Plant-parasitic nematodes (PPNs) are important pests that cause an estimated ten billion dollars of crop loss each year in the United States and over 100 billion dollars globally. The Animal and Plant Health Inspection Service (APHIS) within the U.S. Department of Agriculture maintains and updates the U.S. Regulated Plant Pest list. Currently, the number of PPNs regulated by APHIS includes more than 60 different species. This review focuses on the top ten most economically important regulated and emerging plant-parasitic nematodes and summarizes the diagnostics of morphological and some molecular features for distinguishing them. These ten major previously described nematode species are associated with various economically important crops from around the world. This review also includes their current distribution in the U.S. and a brief historical background and updated systematic position of these species. The species included in this review include three PPNs considered by the U.S. Department of Agriculture as invasive invertebrates Globodera pallida, Globodera rostochiensis, and Heterodera glycines; four regulated PPNs, namely Bursaphelenchus xylophilus, Meloidogyne fallax, Ditylenchus dipsaci, and Pratylenchus fallax; and the three emerging PPNs Meloidogyne chitwoodi, Meloidogyne enterolobii, and Litylenchus crenatae mccannii.
Phylogenetic characters for Heteroderinae Luc. et al., 1988 are evaluated in Meloidodera which is believed to have primarily ancestral characters. Phasmid ultrastructure is observed in second-stage juveniles (J2), third-stage juvenile males, fourth-stage juvenile males, and fifth-stage males of Meloidodera floridensis and M. charis. Phasmid secretion occurs inside the egg before the J1-J2 molt. Before J2 hatch, concentric lamellar membranes occur within the sheath and socket cells. Some membranes become lamellae of the sheath cell plasma membrane; others become multilamellar bodies. During early molting, plasma membrane lamellae disappear and a distal dendrite segment appears in a rudimentary canal. After the molt, the distal dendrite is not present within the canal. The phylogenetic utility of phasmid features is discussed. In both species the ampulla shape and size between molts are stable features in juveniles and males. The posthatch J2 sheath cell receptor cavity may vary in a species specific manner, but comparative morphology requires precise timing after hatch.
The 18S small subunit (SSU) ribosomal DNA sequence is one of the most useful molecular loci for identification and phylogeny reconstruction of agriculturally important nematodes. Various pairs of universal primers have been developed in the past to amplify short and long nematode sequences. However, certain nematode taxa were not readily amplified and/or sequenced with the existing primer tools. Frequently, the center region of a roughly 1,000 nucleotide segment would be lost. Therefore new primers were developed based on a very large 276 taxon alignment of 124 agriculturally important nematode species, and tested on problematic nematode taxa such as Aphelenchoides, Bursaphelenchus, Ditylenchus, and Panagrolaimus. New primers and protocols are provided for successful generation of sequences useful in future investigations of nematode systematics.The 18S small subunit (SSU) ribosomal DNA sequence is one of the most useful molecular loci for identification and phylogeny reconstruction of agriculturally important nematodes. Various pairs of universal primers have been developed in the past to amplify short and long nematode sequences. However, certain nematode taxa were not readily amplified and/or sequenced with the existing primer tools. Frequently, the center region of a roughly 1,000 nucleotide segment would be lost. Therefore new primers were developed based on a very large 276 taxon alignment of 124 agriculturally important nematode species, and tested on problematic nematode taxa such as Aphelenchoides, Bursaphelenchus, Ditylenchus, and Panagrolaimus. New primers and protocols are provided for successful generation of sequences useful in future investigations of nematode systematics.
The invariant development of free-living nematodes combined with the extensive knowledge of Caenorhabditis elegans developmental biology provides an experimental system for an analysis of the evolution of developmental mechanisms. We have collected a number of new nematode species from soil samples. Most are easily cultured and their development can be analyzed at the level of individual cells using techniques standard to Caenorhabditis. So far, we have focused on differences in the development of the vulva among species of the families Rhabditidae and Panagrolaimidae. Preceding vulval development, twelve Pn cells migrate into the ventral cord and divide to produce posterior daughters [Pn.p cells] whose fates vary in a position specific manner [from P1.p anterior to P12.p posterior]. In C. elegans hermaphrodites, P(3-8).p are tripotent and form an equivalence group. These cells can express either of two vulval fates (1 degree or 2 degrees) in response to a signal from the anchor cell of the somatic gonad, or a nonvulval fate (3 degrees), resulting in a 3 degrees-3 degrees-2 degrees-1 degree-2 degrees-3 degrees pattern of cell fates. Evolutionary differences in vulval development include the number of cells in the vulval equivalence group, the number of 1 degree cells, the number of progeny generated by each vulval precursor cell, and the position of VPCs before morphogenesis. Examples of three Rhabditidae genera have a posterior vulva in the position of P9-P11 ectoblasts. In Cruznema tripartitum, P(5-7).p form the vulva as in Caenorhabditis, but they migrate posteriorly before dividing. Induction occurs after the gonad grows posteriorly to the position of P(5-7).p cells. In two other species, Mesorhabditis sp. PS 1179 and Teratorhabditis palmarum, we have found changes in induction and competence with respect to their presumably more C. elegans-like ancestor. In Mesorhabditis, P(5-7).p form the vulva after migrating to a posterior position. However, the gonad is not required to specify the pattern of cell fates 3 degrees-2 degrees-1 degree-2 degrees-3 degrees. Moreover, the Pn.p cells are not equivalent in their potentials to form the vulva. A regulatory constraint in this family thus forces the same set of precursors to generate the vulva, rather than more appropriately positioned Pn.p cells.
Application of Burkholderia cepacia and Trichoderma virens , alone and in combinations, against Meloidogyne incognita on bell pepper. Nematropica 31:75-86. Bell pepper ( Capsicum annuum L.) seeds and seedlings were treated with three potentially beneficial microbes, applied alone and in combinations, to compare effects of these formulations on rootknot nematode ( Meloidogyne incognita ) populations and on plant growth in the greenhouse. Individual treatments (applied as seed coatings and seedling drenches) were formulations of Burkholderia cepacia strains Bc-2 and Bc-F, and of Trichoderma virens strain Gl-3. Combination treatments were Bc- F+Gl-3, Bc-2+Gl-3, Bc-F+Bc-2, and Bc-F+Bc-2+Gl-3. At transplanting, pepper seedlings were each inoculated with 10 000 M . incognita eggs or left uninoculated, and harvested 10 weeks later. Nonviable microbe formulations of each individual strain were also applied; these were tested only on nematodeinoculated plants. No treatment consistently affect
In 2006, a cyst nematode was discovered in tare dirt at a potato (Solanum tuberosum) processing facility in eastern Idaho. The nematode was found during a routine survey conducted jointly by the Idaho State Department of Agriculture and the USDA Animal and Plant Health Inspection Service through the Cooperative Agricultural Pest Survey program. Extensive additional sampling from two suspect fields led to the identification of the same nematode in a 45-acre (18.2-ha) field located in northern Bingham County. The morphology of cysts and second-stage juveniles and molecular analyses established the identity of the species as the pale cyst nematode Globodera pallida (Stone 1973) Behrens 1975. Morphological characters used for identification included cyst shape, characteristics of cyst terminal cone including nature of fenestration, cyst wall pattern, anal-vulval distance, number of cuticular ridges between anus and vulva, and Granek's ratio (1,4). The second-stage juvenile morphologies critical for identification were the following: body and stylet length, shape of stylet knobs, shape and length of tail and hyaline tail terminus, and number of refractive bodies in the hyaline part of tail (1,4). Diagnosis as G. pallida was clearly confirmed by two molecular tests. First, PCR-RFLP (restriction fragment length polymorphism) profiles of a ribosomal DNA fragment using restriction enzymes RsaI, TaqI, and AluI (2) were consistent with a G. pallida control and not G. rostochiensis. Second, the ribosomal DNA region that extends from the 3′ end of the 18S ribosomal subunit and includes all of ITS1, 5.8S, and ITS2 to the 5′ end of the 28S ribosomal subunit was used to generate sequence for the most accurate species determination. Sequences obtained from three individual juveniles were compared with those from several Globodera species (3), revealing unequivocal similarity to G. pallida. This detection represents a new country record for G. pallida in the United States. Collection of additional information regarding distribution of this nematode within the region is underway. References: (1) J. G. Baldwin and M. Mundo-Ocampo. Heteroderinae, Cyst- and Non-cyst-forming Nematodes. Pages 275-362 in: Manual of Agricultural Nematology. W. R. Nickle, ed. Marcel Dekker, New York, 1991. (2) V. C. Blok et al. J. Nematol. 30:262, 1998. (3) L. A. Pylypenko et al. Eur. J. Plant Pathol. 111:39, 2005. (4) A. R. Stone. Nematologica 18:591, 1973.
Absence of the phasmid was demonstrated with the transmission electron microscope in immature third-stage (M3) and fourth-stage (M4) males and mature fifth-stage males (M5) of Heterodera schachtii, M3 and M4 of Verutus volvingentis, and M5 of Cactodera eremica. This absence was supported by the lack of phasmid staining with Coomassie blue and cobalt sulfide. All phasmid structures, except the canal and ampulla, were absent in the postpenetration second-stage juvenile (J2) of H. schachtii. The prepenetration V. volvingentis J2 differs from H. schachtii by having only a canal remnant and no ampulla. This and parsimonious evidence suggest that these two types of phasmids probably evolved in parallel, although ampulla and receptor cavity shape are similar. Absence of the male phasmid throughout development might be associated with an amphimictic mode of reproduction. Phasmid function is discussed, and female pheromone reception ruled out. Variations in ampulla shape are evaluated as phylogenetic character states within the Heteroderinae and putative phylogenetic outgroup Hoplolaimidae.
Abstract A technique is described that refines the standard sugar flotation procedure used to isolate nematodes from their surroundings. By centrifuging nematodes in a number of increasing specific gravity solutions and plotting the fraction floating, the cumulative probability distribution of the population’s specific gravity is generated. By assuming normality, the population mean, μ, and standard deviation, σ, are found by a nonlinear least squares procedure. These density parameters along with their error covariance matrix may be used as a taxonomic physical character. A chi-squared test is derived for comparing populations. Mean and standard deviation pairs (μ, σ) were found for the specific gravities of the adult stage of the plant parasites Pratylenchus agilis (1.068, 0.017), P. scribneri (1.073, 0.028), P. penetrans (1.058, 0.008) and the bacterial-feeder Caenorhabditis elegans (1.091, 0.016). La technique exposée affine le procédure standard par flottation au sucre utilisée pour séparer les nématodes de leur milieu. La centrifugation des nématodes dans une série de solutions de densités spécifiques et la mise en diagramme de la valeur de la fraction surnageante permettent de connaître le répartition de la probabilité cumulée de la densité spécifique de la population en cause. La normalité étant supposée, la moyenne de la population, μ, et la déviation standard, σ, sont calculées par la méthode des moindres carrés non linéaires. Ces paramètres relatifs à la densité ainsi que leur matrice de co-variance d’erreur peuvent être utilisés en taxinomie comme caractère physique. Un test chi2 en est dérivé pour comparer les populations entre elles. Des données en paires — moyenne (μ) et écart-type (σ) — ont été définies pour les densités des adultes des espèces phytoparasites Pratylenchus agilis (1,068; 0,017), P. scribneri (1,073; 0,028), P. penetrans (1,058; 0,008), ainsi que pour l’espèce bactérivore Caenorhabditis elegans (1,091; 0,016).
Abstract Generating DNA markers for microscopic plant parasitic nematodes can be especially difficult if only a few valuable, tiny specimens are available. Providing a reliable maximum amount of unambiguous genetic information from single nematodes is especially important when identifying damaging, regulated nematodes of importance to trade where a few nucleotide differences in diagnostic markers are significant. There are many possible reasons for difficulty amplifying unpurified nematode DNA for long range PCR followed by direct sequencing. Specimen age, proofreading errors and reagent compatibility during PCR are among those problems. While unsuccessful direct amplification of difficult samples may sometimes be overcome by cloning, a more expensive and time-consuming process. Therefore, long segment PCR of a large 3.5 kb segment of ribosomal DNA was optimized for individual difficult-to-amplify young Litylenchus crenatae mccannii (Anguinidae) nematodes by systematically testing thermostable polymerases, proofreading enzymes and buffers. The combination of thermostable DreamTaq™, proofreading Pfu polymerase, and PicoMaxx™ buffer provided the best results. These nematodes are the subject of surveys currently active at many sites in the northeastern United States. This new, optimized PCR protocol will be useful for diagnostic labs associated with the surveys.
