Source: UNIVERSITY OF CALIFORNIA submitted to
A MORPHOLOGICAL AND MOLECULAR EVOLUTIONARY FRAMEWORK/CLASSIFICATION IN SUPPORT OF NOVEL APPROACHES TO MANAGING PLANT-PARASITIC TYLENCHIDA.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
0204174
Grant No.
(N/A)
Project No.
CA-R-NEM-7391-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Mar 1, 2005
Project End Date
Sep 30, 2009
Grant Year
(N/A)
Project Director
Baldwin, J.
Recipient Organization
UNIVERSITY OF CALIFORNIA
(N/A)
RIVERSIDE,CA 92521
Performing Department
Nematology, Riverside
Non Technical Summary
U. S. agriculture is threatened by abundant species of diverse nematode plant pathogens of Tylenchida. This threat is increasing due, in part, to loss of chemical pesticides and increasing global trade/travel that broadens distribution of exotic nematode pests. The most promising and environmentally sound new approaches to managing these agricultural pests (including development of nematode-resistant crops and genomic approaches to interfering with the diverse processes of pathogenesis), requires efficient investigation of genomics and disease-inducing pathways of a few representative model species. Knowledge from models can then be inferred to broader groups, only if the context of each model is understood within a taxonomic/classification system that reliably reflects phylogenetic (evolutionary) relationships. Herein, we propose to develop such a system for Tylenchida through investigating patterns of characteristics found in the morphology and in molecular sequences of genes of hundreds of pathogenic nematode species, and in a framework that extends comparisons to non-pathogenic nematodes. These characteristics will then be used to reconstruct phylogenetic patterns for a reliable classification system. Morphology, researched for the classification system and including sensory, feeding and reproductive structures, will also provide new insight into diverse adaptations for plant parasitism that may then be exploited for novel approaches to pest management. Molecular sequences for phylogenetics will include subsets as suitable tools for rapid species identification.
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2123130103010%
2123130112040%
2163130103010%
2163130112040%
Goals / Objectives
The objective is to gain new knowledge of morphology and molecules for developing a robust Tylenchida phylogeny/classification that will thereby support novel approaches to management of agricultural nematodes. U. S. agriculture is threatened by nematode plant pathogens including many of the more than 2000 species of Tylenchida. This immediate, growing threat is exacerbated by loss of nematicides and increasing global trade/travel, that broadens distribution of exotic nematodes. The result includes urgency for novel approaches to management. However revolutionary approaches (e.g. exploiting genomics, targeting molecular aspects of pathogen biology, GMO resistance) require a solid basis for establishing the origin and maintenance of parasitism genes and morphological/biological specializations for parasitism (e.g. origin, evolution and diversity of the stylet, glandular esophagus; Hussey and Davis, 2004). New management approaches require definition of the distribution of specializations for parasitism across the enormous genetic diversity of Tylenchida, and an understanding of homologies and conservation of these genes/morphological phenotypes in non-plant parasitic outgroups (e.g. Cephalobina, see Blaxter et al. 1998). Apart from an evolutionary framework, the relevant diverse molecular genetic patterns and morphological adaptations cannot be meaningfully and efficiently explored, compared and extended. A reliable phylogeny will also provide the logical basis for a classification of Tylenchida that is informative and predictive of relationships, thus, supporting researchers selecting and inferring from model organisms toward improved nematode management. The practical need for a phylogenetic framework is rooted in the heterogeneity and large number of species of Tylenchida. Tylenchida hosts include invertebrates, fungi and plants; plant parasitic strategies include migratory ectoparasitism and burrowing endoparasitism, sedentary ecto- and endoparasitism, and intermediates; some strategies are associated with little apparent impact on host cells and others induce profound host transformations. For example, preliminary trees based on SSU rRNA, and supported by detailed morphology reject the widely held view that Heteroderinae (cyst) and Meloidogyne (root-knot) sedentary endoparasites collectively are closely related and that genes and even derived morphologies of parasitism are necessarily shared. Alternate hypotheses that Heteroderinae share a clade with Hoplolaimidae (largely migratory ectoparasites) and Meloidogyne with Pratylenchidae (migratory endoparasites) suggests new models that recognize diversity and a basis for informative genetic (with respect to parasitism genomics) comparison (Baldwin et al., 2004). Similarly, non-parasitic Caenorhabditis elegans is often used as a outgroup to hypothesize, through comparisons, genes specific to parasitism in Tylenchida, but a phylogenetic framework points to certain Cephalobina, the immediate outgroup of Tylenchida, as a much more informative point for such comparison (Baldwin et al, 2004ab). A robust Tylenchida phylogeny supports development of novel management strategies.
Project Methods
Current understanding of Tylenchida phylogeny, based largely on small subunit ribosomal RNA gene sequences, has been the basis for family-level classification in which Tylenchida sensu lato (as used in this proposal) is placed within a larger clade, order Rhabditida, suborder Tylenchina, infraorder Tylenchomorpha. Considered monophyletic, this infraorder contains as superfamilies Aphelenchoidea, Criconematoidea, Sphaerularioidea, Tylenchoidea and Myenchoidea. Relationships within and among these groups and their respective families and genera remain undefined. Questions include unresolved relationships of Tylenchida with particular Cephalobina, the controversial questions of monophyly of Aphelenchoidea and Aphelenchoidea+Tylenchida, as well as relationships among major clades of Tylenchida, particularly including plant parasitic Meloidogyninae, Heteroderinae, Hoplolaimidae (lance, spiral, reniform), Pratylenchidae (lesion, burrowing), and Tylenchina (stem and bulb, sting, citrus). These specific questions of Tylenchida are to be addressed through a combination of molecular and morphological tools, with the former often providing a starting point to focus more detailed and technically-challenging morphological questions. Our approach, including PI Baldwin as part of a nematode-phylogeny consortium (see http://nematol.unh.edu/tree/rha2.php), is to expand upon SSU RNA-based phylogenies of Tylenchida to include additional species with broader representation of major clades, additional genes (e.g. large subunit RNA, ribosomal protein genes, heat shock proteins and actins), and with increasingly sophisticated analyses designed to address alignment issues, secondary structure, and, in general more precise models and algorithms (parsimony, liklihood, and Bayesian analysis) for evolution. Digital-video and traditional vouchers will provide an archival link of sequences to phenotype. Preliminary molecular trees and mapping morphology provide a beginning for 1) addressing questions of morphological evolution (e.g. the stylet and glandular basal bulb in Tylenchida from non-plant parasites e.g. Baldwin et al., 2001, 2004) and 2) developing a matrix of morphological characters that can be used in conjunction with molecules for phylogenetic analysis. In each case it is crucial to resolve questions of homology and states of each character to the extent of maximizing the phylogenetic information content of characters. Our approach, in addition to classical light microscopy, is to use a combination of SEM (lip patterns, patterns of sensory organs, male tails), TEM (characters of body wall cuticle layers addressing questions of homology of the stylet both within Tylenchida, between Tylenchida and some Aphelenchida, and with the stoma of non-Tylenchida outgroups including Cephalobina; considering characters of sensory and reproductive systems), 4D microscopy (comparative development e.g. Dolinski et al. 2001) and confocal microscopy, using, for example, florescent antibodies on junctional complexes, to recognize differential cell patterns among taxa. These advancements toward a phylogenetic framework will support novel approaches to nematode management.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Products/innovations during 2008 are in three categories: 1) focusing our molecular-based framework on Tylenchida to address specific questions in clades of high profile to agriculture and to test congruence with morphological analysis. 2) establishing homologies of feeding structures essential to plant parasitism with microbivore outgroups. 3) taxonomic descriptions of new taxa/species of plant parasites with new tools for diagnostics and implications for trade and regulating pests. With regard to category 1, a basis was established in Subbotin et al (2006) indicating lack of support for family Pratylechidae as traditionally defined. This was the impetus for Subbotin et al (2008) in which we focused the molecular analysis to species of the genus Pratylenchus, including an assessment of what traditional and novel morphological characters inform phylogenetics. Using numerous isolates of each species collected worldwide we concluded that most traditionally defined species were monophyletic. It also was clear from the study that classical characters suitable for species descriptions were, for a number of reasons articulated in the manuscript, not suitable for inferring phylogeny. Conversely, a small character set, primarily based on SEM of lip regions, did prove to inform relationships. The approach was novel, using Baysian analysis to depict in a pie chart the strength of each node in the tree and using posterior probability to interpret the strength of each character state at that node. Further contributing to Pratylenchidae, in category 3, and in collaboration with visitor Nguyen from Vietnam, we contributed classical new genus/species descriptions that redefined the limits of traditional Pratylenchidae. Also in category 3 was a reassessment and synonomy, based largely on molecular parameters of the heteroderid nematode, Afenestrata, including new information from isolates received from Central America. In category 2, a major contribution of this period was testing hypothesis of the evolution of the phytoparasite stylet of Tylenchida, using novel approaches to TEM, 3D reconstruction from serial sections and animation software and doing so in the context of comparing each aspect with conserved features in bacterial feeding outgroups (Ragsdale et al, 2008). This work is being extended to additional organs relevant to parasitism and phylogenetics including sensory structures (Bumbarger et al, 2007, 2009; Ragsdale et al in preparation). Additional studies include work on Nacobbus, the false root-knot nematode; the work focuses diagnostics relevant to host specific clades within the genus with the intent to develop practical tools to serve regulatory and pest management agencies. In addition, we have developed new Lucid computer and imaged base keys to assist students and professionals in accurate identification of plant parasitic Tylenchida. These keys will be employed in classroom teaching and upcoming workshops. Throughout Tylenchida molecular and morphological phylogenies are being expanded to include more sophisticated models of evolution, new representative taxa, and refined phylogenetic analyses within each specific clade. PARTICIPANTS: Sergei Subbotin -participating first as a postdoc and research nematologist and more recently working for the CA Dept of Agriculture. Subbotin is a nematode molecular biologist and phylogeneticist. In addition to providing the technical expertise for a phylogenetics overview of Tylenchida, he further applied these approaches to resolution of a number of major clades with the order and collaborated in resolution of Pratylenchus integrating his work with morphological studies by additional experts (Subbotin et al., 2008). Manuel Mundo - curates the UCR taxonomic museum collection that houses vouchers of all the material used in this study and for which he led in publication of a list of type specimens (Mundo and Baldwin, 2009). He further supports the project in all SEM applied to the study and took special initiative in the project leading to synonomy of certain heteroderids (Mundo et al., 2008). Dan Bumbarger - was a graduate student and then a postdoc who was trained in the context of this project. His contribution was to use novel techniques for making 3D reconstructions from serial TEM sections (Bumbarger et al., 2009). This was basic to the morphological component of the study and provided a point of Cephalob comparison for understanding the evolution of the Tylenchid stylet and other key characters via the work of Ragsdale. Erik Ragsdale - is a PhD graduate student who led the widely acknowledged morphological study that resulted in new insight in to the evolution of the stylet of the phytoparasitic Tylenchida (Ragsdale et al., 2008). He is expanding the work to include additional characters and to employ novel molecular tools designed to resolve the base of the Tylenchida tree. Sitara Wijeratne - an undergraduate researcher who gained new skills through participating in the morphological studies leading to co-authorship in publications (e.g. Bumbarger et al., 2009). She is currently a PhD graduate student at Rice University. Cale Carter - an undergraduate researcher who gained new skills through participating in the morphological studies leading to co-authorship in publications (e.g. Bumbarger et al., 2009). Tiago Periera -a laboratory assistant, from Brazil, specialized in molecular techniques and contributing to the project, focusing on groups of plant parasitic Tylenchida that are particularly poorly resolved with respect to phylogeny. Dr. Paolo de-la Rosa -a visiting scholar from Sicily, collaborated with the project in providing specimens collected from Sicily. A portion of this work is part of his PhD dissertation being completed at a university in Sicily. Dr. Larisa Poiras - a visiting scientist and postdoc from Moldova that collaborated with the project in providing specimens collected in Eastern Europe for the project. She is currently completing her DSc in Europe, partly in conjunction with work done at UCR. TARGET AUDIENCES: The target audience is broad spectrum: 1) serving a wide range of investigators in addressing issues of basic science, 2) basic agricultural researchers 3) applied agriculturalists 4) educators and students 5) regulators guarding against invasive pests. The work is primarily focused on priorities of California and US agriculture but it also addresses needs at an international level including issues relevant to international trade. In category 1, publication outlets indicate that our work is recognized as novel and advancing the general science of systematics, phylogenetics and evolution. Novel approaches to 3D reconstruction at the TEM level place our work as an advance among comparative morphologists, as also reflected by publication outlets. Furthermore recent sensory work, published in the Journal of Comparative Neurology, further extends our contributions to basic biology. However a primary target is those using nematodes as model systems, including the C. elegans community, and this specifically brings us to the audience of basic researchers in agriculture. Also within the framework of category 1, in the process of collecting material for phlogenetic analysis we have contributed to knowledge of worldwide biodiversity and distribution of particular species. In category 2 our work addresses the needs of basic agricultural researchers focused on alternatives to pesticides in managing nematodes. By providing such workers with a phylogenetic framework, their efforts on unraveling the genomic pathways of pathogenesis in one nematode can be predicted to be relevant to, for example, sister taxa, and less relevant to distantly related taxa. In this way models can be efficiently developed to represent the full range of strategies for pathogenesis. Our work targets applied agriculturalists (category 3) in that molecular phylogenies allow identifying regions of the genome that are informative for diagnostics. Diagnostics is fundamental to traditional management practices including prediciting disease potential, crop rotation, quarantine, selection of resistant varieties. Similarly we target educators and students (category 4) with new tools for taxonomy and 3D models, including animations that in our experience are extremely effective for conveying key aspects of nematology including morphology and pathogenesis. These are applied in curricula at UCR and in classrooms across the US, as well as in workshops in Mexico, Brazil, Moldova and Belgium. One of the greatest limitations to enforcing quarantine are providing the appropriate tools and skills to regulators. Products from this project including a more meaningful classification system and new morphological and molecular tools, as well as new parameters for predicting pathogenesis will all serve international regulators and implementing quarantine laws. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Phylogenetic analysis employing new knowledge of both molecules and sophisticated morphology provides a basis for improved identification and increasingly reliable classifications of Tylenchida. In the process of developing sequences for phylogenetics we have also recognized regions of the genome that are useful in diagnostics providing an important resource to management including quarantine regulation. In particular, this has been accomplished extensively for species of one of the most important plant parasites worldwide, Pratylenchus, together with establishing the reliability of classical morphological characters for recognizing monophyly and defining most species (Subbotin et al, 2008). The emerging evolutionary framework for Tylenchida is particularly important to agriculture because, as noted, a dependable phylogenetic framework is the basis for developing novel approaches (exploiting genomics, targeting molecular aspects of pathogen biology, GMO resistance) to nematode management. To a large extent understanding (and exploiting for management) the diverse pathways for pathogenesis within Tylenchida require selecting appropriate model systems and it is essential that these systems be based on a reliable phylogeny. Detailed morphological studies including 3D reconstructions from TEM have largely answered questions that arose when morphologists were skeptical with molecular results indicating that plant parasitic Tylenchida had evolved from microbivore Cephalobina. At a detailed (TEM) level, the homologies are clearly established and confirm this relationship, not only with respect to the evolution of the stylet but adaptations of the pharyngeal glands, and sensory structures for parasitism. A practical outcome is that we now recognize the model Caenorhabditis elegans is a less informative basis for applying to phytoparasites, than are models from members of Cephalobina. Our work is providing new knowledge into diverse adaptations for plant parasitism that may then be exploited with novel approaches to management. In this process, previous misunderstandings about morphological characters that resulted in faulty classifications are corrected and classifications refined. One recent focus of this study has been on pathotypes and species of the false root-knot nematode, Nacobbus; results not yet published show that this genus includes two major clades and ongoing work suggests that these clades can be diagnosed with both morphological and molecular tools. Since isolates of Nacobbus are widespread in Mexico and include diverse host ranges and substantial destruction of most vegetable crops, it is a major concern as a potentially invasive pest particularly to California, the work has important practical implications. New knowledge from our studies is being incorporated in to classroom teaching, national and international workshops and further outreach through webpages and CDs with computer-based identification tool. Identification tools include adaptations of Lucid as well as the development of new software for computer image recognition.

Publications

  • Subbotin, S. A. E. J. Ragsdale, T. Mullens, P. A. Roberts, M. Mundo-Ocampo, J. G. Baldwin 2008. A phylogenetic framework for root-lesion nematodes of the genus Pratylenchus (Nematoda): evidence from 18S and D2-D3 expansion segments of 28S ribosomal RNA genes and morphological characters. Molecular Phylogeny and Evolution. 4:491-505.
  • Ragsdale, E. J., Crum, J. Ellisman, M. H., and Baldwin, J. G. 2008. Phylogenetic implications of three dimensional reconstruction of the stomatostylet and anterior epidermis in Aphenchus avenae. Journal of Morphology. 269:1181-1196.
  • Bumbarger, Daniel; Crum, John; Ellisman, Mark; Baldwin, James 2007. Three-dimensional fine structural reconstruction of the nose sensory structures of Acrobeles complexus compared to Caenorhabditis elegans (Nematoda: Rhabditida) Journal of Morphology 268:649-663.
  • Bumbarger, Daniel; Wijeratne, Sitara; Carter, Cale; Crum, John; Ellisman, Mark; Baldwin, James 2009. Three Dimensional Reconstruction of the Amphid Sensilla in the Microbial Feeding Nematode, Acrobeles complexus (Nematoda: Rhabditida). Journal of Comparative Neurology. 512:271-181.
  • Trinh, Phap Q., Lieven Waeyenberge, Chau N. Nguyen, James G. Baldwin, Gerrit Karssen and Maurice Moens 2009. Apratylenchus, a new genus of sedentary Pratylenchidae (Nematoda: Tylenchida) from coffee in Vietnam and a proposal for Apratylenchinae n. subfam. Nematology in press.
  • Mundo-Ocampo and J. G. Baldwin, 2009. A revised list of type specimens deposited at the Univeristy of California, Riverside Nematode Collection. Nematology. In press.


Progress 01/01/07 to 12/31/07

Outputs
We continue toward our goal of using morphology and molecules to develop a robust Tylenchida phylogeny/classification in support of novel approaches to managing plant-parasitic nematodes. Following on our previously reported work with Subbotin in which a molecular phylogeny of Tylenchida was developed, we have now expanded this study to include additional taxa; this update will be presented this year at an International Congress of Nematology in Brisbane, Australia. We have further expanded the work in a detailed phylogenetic analysis of the plant parasitic genus, Pratylenchus (currently submitted and under review in the journal, Molecular Phylogenetics and Evolution) This study is novel in that it provides an overview of the problems of applying diagnostic morphological characters to phylogenetic analysis, and also provides an approach to mapping morphological characters on molecular-based trees, using Baysian methods to show the strength of each node in the phylogeny as well as the strength of each character state at that node. For Pratylenchus, we particularly show the strength of lip pattern characters, as interpreted with SEM. Work on Praylenchidae, is being further expanded with focus on invasive species, including Nacobbus, where ongoing studies recognize two major groups within the species, N. aberrans. Continuing our long-standing work on cyst nematodes of Heteroderidae, we have completed a revision of Afenestrata; this study abolished the genus and placed it, based on morphological and molecular evidence, within a redefined Heterodera (Mundo et al., in press). This Afenestrata project is part of a larger study under contract with Brill to complete a two-volume book, currently in preparation, on the phylogenetics and classification of cyst nematodes. We are continuing morphological research focus to look at Tylenchida in the larger context of microbivorous outgroups. In this capacity, the anterior regions of Acrobeles and Aphelenchus were fully reconstructed through hundreds of serial TEM microgaphs and novel application of a range of computer software for 3D modeling. During the past year, graduate student Ragsdale has followed previous work by Bumbarger to now establish cell-to-cell homologies between the microbivore cephalob outgroups and the tylenchid stomatostylet of plant parasitism (Ragsdale et al. under review in Journal of Morphology); this work resulted in the best student paper award at the 2006 Society of Nematology meetings. Ongoing work is further extending these reconstructions and insight into morphological homologies as they relate to plant parasitism, with specific emphasis on anterior sensory systems and the overall topography of the nervous system (Bumbarger et al., 2007 and Bumbarger et al., submitted to the Journal of Comparative Neurology). Furthermore, molecular phylogenies are being expanded to include more sophisticated models of evolution, new representative taxa, and refined phylogenetic analyses within each specific clade of Tylenchida.

Impacts
Our work on phylogenetic analysis provides a basis for improved identification and increasingly reliable classifications of Tylenchida. We argue that this is particularly crucial to agriculture because a dependable phylogenetic framework is the basis for applying revolutionary approaches (exploiting genomics, targeting molecular aspects of pathogen biology, GMO resistance) to nematode management. We emphasize the importance of understanding the diverse pathways for pathogenesis within Tylenchida; in this regard, we argue that addressing this understanding requires selecting appropriate model systems and it is essential that these systems be based on a reliable phylogeny. To this end our work has shown a number of areas where extant understanding of relationships and classifications have been misleading, not only within Tylenchida but in the inferred relationships to non-parasitic outgroups. Our developing phylogenetic framework provides a basis to select models for understanding evolution and the precise function of the "tools" adapted for plant parasitism; these include the specializations of the stomatostylet, pharyngeal glands, sensory and reproductive systems. Resulting new knowledge into diverse adaptations for plant parasitism may then be exploited with novel approaches to management. These studies will result in correcting faulty and misleading classifications and thereby it will support a wide range of projects from genomics to directly applied management oriented projects on agricultural nematode pests.

Publications

  • Bumbarger, D. J., J. Crum, M. H. Ellisman, and James G. Baldwin, 2007. Three-dimensional fine structural reconstruction of the nose sensory structures of Acrobeles complexus compared to Caenorhabdtis elegans (Nematoda: Rhabditida). J. Morphology 268: 649-663.
  • Mundo-Ocampo, Manuel, A. Trocolli, S. A. Subbotin, J. Del Cid, J. G. Baldwin and R. N. Inserra, 2008. Synonomy of Afenestrata with Heterodera supported by phylogenetics with molecular and morphological characterization of H. koreana comb. n. and H. orientalis comb. n. (Tylenchida: Heteroderidae. Nematology in press.


Progress 01/01/06 to 12/31/06

Outputs
During 2006, we made major progress toward our goal of using morphology and molecules to develop a robust Tylenchida phylogeny/classification in support of novel approaches to managing plant-parasitic nematodes. The most important publication directly representing this project is Subbotin et al (2006 a) in which a molecular phylogeny of Tylenchida was developed based on 82 representative species. Results generally agree with classical systems but demonstrate a basal position of groups that include entomoparasitic nematodes, paraphyly of Dolichodoroidea sensu Siddiqi (2000), evidence for a Pratylenchus-Hirschmanniella-Meloidogyne clade and lack of support for widely held traditional placement of Radopholus within Pratylenchidae. In this paper we also discuss a range of discoveries with respect to congruence and incongruence of molecular phylogeny and traditional morphology-based views of classification. This work was further detailed in separate publications using novel approaches to molecular phylogenetic analysis in the tylenchid families Hoplolaimidae (Subbotin 2006b). Additional work (Chizhov, 2006; Giblin-Davis et al, 2006) focused on phylogenetic questions and species descriptions of Aphelenchida, a group of plant parasites of questionable paraphyly with Tylenchida. A recent morphological research focus has been to look at Tylenchida in the larger context of microbivorous outgroups and particularly to address the question of evolution of the stomatostylet, an organ associated with most plant parasitism. In this regard the hypodermal syncytia responsible for stoma-formation of a representative cephalob (the sister group of Tylenchida, see Nadler et al., 2006) was fully reconstructed through hundreds of serial TEM microgaphs and novel application of a range of computer software for 3D modeling (Bumbarger et al.2006). This work is currently being extended to Aphelenchus, a taxon selected as near the base of the tylenchid clade, and cell-to-cell homologies have been fully established between the stomatostylet and the stoma of microbivore outgroups. Ongoing work is further extending these reconstructions and insight into morphological homologies as they relate to plant parasitism, with specific emphasis on sensory systems. Furthermore, molecular phylogenies are being expanded to include more sophisticated models of evolution, new representative taxa, and refined phylogenetic analyses within each specific clade of Tylenchida.

Impacts
Advances in phylogenetic analysis provide a basis for improved identification and increasingly reliable classifications of Tylenchida. This is particularly important to agriculture because (as we have noted previously "a dependable phylogenetic framework is the basis for applying revolutionary approaches (exploiting genomics, targeting molecular aspects of pathogen biology, GMO resistance) to nematode management. To a large extent understanding (and exploiting for management) the diverse pathways for pathogenesis within Tylenchida require selecting appropriate model systems and it is essential that these systems be based on a reliable phylogeny". Our developing phylogenetic framework (e.g. Subbotin, 2006a) also provides a basis to select models for testing hypotheses about evolution (e.g. through mapping on molecular-based trees) of the "tools" adapted for plant parasitism; these include the specializations of the stomatostylet, pharyngeal glands, sensory and reproductive systems. This work is providing new knowledge into diverse adaptations for plant parasitism that may then be exploited with novel approaches to management. In this process, previous misunderstandings about morphological characters that resulted in faulty classifications are corrected and classifications refined. Beyond phylogeny one benefit of developing molecular sequences to plant parasitic Tylenchida is that the work is basic to identifying regions of the genome promising for species (and sometimes "race" or pathotype) diagnostics with important implications for management and regulation.

Publications

  • Abebe, Eyualem, James G. Baldwin, Bryon Adams, Duane Hope, Scott Gardner, Robin Huettel, Peter Mullin, Tom Powers, Jyotsna Sharma, Weimin Ye, W. Kelley Thomas. 2006. A position paper on the electronic publication of nematode taxonomic manuscripts. Journal of Nematology 38:305-311.
  • Bumbarger, Daniel J. John Crum, Mark H. Ellisman and James G. Baldwin 2006. Three dimensional reconstruction of the nose epidermal cells in the microbial feeding nematode, Acrobeles complexus (Nematoda: Rhabditida). Journal of Morphology 267:1257-1272.
  • Giblin-Davis, Robin M., N. Kanzaki, Weimin Ye, Manuel Mundo-Ocampo, James G. Baldwin, W. Kelley Thomas. 2006. Morphology and description of Bursaphelenchus platzeri n. sp. (Nematoda: Parasitaphelnenchidae), and associate of nitidulid beetles. Journal of Nematology 38:150-157.
  • Nadler Steve, Paul De Ley, Manuel Mundo-Ocampo, Ashleigh B Smythe, S. Patricia Stock, Ph.D, Dan Bumbarger, Byron J Adams, Irma De Ley, Oleksandr Holovachov; James G Baldwin, 2006. Phylogeny of Cephalobina (Nematoda): Molecular evidence for recurrent evolution of probolae and incongruence with traditional classifications. Molecular Phylogenetics and Evolution. 40:696-711.
  • Sergei A. Subbotin, Dieter Sturhan, Vladimir N. Chizhov, Nicola Vovlas and James G. Baldwin 2006a. Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2 and D3 expansion fragments of the 28S rRNA gene sequences. Nematology 8:455-474.
  • Subbotin, Sergei. Dieter Sturhan, Nicola Vovlas, Pablo Castillo, James Tanyi Tamber; Maurice Moens, James G Baldwin 2006b. Application of secondary structure model of rRNA for phylogeny: D2-D3 expansion segments of the LSU gene of plant-parasitic nematodes from the family Hoplolaimidae Filipjev, 1934. Molecular Phylogenetics and Evolution. In press.
  • Vladimir N. Chizhov, Olga A. Chumakova, Sergei A. Subbotin and James G. Baldwin, 2006. Morphological and molecular characterization of foliar nematodes of the genus Aphelenchoides: A. fragariae and A. ritzemabosi (Nematoda: Aphelenchoididae) from the main Botanical Garden of the Russian Academy of Sciences, Moscow. Russian Journal of Nematology. 14:179-184.


Progress 01/01/05 to 12/31/05

Outputs
The overall goal of our work is to gain knowledge of morphology and molecules to develop a robust Tylenchida phylogeny/classification in support of novel approaches to managing plant-parasitic Tylenchida. During 2005, working with Dr. Subbotin, a member of my research lab, we enlisted worldwide collaborative help in providing specimens to develop a molecular phylogeny of the subfamily, Criconematina (Subbotin et al., 2005). Whereas in many respects this molecular phylogeny is congruent with extant classifications, we also demonstrated misinterpretations in key areas that suggest a basis for re-evaluation and revised classification. Additional work has lead to the final stages of manuscript preparation for a preliminary molecular/morphological phylogenetic analysis of Tylenchida. Surprising results including underscoring the independent evolution of Meloidogyne (root-knot nematodes) from Heteroderinae (including cyst nematodes) and instead strongly suggesting a sister relationship between Meloidogyne and Pratylenchus spp. (lesion nematodes). This work has major implications for selecting model systems to understand diverse pathways of pathogenesis in parasitic nematodes. Additional evidence from our work indicates paraphyly of Pratylenchidae including the exclusion of Radopholus, which is traditionally placed within Pratylenchidae. Further publications related to subgroups of Tylenchida and nearing completion include a phylogenetic analysis of Hoplolaiminae and a separate paper on phylogeny of Pratylenchinae. The work on Hoplolaiminae not only includes important new insight on phylogenetic relationships but integrates novel methodologies (including new software developed in our lab) in phylogenetic analysis based on secondary structure of DNA. Broader studies are addressing outgroups in Cephalobina (Waceke et al. 2005); this sister group of Tylenchida is being investigated to understand the origin and evolution of plant parasitism and most specifically the evolution of the stylet (a structure not present in cephalobids and apparently necessary for plant parasitism) via UCR graduate students Bumbarger and Ragsdale. Using electron microscopy and computer-based tools for 3-D reconstruction important new information is being developed to provide new morphological data for interpreting evolution and particularly to establish homologies of feeding structures between these nonparasitic outgroups and parasitic tylenchids. In addition graduate student Ragsdale is using molecular and morphological tools to address questions of monoplyly of Tylenchida with Aphelenchida and to develop a clearer phylogenetic perspective of taxa basal to Tylenchida including several groups that primarily are parasites or associates of insects. To advance nematology in the area of molecular identification of species we have also collaborated with the De Ley lab at UCR to provide novel approaches for barcoding (De Ley et al., 2005).

Impacts
Work on phylogenetic analysis is providing a basis for improved identification and increasingly reliable classifications of Tylenchida. We have noted that such a dependable phylogenetic framework is the basis for applying revolutionary approaches (exploiting genomics, targeting molecular aspects of pathogen biology, GMO resistance) to nematode management. To a large extent understanding (and exploiting for management) the diverse pathways for pathogenesis within Tylenchida requires selecting appropriate model systems and it is essential that these systems be based on a reliable phylogeny. Thus are addressing both an overview of Tylenchida phylogeny and in addition we are focusing on detailed resolution of subgroups of major agricultural significance including Criconematina, Hoplolaiminae and Pratylenchidae. In the process molecular sequences also provide a basis to identify regions of the genome that are promising for diagnostics and thus support simplified, barcoding, approaches to identification of pathogens with important implications for management and regulation. Morphological work including detailed fine structure studies complements molecular work by unraveling cases that might otherwise, at a superficial level, appear to be incongruent; at some levels morphological data provides phylogenetic resolution where molecules being considered are less informative. Morphological work on sensory, feeding and reproductive structures will also provide insight into diverse adaptations for plant parasitism that may then be exploited with novel approaches to management.

Publications

  • DeLey, P., I. Tandingan DeLey, K. Morris, E. Abebe, M. Mundo, M. Yoder, J. Heras, D. Waumann, A. Rocha-Olivares, J. Burr, J.G. Baldwin and W.K. Thomas. 2005. An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding. Philosophical Transactions of the Royal Society of London B, 1726 : 1-14.
  • Subbotin, Sergei A., Nicola Vovlas, Renato Crozzoli, Dieter Sturhan, Franco Lamberti Maurice Moens and James G. Baldwin 2005. . Phylogeny of the suborder Criconematina Siddiqi, 1980 (Nematoda: Tylenchida) based on morphology and the D2-D3 expansion segments of the 28S ribosomal DNA gene sequences using maximum parsimony, maximum likelihood and Bayesian analyses. Nematology. In Press.
  • Waceke, J. W., D. J. Bumbarger, M. Mundo-Ocampo, S. A. Subbotin and J. G. Baldwin. 2005. Zeldia spannata n. sp. (Nematoda: Cephalobidae) from the Mojave Desert, California. Journal of Nematode Morphology and Systematics: 8:57-67.