Nematicidal Metabolites Research Articles

Nematicidal activity of grammicin produced by Xylaria grammica KCTC 13121BP against Meloidogyne incognita.

The endolichenic fungus Xylaria grammica KCTC 13121BP showed strong nematicidal activity against Meloidogyne incognita. This study aimed to identify the nematicidal metabolites and to evaluate the efficacy of the strain as a biocontrol agent under pot and field conditions. Bioassay-guided fractionation and instrumental analyses led to grammicin being identified as the nematicidal metabolite. Because patulin is a mycotoxic isomer of grammicin and is known to have strong antibacterial and cytotoxic activities, several biological activities of the two compounds were compared. Grammicin showed strong second-stage juvenile killing and egg-hatching inhibitory effects, with a 50% effective concentration at 72 h (EC50/72 h ) of 15.9 µg/mL and a 50% effective concentration at 14 days (EC50/14 days ) of 5.87 µg/mL, respectively, whereas patulin was virtually inactive in both respects. Patulin was strongly active toward various phytopathogenic bacteria in vitro, whereas grammicin was weakly so. Patulin at the concentration range of 0.1-10 µg/mL also showed dose-dependent cytotoxicity toward the human first-trimester trophoblast cell line SW.71, whereas grammicin was not toxic toward this cell line. In pot and field experiments, a wettable powder-type formulation and fermentation broth filtrate of X. grammica KCTC 13121BP effectively suppressed the development of root-knot nematode disease on tomato and melon plants. The results suggest that X. grammica and grammicin may have potential applications for control of root-knot nematode disease of various crops. © 2017 Society of Chemical Industry.

The Edible Mushroom Pleurotus djamor Produces Metabolites with Lethal Activity Against the Parasitic Nematode Haemonchus contortus.

Pleurotus djamor is an edible mushroom that has medicinal properties. This study aimed to assess the in vitro activity of P. djamor extracts and fractions against Haemonchus contortus eggs and exsheathed infective larvae (L3). Crude hydroalcoholic extracts were obtained by maceration and fractions were obtained through chromatography. Metabolite identity was determined using gas chromatography/mass spectrometry (GC-MS) analysis. The results showed that P. djamor extracts had no significant activity against eggs at the concentrations used. However, the extract showed 98.7% and 77% larval mortality 72 h postconfrontation at 320 and at 160 mg/mL, respectively. The chromatography analysis resulted in 23 fractions that were eventually grouped into three fractions (E1, E2, and E3). These fractions showed the following egg hatching inhibition percentages: E1 = 100, E2 = 38.7, and E3 = 5.5 at 10 mg/mL concentration 72 h postexposure. Likewise, larval mortality percentages after this period were 90.6, 100, and 0.44 at 40 mg/mL (P < .05), respectively. The GC-MS showed five major compounds in E1 fraction, including four fatty acids: (i) pentadecanoic, (ii) hexadecanoic, (iii) octadecadienoic, (iv) octadecanoic acid, and one terpene identified as β-sitosterol. We concluded that the edible mushroom P. djamor possesses nematicidal metabolites, which could be used as an alternative anthelmintic treatment.

Identification of 2-methylbutyric Acid as a Nematicidal Metabolite, and Biocontrol and Biofertilization Potentials of Bacillus pumilus L1

The present study described the isolation of 2-methylbutyric acid (2-MBA) produced from Bacillus pumilus L1, to subsequently investigate its nematicidal activity for the control of the root-knot nematode. The results showed that 2-MBA could be purified by chromatographic techniques and was identified using nuclear magnetic resonance and liquid chromatography-mass spectrometry. Crude extract and partially purified compounds had a significant effect on the inhibition of egg hatchability and second-stage juvenile (J2) mortality. A dose-dependent effect of 2-MBA was observed for J2 mortality and egg hatchability. Egg hatchability was 69.2%, 59.9%, 32.7%, and 0.0% at 125, 250, 500, and 1000 μg mL -1 of 2-MBA after 4 d of incubation, respectively. Meanwhile, J2 mortality was in the range of 24.4%-100.0% after 2 d of incubation, depending on the concentrations of 2-MBA used. A pot experiment also demonstrated that treatment of B. pumilus L1 culture caused a significant reduction in the number of galls, egg masses, and J2 population than that of the tap water (TW) control. However, as the B. pumilus L1 culture concentration was decreased, the efficacy of nematode control by treatment of B. pumilus L1 culture was reduced compared to that of TW. B. pumilus L1 inoculation at different concentrations also promoted cucumber plant growth. Therefore, our study demonstrated the potential of 2-MBA from B. pumilus L1 as a biocontrol agent against the root-knot nematode and a plant growth promoter for cucumber plants.

Nematicidal Activity of Kojic Acid Produced by Aspergillus oryzae against Meloidogyne incognita.

The fungal strain EML-DML3PNa1 isolated from leaf of white dogwood (Cornus alba L.) showed strong nematicidal activity with juvenile mortality of 87.6% at a concentration of 20% fermentation broth filtrate at 3 days after treatment. The active fungal strain was identified as Aspergillus oryzae, which belongs to section Flavi, based on the morphological characteristics and sequence analysis of the ITS rDNA, calmodulin (CaM), and β-tubulin (BenA) genes. The strain reduced the pH value to 5.62 after 7 days of incubation. Organic acid analysis revealed the presence of citric acid (515.0 mg/kg), malic acid (506.6 mg/kg), and fumaric acid (21.7 mg/kg). The three organic acids showed moderate nematicidal activities, but the mixture of citric acid, malic acid, and fumaric acid did not exhibit the full nematicidal activity of the culture filtrate of EML- DML3PNa1. Bioassay-guided fractionation coupled with (1)H- and (13)C-NMR and EI-MS analyses led to identification of kojic acid as the major nematicidal metabolite. Kojic acid exhibited dose-dependent mortality and inhibited the hatchability of M. incognita, showing EC50 values of 195.2 µg/ml and 238.3 µg/ml, respectively, at 72 h postexposure. These results suggest that A. oryzae EML-DML3PNa1 and kojic acid have potential as a biological control agent against M. incognita.

Nematicidal metabolites from Gliocladium roseum YMF1.00133

A strain of the fungus Gliocladium roseum YMF1.00133 was found to secrete nematicidal metabolites against nematodes Panagrellus redivivus, Caenothabditis elegans and Bursaphelenchus xylophilus in experiments searching for nematicidal fungi. Through bioassay-guided fractionations, a unique trioxopiperazine alkaloid, gliocladin C (compound 1), and an alkylane resorcinol, 5-n-heneicosylresorcinol (compound 2) were obtained from the methanol extract of the fungus and determined by single-crystal X-ray analysis and spectroscopic data. In vitro immersion experiments showed that the ED50 values of compounds 1 and 2 after 24 h incubation were 15 and 30 μg/mL against C. elegans, 50 and 80 μg/mL against P. redivivus, and 200 and 180 μg/mL against B. xylophilus, respectively. The X-ray diffraction data of compound 1 and the nematicidal activity of compounds 1 and 2 were reported for the first time.

Metabolites from nematophagous fungi and nematicidal natural products from fungi as alternatives for biological control. Part II: metabolites from nematophagous basidiomycetes and non-nematophagous fungi.

In this second section of a two-part mini-review article, we introduce 101 further nematicidal and non-nematicidal secondary metabolites biosynthesized by nematophagous basidiomycetes or non-nematophagous ascomycetes and basidiomycetes. Several of these compounds have promising nematicidal activity and deserve further and more detailed analysis. Thermolides A and B, omphalotins, ophiobolins, bursaphelocides A and B, illinitone A, pseudohalonectrins A and B, dichomitin B, and caryopsomycins A–C are excellent candidates or lead compounds for the development of biocontrol strategies for phytopathogenic nematodes. Paraherquamides, clonostachydiol, and nafuredins offer promising leads for the development of formulations against the intestinal nematodes of ruminants.

Nematicidal metabolites from roots of Stellera chamaejasme against Bursaphelenchus xylophilus and Bursaphelenchus mucronatus

Bursaphelenchus xylophilus, the causal agent of pine wilt disease, severely damages pine forests, and Bursaphelenchus mucronatus, a species related to B. xylophilus, is weakly pathogenic. Because the use of synthetic pesticides to control pine wilt disease has raised concerns for human and environmental health, a search for useful botanically based compounds is needed. The ethyl acetate layer from an ethanol extract of Stellera chamaejasme L. roots showed significant nematicidal activity against B. xylophilus and B. mucronatus, with LC50 values of 169.7 and 37.7 µg mL(-1), respectively, at 72 h. Further purification of the active layer yielded eight effective nematicidal compounds, which were identified by mass and nuclear magnetic resonance spectral data. Among the eight bioactive compounds, chamaejasmenin C induced the highest mortality against B. xylophilus, with an LC50 value of 2.7 µM at 72 h, and chamaechromone exhibited strong nematicidal activity against B. mucronatus, with an LC50 value of 0.003 µM at 72 h. The nematicidal metabolites from S. chamaejasme roots could be used as lead compounds for developing botanically based nematicides for the management of pine wilt disease.

Isolation of an Acremonium sp. from a screening of 52 seawater fungal isolates and preliminary characterization of its growth conditions and nematicidal activity

Fifty-two fungal isolates obtained from seawater were screened for anti-nematode activity. One isolate, an Acremonium sp., exhibited the highest activity against the pinewood nematode, Bursaphelenchus xylophilus. When it was grown at pH 7.0 and 24-26 °C, nematicidal metabolites were produced. These were heat-stable, water-soluble molecules with MWs <6 kDa. Nematicidal activity was highest at neutral pH.

In vivo efficacy of the anthelmintic tribendimidine against the cestode Hymenolepis microstoma in a controlled laboratory trial

Tribendimidine has been registered for the treatment of human soil transmitted helminthiases in China. In the model nematode Caenorhabditis elegans it is an agonist of L-subtype nicotinic acetylcholine receptors and therefore shares its mode of action with levamisole and pyrantel. Besides its broad spectrum of nematicidal efficacy, tribendimidine is efficacious against several trematodes and has been attributed to have anti-cestodal effects. However, there are few published data available for the latter. The efficacy of tribendimidine and its nematicidal metabolite deacylated amidantel against Hymenolepis microstoma were examined for their anti-cestodal potential. Doses of 50 and 100mg/kg body weight deacylated amidantel and 10, 25, 50, and 100mg/kg tribendimidine were administered orally on three consecutive days to mice experimentally infected with eight cysticercoids. Necropsy was performed and the worm burdens were determined one day after the last treatment. Furthermore, levamisole was used in combination with tribendimidine (100mg/kg levamisole plus 10 and 25mg/kg tribendimidine, respectively) and alone (50 and 100mg/kg) to investigate any possible interactions of the partner compounds against cestodes. Tribendimidine showed a very high efficacy at dosages of 50mg/kg or higher. Surprisingly, deacylated amidantel led to no reduction of the worm burden in any of the treatments. Combinations of levamisole with tribendimidine did not augment the effects of tribendimidine alone and as expected levamisole alone also showed no anti-cestodal activity. To our knowledge, this study shows for the first time activity of tribendimidine against a cestode in a controlled laboratory study. Due to the excellent cure rates observed here, multiple tribendimidine treatments might be considered as useful scheme for treatments of cestode, nematode and trematode infections although this would significantly increase both costs and management efforts. Moreover, the differences between tribendimidine and deacylated amidantel indicate at least a strong difference in sensitivity of H. microstoma or a strong difference in drug availability.

English

In the present investigation, antagonistic effect of culture filtrates of&nbsp;Gymnoascus reesii&nbsp;at different concentrations were tested on eggs and juveniles of root knot nematode&nbsp;(Meloidogyne incoginta),&nbsp;invitro. The percentage mortality and inhibition of hatching of root-knot nematode were directly proportional to the concentration of culture filtrates of&nbsp;G. reesii. Compared with the nematicides treatments of Cadusafos and Avermectins, the effects of higher concentration of culture filtrates of&nbsp;G. reesii&nbsp;were in some cases not statistically different. To our knowledge, this is the first report of culture filtrates of&nbsp;G. reesii&nbsp;observed to have nematicidal activity toward root-knot nematode and a&nbsp; nematicidal metabolite (3E,5E)-2,5-dihydroxy-2,7-dihydrooxepine-3-carboxylic anhydride was isolated based on bioassay-guided fractionation from the extracts of the fungus&nbsp;G. reessii. &nbsp; Key words:&nbsp;Gymnoascus reesii,&nbsp;nematicidal activity,&nbsp;Meloidogyne incoginta.

Nematicidal endophytic bacteria obtained from plants

Two hundred and six bacterial isolates were obtained from leaf, flower and stem of three healthy plants,Euphorbia pulcherrima Willd,Pyrethrum cinerariifolium Trev. andHeracleum candicans Wall. The nematicidal activity experiment showed that a total of 92 isolates displayed activity againstCaenorhabditis elegans (Maupas) Dougherty, and 70 isolates resulted active againstBursaphelenchus xylophilus (Steiner & Buhrer) Nickle. Strain LCB-3 exhibited the strongest nematicidal activity against both two nematodes. According to the 16S rDNA, the strain LCB-3 was identified asBrevundimonas diminuta. Based on bioassay-guided fractionation, a nematicidal metabolite (R)-(−)-2-ethylhexan-1-ol was obtained from LCB-3. The median lethal concentrations (LC50) of the compound were 542.0 mg l−1 againstC. elegans and 168.1 mg l−1 againstB. xylophilus 48 h after treatment.

Biologically active secondary metabolites from the ascomycete A111-95. 2. Structure elucidation.

Eight secondary metabolites were isolated from submerged cultures of the ascomycete A111-95 during a search for new nematicidal metabolites. (-)-Galiellalactone (7) and compound 2 are metabolites previously obtained from cultures of Galiella rufa while the compounds 1, 3, 4, 5, 6 and 8 (3 and 4 were obtained as an unseparable mixture), were isolated as natural products for the first time. Compound 2, pregaliellalactone (5) and the mixture of 3 and 4 showed nematicidal activities towards Caenorhabditis elegans and Meloidogyne incognita. All compounds showed moderate or weak cytotoxic activities.

Biologically active secondary metabolites from the ascomycete A111-95. 1. Production, isolation and biological activities.

Eight secondary metabolites were isolated from submerged cultures of the ascomycete A111-95 during a search for new nematicidal metabolites. (-)-Galiellalactone (7) and compound 2 are metabolites previously obtained from cultures of Galiella rufa while the compounds 1, 3, 4, 5, 6 and 8 (3 and 4 were obtained as an unseparable mixture), were isolated as natural products for the first time. Compound 2, pregaliellalactone (5) and the mixture of 3 and 4 showed nematicidal activities towards Caenorhabditis elegans and Meloidogyne incognita. All compounds showed moderate or weak cytotoxic activities.

Nematicidal Metabolites From Higher Fungi

The adverse effects on human health and well being and the high economic losses inflicted by pathogenic nematodes are important stimulants for the search of new, safe and effective nematicides and anthelminthics. The pool of natural products is an important source for such compounds, and several of the most potent nematicides used today originate from streptomycetes. Higher fungi have an extensive secondary metabolism at their disposal, and obviously some benefit from the capability to kill nematodes. Surprisingly little is known about the production of nematicidal metabolites by fungi. Examples of compounds isolated from ascomycetes are the cochlioquinones and the mycorrhizins, which are strongly active against the saprophytic Caenorhabditis elegans but not towards the economically important plant pathogen Meloidogyne incognita. From basidiomycetes, the cyclic dodecapeptide omphalotin was obtained which seems to be highly selective for M. incognita. Compounds with good activity against a abomasum nematode in vivo are the macrodiolides clonostachydiol and helminidiol. When screening for nematicidal activity, it is important to use the proper test system as the sensitivity of different nematodes may vary considerably. Consequently, the search for agents active against M. incognita may be futile, if saprophytic nematodes are used as test organisms. The importance to develop relevant screening systems that are easy to use and have an acceptable capacity is therefore emphasized.