crystal protein in the interaction of B. thuringiensis-C. elegans. These findings could shed light on understanding the interaction of C. elegans with B. thuringiensis and provide a perfect model to study the role of pathogenic factor in the interaction of pathogen-host.

Synthesis and Quantitative Structure–Activity Relationship (QSAR) Study of Novel N-Arylsulfonyl-3-acylindole Arylcarbonyl Hydrazone Derivatives as Nematicidal Agents

In continuation of our program aimed at the discovery and development of natural-product-based pesticidal agents, 54 novel N-arylsulfonyl-3-acylindole arylcarbonyl hydrazone derivatives were prepared, and their structures were well characterized by ¹H NMR, ¹³C NMR, HRMS, ESI-MS, and mp. Their nematicidal activity was evaluated against that of the pine wood nematode, Bursaphelenchus xylophilus in vivo. Among all of the derivatives, especially V-12 and V-39 displayed the best promising nematicidal activity with LC₅₀ values of 1.0969 and 1.2632 mg/L, respectively. This suggested that introduction of R¹ and R² together as the electron-withdrawing substituents, R³ as the methyl group, and R⁴ as the phenyl with the electron-donating substituents could be taken into account for further preparation of these kinds of compounds as nematicidal agents. Six selected descriptors are a WHIM descriptor (E1m), two GETAWAY descriptors (R1m+ and R3m+), a Burden eigenvalues descriptor (BEHm8), and two edge-adjacency index descriptors (EEig05x and EEig13d). Quantitative structure-activity relationship (QSAR) studies demonstrated that the structural factors, such as molecular mass (a negative correlation with the bioactivity) and molecular polarity (a positive correlation with bioactivity), are likely to govern the nematicidal activities of these compounds. For this model, the correlation coefficient (R²(training set)), the leave-one-out cross-validation correlation coefficient (Q²(LOO)), and the 7-fold cross-validation correlation coefficient (Q²(7-fold)) were 0.791, 0.701, and 0.715, respectively. The external cross-validation correlation coefficient (Q²ext) and the root-mean-square error for the test set (RMSE(test set)) were 0.774 and 3.412, respectively. This study will pave the way for future design, structural modification, and development of indole derivatives as nematicidal agents.

Nematicidal Activity of 2-Thiophenecarboxaldehyde and Methylisothiocyanate from Caper (Capparis spinosa) against Meloidogyne incognita

New pesticides based on plant extracts have recently gained interest in the development of nontoxic crop protection chemicals. Numerous research studies are focused on the isolation and identification of new active compounds derived from plants. In this manuscript we report about the use of the Mediterranean species Capparis spinosa as a potent natural nematicidal agent against the root knot nematodes Meloidogyne incognita. Leaves, stems, and caper buds of Capparis spinosa were used to obtain their methanol extracts (LME, SME, BME) that were successively in vitro tested against second stage nematode juveniles (J2). In terms of paralysis induction, the methanol extract of the stem part (SME) was found more effective against M. incognita and then the caper methanol buds and leaves extracts. The chemical composition analysis of the extracts carried out by GC/MS and LC/MS techniques showed that methylisothiocyanate was the main compound of SME. The EC50 for SME after 3 days of immersion was 215 ± 36 mg/L. The constituent components of SME such as 2-thiophenecarboxaldehyde and methylisothiocyanate were successively in vitro tested for their nematicidal activity against J2. Both compounds induced paralysis on root knot nematodes ranking first (EC50 = 7.9 ± 1.6, and 14.1 ± 1.9 mg/L respectively) for M. incognita. Moreover, 2-thiophenecarboxaldehyde showed a strong fumigant activity.

Inhibition properties of three acetylcholinesterases of the pinewood nematode Bursaphelenchus xylophilus by organophosphates and carbamates

The pinewood nematode, Bursaphelenchus xylophilus, causes severe damage to pine species by transmitting the pine wilt disease, and the injection of nematicides, such as emamectin benzonate and milbemectin, is the most common practice to control this pest. However, despite their high efficacy, these macrocyclic lactone nematicides are expensive, limiting their practicability. In an attempt to screen affordable alternative nematicidal agents, we expressed three recombinant acetylcholinesterases (ACEs, EC 3.1.1.7) of B. xylophilus using an in vitro baculovirus expression system and evaluated the effects of 11 organophosphates (OPs) and three carbamates (CBs) on the toxicological properties of the enzymes. Of the three recombinant B. xylophilus ACEs (BxACEs; BxACE-1, BxACE-2 and BxACE-3), BxACE-1 and BxACE-2 were highly inhibited by OPs and CBs, including paraoxon, dichlorvos, chlorpyrifos-oxon, chlorpyrifos-methyl-oxon, mevinphos and carbofuran, as demonstrated by an inhibition assay. In contrast, BxACE-3 was insensitive to most of the pesticides tested, with the exception of mevinphos. BxACE-2 was more sensitive than BxACE-1 and BxACE-3 to most of the OPs, whereas BxACE-1 was more sensitive to the CBs than BxACE-2 and BxACE-3. An in vivo toxicity assay revealed that some compounds that were rarely employed as nematicides exhibited higher toxicities than those chemicals commonly used as nematicides. The inhibition kinetic data and in vivo toxicity assay obtained in this study should provide essential information for the development of OP- and CB-based nematicides against B. xylophilus. The availability of recombinant ACEs will also facilitate the development of an in vitro screening system to develop potential OP- and CB-based nematicides.

Nematicidal Activity of (E,E)-2,4-Decadienal and (E)-2-Decenal from Ailanthus altissima against Meloidogyne javanica

Methanol extracts of various plant parts of Ailanthus altissima were tested against the root knot nematode Meloidogyne javanica . Extracts of bark (ABE), wood (AWE), roots (ARE), and leaves (ALE) from A. altissima were investigated against freshly hatched second-stage juveniles (J(2)). AWE was the most active extract, with EC(50/3d) of 58.9 mg/L, while ALE, ARE, and ABE did not show nematicidal activity. The chemical composition of the extracts of A. altissima was determined by gas chromatography-mass spectrometry, and (E,E)-2,4-decadienal, (E)-2-undecenal, (E)-2-decenal, hexanal, nonanal, and furfural were the most prominent constituents. (E,E)-2,4-Decadienal, (E)-2-decenal, and furfural showed the highest nematicidal activity against M. javanica , with EC(50/1d) = 11.7, 20.43, and 21.79 mg/L, respectively, while the other compounds were inactive at the concentrations tested. The results obtained showed that AWE and its constituents (E,E)-2,4-decadienal and (E)-2-decenal could be considered as potent botanical nematicidal agents.

ChemInform Abstract: Synthesis and in vitro Study of Methylene‐bis‐tetrahydro[1,3]thiazolo[4,5‐c]isoxazoles as Potential Nematicidal Agents.

AbstractThe title compounds (VII) are prepared via key intermediate (IV) which is obtained under microwave irradiation as well as under conventional heating conditions.

Synthesis and in vitro study of methylene-bis-tetrahydro[1,3]thiazolo[4,5- c]isoxazoles as potential nematicidal agents

A series of methylene-bis-tetrahydro[1,3]thiazolo[4,5- c]isoxazoles 6 were synthesized by the reaction of chalcone derivative of methylene-bis-thiazolidinone 5 with hydroxylamine hydrochloride. The chemical structures of newly synthesized compounds were elucidated by IR, 1H, 13C NMR, MS and elemental analyses. The compounds 6a– g were evaluated for their nematicidal activity against Ditylenchus myceliophagus and Caenorhabditis elegans, compound 6e and 6f showed appreciable nematicidal activity. Further the compounds 6a– g were screened for their antifungal activity against Candida albicans (ATCC 10231), Aspergillus fumigatus (HIC 6094), Trichophyton rubrum (IFO 9185) and Trichophyton mentagrophytes (IFO 40996). The compounds 6b and 6f displayed notable antifungal activity against all the microorganisms employed. The activity of these two compounds is almost equal to the standard. It is also interesting to note that the compounds 6b, 6f and 6g showed activity towards C. albicans at the concentration of 3.75 μM, which is less than the concentration of the standard Amphotericin B.

Suppression of pine wilt disease by an antibacterial agent, oxolinic acid

Pine wilt disease (PWD) is very complex and has been reported to be caused by pine wood nematode, Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, and its accompanying bacteria. However, there is no report on the control of PWD by antibacterial agent. The present study was performed to investigate disease control efficacy of antibacterial agents against PWD. Among six antibacterial antibiotics tested, oxolinic acid (OA) showed the strongest antibacterial activity against five bacteria isolated from three strains of pine wood nematode. In in vivo assay, it effectively suppressed the development of PWD in three-year-old seedlings of Pinus densiflora Sieb. & Zucc.; it showed 71% control when injected at 3 mg per seedling. A mixture of OA and the nematicidal agent abamectin (Ab) showed higher disease control efficacy against PWD than either OA or Ab alone. In addition, OA alone and a mixture of OA and Ab also controlled PWD in approximately 20-year-old pine trees under field conditions. This is the first report on the suppression of PWD by OA. The result strongly indicates that PWD could be controlled by antibacterial antibiotic alone and a combination of antibacterial and nematicidal agents.

Nematicidal activity against Bursaphelenchus xylophilus of isoobtusilactone A isolated from Persea americana

Abstract Methanol extracts of 50 plant species were assessed for nematicidal activity against the pine wood nematode (PWN), Bursaphelenchus xylophilus. Out of these, five extracts: Justicia diclipteroides subsp. usambarica; Nerium oleander; Persea americana; Scadoxus multiflorus subsp. multiflorus; and Thespesia populnea, caused 96-100% mortalities of pine wood nematode at 1000 μg ml–1 for 48 h. Methanol extract of the stem bark of P. americana (avocado) and the n-hexane layer obtained from the methanol extract by solvent partitioning caused 100% mortality of PWN at low concentrations of 125 and 63 μg ml–1, respectively. One potent nematicidal compound was isolated from the stem bark of P. americana and its chemical structure was determined to be isoobtusilactone A (1) by mass and NMR spectral analyses. The chemical showed a very strong nematicidal activity against PWN; it caused mortalities of over 97% at concentrations higher than 50 μg ml–1. Compared with a commercial synthetic nematicide, morantel tartrate, compound 1 showed a little more potent activity. The results suggested that compound 1 may have the potential to be explored as a natural nematicide or be useful as a lead molecule for development of new nematicidal agents for controlling the pine wilt disease caused by B. xylophilus.

Identification and Characterization of a Dual-Acting Antinematodal Agent against the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a mycophagous and phytophagous pathogen responsible for the current widespread epidemic of the pine wilt disease, which has become a major threat to pine forests throughout the world. Despite the availability of several preventive trunk-injection agents, no therapeutic trunk-injection agent for eradication of PWN currently exists. In the characterization of basic physiological properties of B. xylophilus YB-1 isolates, we established a high-throughput screening (HTS) method that identifies potential hits within approximately 7 h. Using this HTS method, we screened 206 compounds with known activities, mostly antifungal, for antinematodal activities and identified HWY-4213 (1-n-undecyl-2-[2-fluorphenyl] methyl-3,4-dihydro-6,7-dimethoxy-isoquinolinium chloride), a highly water-soluble protoberberine derivative, as a potent nematicidal and antifungal agent. When tested on 4 year-old pinewood seedlings that were infected with YB-1 isolates, HWY-4213 exhibited a potent therapeutic nematicidal activity. Further tests of screening 39 Caenorhabditis elegans mutants deficient in channel proteins and B. xylophilus sensitivity to Ca2+ channel blockers suggested that HWY-4213 targets the calcium channel proteins. Our study marks a technical breakthrough by developing a novel HTS method that leads to the discovery HWY-4213 as a dual-acting antinematodal and antifungal compound.

ChemInform Abstract: Synthesis and Biological Evaluation of Novel Methylene‐bis‐thiazolidinone Derivatives as Potential Nematicidal Agents.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis and biological evaluation of novel methylene‐bisthiazolidinone derivatives as potential nematicidal agents

Abstractmagnified imageIn a one pot procedure, a series of novel methylene‐bis‐thiazolidinone derivatives 5 and 6 was prepared by condensation of 5‐(3‐formyl‐4‐methoxybenzyl)‐2‐methoxybenzaldehyde 3 with mercapto acids and primary aromatic amines 4 in presence of ZnCl2 under both microwave irradiation and conventional heating conditions. High yields are achieved even on a gram scale, while reaction times are considerably shortened under microwave irradiation compared to conventional heating conditions. Characterization of new compounds has been done by means of IR, NMR, MS and elemental analysis. The nematicidal and antibacterial activity of the compounds has also been evaluated.

Nematicidal Substances from Fungi

This review summarizes the 179 compounds from fungi that have shown to possess nematicidal activities. These compounds belong to diverse chemical groups and they are mainly isolated from a variety of deuteromycete, ascomycete and basidiomycete fungi. Some of them have been patented as nematicidal agents. We review and classify these compounds based on their structural types. Their nematicidal activities are described and their potential roles in natural environments and in the biocontrol of nematodes are discussed.

Proteases from Bacillus: a new insight into the mechanism of action for rhizobacterial suppression of nematode populations

The aim of this study was to investigate the role of proteases in Bacillus spp. of rhizobacteria in suppressing nematode populations and to understand their mechanism of action. Rhizobacteria with nematicidal activity were isolated from soil samples of five root knot nematode-infested farms. Among these strains, nematotoxicities of Bacillus strains were intensively analysed. Further assays of nematicidal toxins from Bacillus sp. strain RH219 indicated an extracellular cuticle-degrading protease Apr219 was an important pathogenic factor. The Apr219 shared high similarity with previously reported cuticle-degrading proteases from Brevibacillus laterosporus strain G4 and Bacillus sp. B16 (Bacillus nematocida). The cuticle-degrading protease genes were also amplified from four other nematicidal Bacillus strains isolated from the rhizosphere. In addition to Apr219, a neutral protease Npr219 from Bacillus sp. RH219 was also investigated for activity against nematodes. The wide distribution of cuticle-degrading proteases in Bacillus strains with nematicidal activity suggested that these enzymes likely play an important role in bacteria-nematode-plant-environment interactions and that they may serve as important nematicidal factors in balancing nematode populations in the soil. Increased understanding of the mechanism of action of Bacillus spp. against nematodes could potentially enhance the value of these species as effective nematicidal agents and develop new biological control strategies.

Pyrrolizidine alkaloids of Chromolaena odorata act as nematicidal agents and reduce infection of lettuce roots by Meloidogyne incognita

Abstract 1,2-dehydropyrrolizidine alkaloids (PAs) represent a class of secondary plant compounds that are active in defence against herbivory. They are present in Chromolaena odorata, one of the most invasive weeds of Asia and Africa. In vitro studies demonstrate that pure PAs from C. odorata roots have nematicidal effects on the root-knot nematode Meloidogyne incognita, even at concentrations of 70-350 ppm. In vivo experiments show that mulch or aqueous crude extracts from C. odorata roots reduce the infection of lettuce by M. incognita. Thus, the use of PA-containing plants appears to be a valuable element for integrated nematode management.