Nematotoxicity of Paeonia spp. Extracts and Camellia oleifera Tea Seed Cake and Extracts to Heterodera glycines and Meloidogyne incognita.

Tea-oil camellia (Camellia oleifera) is grown for tea seed oil production, with tea seed cake produced as a byproduct. Rather than disposing of the cake, agricultural uses increase the value of oil production. Constituents of C. oleifera are also utilized for traditional Chinese medicine, as are compounds produced by tree peony roots. Consequently, the unused C. oleifera cake, and stems from two tree peony species, Paeonia rockii and Paeonia suffruticosa, were studied for compounds antagonistic to soybean cyst nematode (Heterodera glycines) and root-knot nematode (Meloidogyne incognita). Extracts from C. oleifera cake and P. rockii stems suppressed hatch and were nematotoxic to second-stage juveniles (J2) of both nematode species. P. rockii extracts were more effective than P. suffruticosa extracts for decreasing M. incognita hatch and J2 viability. In greenhouse trials with soybean (Glycine max 'Essex'), powdered C. oleifera cake applied as a soil amendment suppressed H. glycines cysts/g root by up to 66% compared with nonamended controls. These results indicate that the extracts and cake contain compounds active against H. glycines and M. incognita, with activity varying between the two Paeonia species. C. oleifera tea seed cake, and constituents of the cake or of P. rockii, are candidates for further studies on management of these nematodes.

List of Type Specimens Deposited Since 1998 in the United States Department of Agriculture Nematode Collection, Beltsville, Maryland.

The United States Department of Agriculture Nematode Collection (USDANC) is one of the largest and most valuable in existence and includes millions of specimens housed in over 39,800 permanent slides and 9,300 vials. This collection preserves type specimens of nematodes to serve as a reference for identifications and future taxonomic revisions. Also, the collection provides useful information on nematode hosts, occurrence, and distribution. The present list includes type specimens added to the USDANC since 1998. Since that time, the collection has expanded, with 474 type species mounted and preserved on 2,564 glass slides and 180 vials. We encourage nematologists throughout the world to deposit type specimens in the USDANC for use by future generations.

Activity of Vetiver Extracts and Essential Oil against Meloidogyne incognita.

Vetiver, a nonhost grass for certain nematodes, was studied for the production of compounds active against the southern root-knot nematode, Meloidogyne incognita . In laboratory assays studying the effects on second-stage juvenile (J2) activity and viability, crude vetiver root and shoot extracts were nematotoxic, resulting in 40% to 70% J2 mortality, and were also repellent to J2. Vetiver oil did not exhibit activity against J2 in these assays. Gas chromatography-mass spectrometry analyses of three crude vetiver root ethanol extracts and a commercial vetiver oil determined that two of the major components in each sample were the sesquiterpene acid 3,3,8,8-tetramethyltricyclo[5.1.0.0(2,4)]oct-5-ene-5-propanoic acid and the sesquiterpene alcohol 6-isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-2-ol. The acid was present in higher amounts in the extracts than in the oil. These studies demonstrating nematotoxicity and repellency of vetiver-derived compounds to M. incognita suggest that plant chemistry plays a role in the nonhost status of vetiver to root-knot nematodes, and that the chemical constituents of vetiver may be useful for suppressing nematode populations in the soil.

Expression of a cystatin transgene can confer resistance to root lesion nematodes in Lilium longiflorum cv. 'Nellie White'.

Lilium longiflorum cv. 'Nellie White' assumes a great economic importance as cut flowers, being one of the most valuable species (annual pot plants value above $20,000,000) in terms of wholesales in the US. The root lesion nematode Pratylenchus penetrans (RLN) constitutes one of the main pests for lily producers due to the significant root damage it causes. Our efforts have focused on the generation of soybean hairy roots (as a transient test model) and stable transgenic lilies overexpressing a modified rice cystatin (Oc-IΔD86) transgene and challenged with root lesion nematodes. Lily transformation was achieved by gene gun co-bombardment using both a pBluescript-based vector containing the cystatin gene and pDM307 that contains a bar gene for phosphinothricin selection. Both soybean hairy roots and lilies overexpressing the OcIΔD86 transgene exhibited enhanced resistance to RLN infection by means of nematode reduction up to 75 ± 5% on the total number of nematodes. In addition, lily plants overexpressing OcIΔD86 displayed an increase of plant mass and better growth performance in comparison to wild-type plants, thereby demonstrating an alternative strategy for increasing the yield and reducing nematode damage to this important floral crop.

A potential biochemical mechanism underlying the influence of sterol deprivation stress on Caenorhabditis elegans longevity.

To investigate the biochemical mechanism underlying the effect of sterol deprivation on longevity in Caenorhabditis elegans, we treated parent worms (P0) with 25-azacoprostane (Aza), which inhibits sitosterol-to-cholesterol conversion, and measured mean lifespan (MLS) in F2 worms. At 25 µM (∼EC(50)), Aza reduced total body sterol by 82.5%, confirming sterol depletion. Aza (25 µM) treatment of wild-type (N2) C. elegans grown in sitosterol (5 µg/ml) reduced MLS by 35%. Similar results were obtained for the stress-related mutants daf-16(mu86) and gas-1(fc21). Unexpectedly, Aza had essentially no effect on MLS in the stress-resistant daf-2(e1370) or mitochondrial complex II mutant mev-1(kn1) strains, indicating that Aza may target both insulin/IGF-1 signaling (IIS) and mitochondrial complex II. Aza increased reactive oxygen species (ROS) levels 2.7-fold in N2 worms, but did not affect ROS production by mev-1(kn1), suggesting a direct link between Aza treatment and mitochondrial ROS production. Moreover, expression of the stress-response transcription factor SKN-1 was decreased in amphid neurons by Aza and that of DAF-28 was increased when DAF-6 was involved, contributing to lifespan reduction.

Description of Globodera ellingtonae n. sp. (Nematoda: Heteroderidae) from Oregon.

A new species of cyst nematode, Globodera ellingtonae, is described from soil collected from a field in Oregon. Second-stage juveniles (J2) of the species are characterized by body length of 365-515 µm, stylet length of 19-22.5 µm, basal knobs rounded posteriorly and pointed anteriorly, tail 39-55 µm, hyaline tail terminus 20-32.5 µm, and tail tapering uniformly but abruptly narrowing and constricted near the posterior third of the hyaline portion, ending with a peg-like, finely rounded to pointed terminus. Cysts are spherical to sub-spherical, dark to light brown and circumfenestrate and cyst wall pattern is ridge-like with heavy punctations. Males have a stylet length of 21-25 µm and spicule length of 30-37 µm with a pointed thorn-like tip. Females have a stylet length of 20-22.5 µm, one head annule and labial disc, heavy punctations on the cuticle, and short vulval slit 7.5-8 µm long. Morphologically this new, round-cyst species differs from the related species G. pallida, G. rostochiensis, G. tabacum complex and G. mexicana by its distinctive J2 tail, and by one or another of the following: shorter mean stylet length in J2, females and males; number of refractive bodies in the hyaline tail terminus of J2; cyst morphology including Granek's ratio; number of cuticular ridges between the anus and vulva; and in the shape and length of spicules in males. Its relationship to these closely related species are discussed. Based upon analysis of ribosomal internal transcribed spacer (ITS) sequences, G. ellingtonae n. sp. is distinct from G. pallida, G. rostochiensis, G. tabacum and G. mexicana. Bayesian and Maximum Parsimony analysis of cloned ITS rRNA gene sequences indicated three clades, with intraspecific variability as high as 2.8%. In silico analysis revealed ITS restriction fragment length polymorphisms for enzymes Bsh 1236I, Hinf I, and Rsa I that overlap patterns for other Globodera species.

Poly(T) variation in heteroderid nematode mitochondrial genomes is predominantly an artefact of amplification.

We assessed the rate of in vitro polymerase errors at polythymidine [poly(T)] tracts in the mitochondrial DNA (mtDNA) of a heteroderid nematode (Heterodera cajani). The mtDNA of these nematodes contain unusually high numbers of poly(T) tracts, and have previously been suggested to contain biological poly(T) length variation. However, using a cloned molecule, we observed that poly(T) variation was generated in vitro at regions containing more than six consecutive Ts. This artefactual error rate was estimated at 7.3 × 10(-5) indels/poly(T) tract >6 Ts/cycle. This rate was then compared to the rate of poly(T) variation detected after the amplification of a biological sample, in order to estimate the 'biological + artefactual' rate of poly(T) variation. There was no significant difference between the artefactual and the artefactual + biological rates, suggesting that the majority of poly(T) variation in the biological sample was artefactual. We then examined the generation of poly(T) variation in a range of templates with tracts up to 16 Ts long, utilizing a range of Heteroderidae species. We observed that T deletions occurred five times more frequently than insertions, and a trend towards increasing error rates with increasing poly(T) tract length. These findings have significant implications for studies involving genomes with many homopolymer tracts.

The mitochondrial genome of the soybean cyst nematode, Heterodera glycines.

We sequenced the entire coding region of the mitochondrial genome of Heterodera glycines. The sequence obtained comprised 14.9 kb, with PCR evidence indicating that the entire genome comprised a single, circular molecule of approximately 21-22 kb. The genome is the most T-rich nematode mitochondrial genome reported to date, with T representing over half of all nucleotides on the coding strand. The genome also contains the highest number of poly(T) tracts so far reported (to our knowledge), with 60 poly(T) tracts ≥ 12 Ts. All genes are transcribed from the same mitochondrial strand. The organization of the mitochondrial genome of H. glycines shows a number of similarities compared with Radopholus similis, but fewer similarities when compared with Meloidogyne javanica. Very few gene boundaries are shared with Globodera pallida or Globodera rostochiensis. Partial mitochondrial genome sequences were also obtained for Heterodera cardiolata (5.3 kb) and Punctodera chalcoensis (6.8 kb), and these had identical organizations compared with H. glycines. We found PCR evidence of a minicircular mitochondrial genome in P. chalcoensis, but at low levels and lacking a noncoding region. Such circularised genome fragments may be present at low levels in a range of nematodes, with multipartite mitochondrial genomes representing a shift to a condition in which these subgenomic circles predominate.

Caenorhabditis elegans utilizes dauer pheromone biosynthesis to dispose of toxic peroxisomal fatty acids for cellular homoeostasis.

Caenorhabditis elegans excretes a dauer pheromone or daumone composed of ascarylose and a fatty acid side chain, the perception of which enables worms to enter the dauer state for long-term survival in an adverse environment. During the course of elucidation of the daumone biosynthetic pathway in which DHS-28 and DAF-22 are involved in peroxisomal beta-oxidation of VLCFAs (very long-chain fatty acids), we sought to investigate the physiological consequences of a deficiency in daumone biosynthesis in C. elegans. Our results revealed that two mutants, dhs-28(tm2581) and daf-22(ok693), lacked daumones and thus were dauer defective; this coincided with massive accumulation of fatty acyl-CoAs (up to 100-fold) inside worm bodies compared with levels in wild-type N2 worms. Furthermore, the deficiency in daumone biosynthesis and the massive accumulation of fatty acids and their acyl-CoAs caused severe developmental defects with reduced life spans (up to 30%), suggesting that daumone biosynthesis is be an essential part of C. elegans homoeostasis, affecting survival and maintenance of optimal physiological conditions by metabolizing some of the toxic non-permissible peroxisomal VLCFAs from the worm body in the form of readily excretable daumones.

Effects of sterols on the development and aging of Caenorhabditis elegans.

Although Caenorhabditis elegans lacks several components of the de novo sterol biosynthetic pathway, it requires sterols as essential nutrients. Supplemental cholesterol undergoes extensive enzymatic modification in C. elegans to form certain sterols of unknown function. Since sterol metabolism in C. elegans differs from that in other species, such as mammals and yeast, it is important to examine how sterols regulate worm physiology. To examine the functions of sterols in C. elegans, a sterol-feeding experiment was carried out and several critical parameters, such as brood size, growth rate, and life span, were measured. In addition, the change in lipid distribution in C. elegans can be both qualitatively and quantitatively determined by various methods, including staining and chromatographic techniques. Taken together, the effects of sterols on C. elegans are very prominent and can be easily assessed using the techniques described here.

Proteomic analysis of the sterol-mediated signaling pathway in Caenorhabditis elegans.

Since Caenorhabditis elegans is incapable of de novo cholesterol biosynthesis, it must utilize other nonpermissive sterols that are present in the environment by converting them into cholesterol for cellular function. The inhibition of sterol conversion to cholesterol in C. elegans by various sterol biosynthesis inhibitors (SBIs) is known to cause serious defects in the development of these worms. To determine the biochemical consequences of these physiological abnormalities, one can perform a proteomic analysis of worms of a certain stage that are grown in the presence of SBIs in order for the differential expression of proteins involved in the sterol-mediated signaling pathway to be identified. For example, reductions in the expression of lipoprotein family members, such as vitellogenin-2 and vitellogenin-6, are prominent in azacoprostane-treated worms. This phenomenon is also seen in worms treated with AY-9944, which blocks the conversion of 7-dehydrocholesterol, a major sterol present in C. elegans, to cholesterol.

Dose-response effects of clove oil from Syzygium aromaticum on the root-knot nematode Meloidogyne incognita.

BACKGROUND: Clove oil, derived from the plant Syzygium aromaticum (L.) Merr. & Perry, is active against various organisms, and was prepared in a soy lecithin/detergent formulation to determine concentrations active against the root-knot nematode Meloidogyne incognita (Kofoid and White) Chitwood. RESULTS: In microwell assays, the mean effective clove oil concentration that reduced egg hatch by 50% (EC(50)) was 0.097% (v/v) clove oil; the EC(50) for second-stage juvenile (J2) viability was 0.145% clove oil (compared with carrier control treatments). Volatiles from 5.0% clove oil reduced nematode egg hatch in water by 30%, and decreased viability of hatched J2 by as much as 100%. Reductions were not as large with nematodes in carrier. In soil trials with J2 recovered from Baermann funnels, the EC(50) = 0.192% clove oil (compared with water controls). CONCLUSION: The results demonstrated that the tested formulation is active against M. incognita eggs and J2, that the EC(50) values for J2 in the microwell studies and the soil recovery tests were similar to each other and that direct contact with the clove oil is needed for optimal management results with this natural product.

New Nematotoxic Indoloditerpenoid Produced by Gymnoascus reessii za-130.

Chemical investigation of the fungal strain Gymnoascus reessii za-130, which was previously isolated from the rhizosphere of tomato plants infected by the root-knot nematode Meloidogyne incognita, led to the isolation and identification of a new indoloditerpenoid metabolite designated gymnoascole acetate. Its structure was established by spectroscopic methods including 1D- and 2D-NMR and MS analyses. Gymnoascole acetate demonstrated strong adverse effects on M. incognita second-stage juvenile (J2) viability; exposure to 36 µg/mL for 24 h induced 100% paralysis of J2 (EC50 = 47.5 µg/mL). Gymnoascole acetate suppressed M. incognita egg hatch relative to controls by >90% at 133 µg/mL after 7 days of exposure. The numbers of root galls and J2 in both soil and roots were significantly reduced (p = 0.05) by treatment with 2-200 µg/mL gymnoascole acetate/kg soil, compared to untreated control plants; nematode suppression increased with gymnoascole acetate concentration. This study demonstrated the nematotoxicity of gymnoascole acetate and indicates that it might be a potential biobased component in integrated management of M. incognita.

Phytochemical based strategies for nematode control.

This review examines the discovery of naturally occurring phytochemicals antagonistic toward plant-parasitic and other nematodes. Higher plants have yielded a broad spectrum of active compounds, including polythienyls, isothiocyanates, glucosinolates, cyanogenic glycosides, polyacetylenes, alkaloids, lipids, terpenoids, sesquiterpenoids, diterpenoids, quassinoids, steroids, triterpenoids, simple and complex phenolics, and several other classes. Many other antinematodal compounds have been isolated from biocontrol and other fungi. Natural products active against mammalian parasites can serve as useful sources of compounds for examination of activity against plant parasites. The agricultural utilization of phytochemicals, although currently uneconomic in many situations, offers tremendous potential.

Plantago lanceolata and Plantago rugelii Extracts are Toxic to Meloidogyne incognita but not to Certain Microbes.

Extracts from the plants Plantago lanceolata and P. rugelii were evaluated for toxicity to the root-knot nematode Meloidogyne incognita, the beneficial microbes Enterobacter cloacae, Pseudomonas fluorescens and Trichoderma virens, and the plant-pathogenic fungi Fusarium oxysporum f. sp. gladioli, Phytophthora capsici, Pythium ultimum, and Rhizoctonia solani. Wild plants were collected, roots were excised from shoots, and the plant parts were dried and ground to a powder. One set of extracts (10% w/v) was prepared in water and another in methanol. Treatments included extract concentrations of 25%, 50%, 75% and 100%, and water controls. Meloidogyne incognita egg hatch was recorded after 7-day exposure to the treatments, and second-stage juvenile (J2) activity after 48 hours. All extracts were toxic to eggs and J2, with P. lanceolata shoot extract tending to have the most activity against M. incognita. Numbers of active J2 remained the same or decreased in a 24-hour water rinse following the 48-hour extract treatment, indicating that the extracts were lethal. When data from water- and methanol-extracted roots and shoots of both plant species were combined for analysis, J2 tended to be more sensitive than eggs to the toxic compounds at lower concentrations, while the higher concentrations (75% and 100%) were equally toxic to both life stages. The effective concentrations causing 50% reduction (EC(50)) in egg hatch and in J2 viability were 44.4% and 43.7%, respectively. No extract was toxic to any of the bacteria or fungi in our assays.

Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi.

Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in all stages. The highest expression level of Ha-far-1 was observed in fourth-stage juvenile (J4), whereas the highest expression level of Ha-far-2 occurred in second-stage juvenile (J2). In conclusion, we have identified two novel far genes from H. avenae and one from H. filipjevi and have provided further indication that nematode far genes are present in a variety of nematode species, where the FAR proteins share similar basic structure, expression pattern and biochemical activities.

Human proprotein convertase 2 homologue from a plant nematode: cloning, characterization, and comparison with other species.

Proprotein convertases (PCs) are evolutionarily conserved enzymes responsible for processing the precursors of many bioactive peptides in mammals. The invertebrate homologues of PC2 play important roles during development that makes the enzyme a good target for practical applications in pest management. Screening of a plant nematode Heterodera glycines cDNA library resulted in isolation of a full-length clone encoding a PC2-like precursor. The deduced protein (74.2 kD) exhibits strong amino acid homology to all known PC2s, including human, and shares the main structural characteristics: signal peptide; prosegment; catalytic domain, with D/H/S catalytic triad, PC2-specific residues, and 7B2 binding sites; P domain (with RRGDT pentapeptide); and carboxyl terminus. Comparative analysis of PC2s from 15 species discloses the presence of an insert in the catalytic domain unique to nematodes. Expression of PC2-like mRNA found in eggs and juveniles was undetectable in adult stages of H. glycines. Nucleotide analysis reveals distinctive differences in base composition and codon usage between H. glycines and Caenorhabditis elegans PC2s. The H. glycines cDNA clone encoding PC2 is the first one isolated from plant-parasitic nematodes.

Research on plant-parasitic nematode biology conducted by the United States Department of Agriculture-Agricultural Research Service.

The recent de-registration of several chemical nematicides and the impending loss of methyl bromide from the pest-control market necessitate the development of new methods for controlling nematode-induced crop damage. One approach for developing novel target-specific controls is by exploiting fundamental differences between the biological processes of nematodes and their host plants. Researchers of the Agricultural Research Service (ARS) of the US Department of Agriculture are actively exploring these differences. Research accomplishments include the discovery of heat shock protein genes possibly involved in developmental arrest of the soybean cyst nematode, the identification of neuropeptides and female-specific proteins in the soybean cyst nematode, the disruption of nematode reproduction with inhibitors of nematode sterol metabolism, the development of novel morphological and molecular (heat shock protein genes and the D3 segment of large subunit ribosomal DNA) features useful for nematode identification and classification, and the elucidation of the population genetics of potato cyst nematode pathotypes. In addition, several ARS researchers are investigating biological determinants of nematode response to management strategies utilized in agricultural fields. These collective efforts should lead to new chemical and non-chemical alternatives to conventional nematode control strategies.

Nematotoxicity of drupacine and a Cephalotaxus alkaloid preparation against the plant-parasitic nematodes Meloidogyne incognita and Bursaphelenchus xylophilus.

BACKGROUND: Species of Cephalotaxus (the plum yews) produce nematotoxic compounds of unknown identity. Consequently, bioassay-guided fractionation was employed to identify the compound(s) in Cephalotaxus fortunei twigs and leaves with activity against plant-parasitic nematodes. RESULTS: A crude alkaloid extract, particularly drupacine, was responsible for much of the nematotoxicity. The ED50 of drupacine for Bursaphelenchus xylophilus was 27.1 µg mL⁻¹, and for Meloidogyne incognita it was 76.3 µg mL⁻¹. Immersion of M. incognita eggs in 1.0 mg mL⁻¹ crude alkaloid extract (the highest tested concentration) reduced hatch by 36%; immersion of second-stage juveniles (J2) resulted in 72-98% immobility. Crude alkaloid extract and drupacine suppressed protease activity in extracts of the microbivorous nematode Panagrellus redivivus by 50% and 80%, respectively. Application of 0.02-0.5 mg mL⁻¹ crude alkaloid extract to soil with M. incognita inoculum did not significantly reduce pepper plant shoot length or weight, compared with nematode-inoculated, water-treated controls, but the number of eggs and J2 per root system respectively decreased by 69% and 73% at 0.5 mg mL⁻¹. CONCLUSION: Drupacine and a crude alkaloid extract suppress nematode hatch, activity of mixed life stages, and population numbers on plant roots. This is the first demonstration of nematotoxicity of crude Cephalotaxus alkaloids and drupacine.