Progress 11/01/00 to 09/30/05

Outputs
(i) Nematode survey of vernal pools from the Santa Rosa Plateau Ecological Reserve (Murietta - support by UC Water Resources Center): We completed the first nematode survey on record for this ecologically important habitat and found an estimated fifty-one nematode genera representing sixty-two species. An estimated fourteen species are new to science, including a cryptic species of Hirschmanniella that is morphologically similar to H. pomponiensis but clearly different in ribosomal DNA sequence. Soils from both pools differ substantially in composition and dynamics of their respective nematode communities. During the dry summer phase, roots and plant debris appear to play an important role in both pools as refuges for omnivorous, bacterivorous and phytoparasitic nematodes. First feeding assays of live omnivore/predator nematodes do not suggest that these feed on fairy shrimp cysts, although assays may need to be improved in order to better simulate natural conditions. Nematode fragments have been frozen for follow-up analyses to check for shrimp DNA in nematode gut contents. (ii) Application of organic mulches for thrips control in avocado orchards (Temecula - support by UC Specialty Crops and Exotic Pests and Diseases programs): Pathogenicity of Steinernema feltiae towards avocado thrips has been established in two series of assays, one conducted in 2004 and a second one in 2005. Mesocosm experiments have shown that antagonistic effects against the thrips occur in mulch regardless of the presence or absence of fungal or nematode entomopathogens (resp. Beauveria bassiana and S. feltiae) and/or predatory mites (Hypoaspis spp.). plates and 8 multiwell control plates with over 1000 individually contained avocado thrips larvae and pupae. (iii) Genetic diversity and pathogenicity of ring nematodes from five vineyards in central and southern California (support by DANR): Four populations of M. xenoplax segregate clearly into two haplotypes, based on Internal Transcribed Spacer sequences. A fifth population was found to be mixed with other ring nematode species. Field trials on two different grapevine rootstock varieties indicate that the two haplotypes of M. xenoplax behave quite differently in terms of reproductive success, and that diagnostic tests are needed to assess potential yield loss depending on the exact nematode haplotype. (iv) National and international collaboration on nematode phylogeny (support by NSF Assembling the Tree of Life program): Specimens from all these projects are being archived with video capture tools prior to sequencing of informative ribosomal DNA loci. Over 500 individual nematodes have been processed in this way, providing phylogenetic and diagnostic information about a wide range of previously unavailable species. We also developed new methodological improvements that make it much easier to conduct combined molecular and morphological inventories of nematodes.

Impacts
This project set out to explore nematode diversity and to analyse its relevance to rhizosphere ecology. With the techniques we are developing and applying, cryptic species and/or phytopathologically different nematode populations can be detected and accurately characterized. Our results indicate that natural habitats in southern California clearly contain many additional, as yet unknown nematode species, including some that are cryptic new members of agriculturally important families and genera of plant parasites. Interesting habitats with unexplored diversity include ephemeral pools and mulched orchards. Mulches are especially promising for potential applications in pest control in orchards.

Publications

• De Ley, P., W. Decraemer and E. Abebe. 2006 (in press). Introduction: summary of present knowledge and research addressing the ecology and taxonomy of freshwater nematodes. In Freshwater Nematodes: Ecology and Taxonomy (eds. E. Abebe, W. Traunspurger, and I. Andrassy). CAB International, Wallingford UK: - .
• Holovachov, O. and P. De Ley. 2006 (in press). Order Plectida. In Freshwater Nematodes: Ecology and Taxonomy (eds. E. Abebe, W. Traunspurger, and I. Andrassy). CAB International, Wallingford UK: - .
• De Ley, P., I. Tandingan De Ley, K. Morris, E. Abebe, M. Mundo-Ocampo, M. Yoder, J. Heras, D. Waumann, A. Rocha-Olivares, A.H.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, 360 : 1945-1958.
• De Ley, P. 2005. A quick tour of nematode diversity and the backbone of nematode phylogeny. In WormBook (eds. The C.elegans Research Community). WormBook. http://www.wormbook.org

Progress 01/01/04 to 12/31/04

Outputs
(i) Nematode survey of vernal pools from the Santa Rosa Plateau Ecological Reserve (Murietta - support by UC Water Resources Center): We conducted the first nematode survey on record for this ecologically important habitat. At least fifty-one nematode genera were isolated, respresenting a minimum estimate of sixty-two species. An estimated fourteen species are new to science, including a cryptic species of Hirschmanniella that is morphologically similar to H. pomponiensis but clearly different in ribosmal DNA sequence. Soils from both pools were substantially different in the composition and dynamics of their respective nematode communities. During the dry summer phase, roots and plant debris appear to play an important role in both pools as refuges for omnivorous, bacterivorous and phytoparasitic nematodes. Analyses of nematode abundances yielded significant differences indicative of interactions between sample dates and extracted sample fractions. Significant differences in abundance patterns were also detected across different nematode genera. (ii) Application of organic mulches for thrips control in avocado orchards (Temecula - support by UC Specialty Crops and Exotic Pests and Diseases programs): Pathogenicity of Steinernema feltiae towards avocado thrips has been confirmed using a series of 13 multiwell assay plates and 8 multiwell control plates with over 1000 individually contained avocado thrips larvae and pupae. Nematodes extracted from an infected thrips had neither released all their entomopathogenic bacterial symbionts nor commenced feeding, suggesting that thrips mortality could have resulted from nematode penetration rather than from bacterial toxicity. Additionally, a strain of Tricephalobus sp. was newly isolated from infected waxmoth larvae; it will also be investigated for thrips pathogenicity. First data from nematode community analysis show that total nematode density is clearly higher in mulched treatments, but is also subject to extreme fluctuations, confirming that populations must be sampled frequently and that numerous replicate samples must be collected for studies of this type of substrate. (iii) Genetic diversity and pathogenicity of ring nematodes from five vineyards in central and southern California (support by DANR): Four populations of M. xenoplax segregate clearly into two haplotypes, based on Internal Transcribed Spacer sequences. A fifth population was found to be mixed with other ring nematode species. Field trials on two different grapevine rootstock varieties indicate that the two haplotypes of M. xenoplax behave quite differently in terms of reproductive success, and that diagnostic tests are needed to assess potential yield loss depending on the exact nematode haplotype. (iv) National and international collaboration on nematode phylogeny (support by NSF Assembling the Tree of Life program): Specimens from the above projects are being archived with video capture tools prior to sequencing of phylogenetically and diagnostically informative sites. Over 300 individual nematodes have been processed in this way, providing phylogenetic information about a wide range of previously unavailable species.

Impacts
This project sets out to explore nematode diversity and to analyse its relevance to rhizosphere ecology. In the past year, it has become evident that cryptic species and/or phytopathologically different nematode populations can be detected and accurately characterized through the combined morphological and molecular techniques we have developed in the two preceding years. Our results indicate that natural habitats in southern California clearly contain many additional, as yet unknown nematode species, including some that are cryptic new members of agriculturally important families and genera of plant parasites.

Publications

• Tenente, G.C.M.V., P. De Ley, I. Tandingan De Ley, G. Karssen and J.R. Vanfleteren. 2004. Sequence analysis of the D2/D3 region of the large subunit rDNA from different Meloidogyne isolates. Nematropica, 34: 1-12.
• Blaxter, M., R. Floyd, M. Dorris, E. Abebe & P. De Ley. 2004. Utilising the new nematode phylogeny for studies of parasitism and diversity. Nematology Monographs and Perspectives, 2: 615-632.
• De Ley P., R. Van Driessche and A. Coomans. 2005. Terrestrial nematodes of the Galapagos Archipelago. 10. Morphological and taxonomical analysis of Carcharolaimus ramirezi Thorne, 1967 (Dorylaimida: Qudsianematidae). Nematology, : In press.

Progress 01/01/03 to 12/31/03

Outputs
The purpose of my research program is to apply new and traditional methods to the ecological characterisation of a taxonomically diverse range nematode species, in order to characterize their feeding behavior, dispersal and reproduction. This will allow more factual approaches to nematode community analysis. Because of the enormous diversity and numbers of nematodes occurring in rhizospheres, both aspects are essential to an improved understanding of rhizosphere ecology. In the past year, I have focused on research projects addressing specific questions of nematode diversity while providing sources of a wide range of nematode species from natural and agricultural ecosystems. (i) Nematode survey of vernal pools from the Santa Rosa Plateau Ecological Reserve (Murietta - support by UC Water Resources Center): First results suggest that nematode communities in the vernal pools of Santa Rosa Plateau consist of a mixture a plant parasites and aquatic species. Nematodes persist throughout dry periods and larger species appear to congregate inside accumulated plant litter. (ii) Application of organic mulches for thrips control in avocado orchards (Temecula - support by UC Specialty Crops and Exotic Pests and Diseases programs): Nematode communities of avocado orchards are found to be quite diverse, and include some species likely to cause thrips mortality. Assays with Steinernema feltiae have just begun and first results are ambiguous - thrips pupae or larvae appear to die prematurely in the presence of S. feltiae but are not found to contain nematodes. More extensive assays will be conducted to determine whether nematodes might kill thrips without fully penetrating into their body cavity. (iii) Genetic diversity and pathogenicity of ring nematodes from five vineyards in central and southern California (support by DANR): Four populations of M. xenoplax segregate clearly into two haplotypes, based on Internal Transcribed Spacer sequences. Additional numbers of individuals are being analysed to quantify sequence polymorphisms and assess gene flow between both ITS haplotypes. (iv) National and international collaboration on nematode phylogeny (support by NSF Assembling the Tree of Life program): Specimens from the above projects are being archived with video capture tools prior to sequencing of phylogenetically informative sites. Over 200 individual nematodes have been processed in this way, providing phylogenetic information about a wide range of previously unavailable species.

Impacts
This project sets out to explore nematode diversity and to analyse its relevance to rhizosphere ecology. In the past year, I have started applying video capture methods developed to record and distribute nematode morphology as seen through the light microscope, preliminary to molecular analysis of each individual nematode archived in this manner. This new approach is beginning to produce interesting results in a wide range of research projects with potential applications to control of pests of grapevine and avocado, and to management of vernal pools and their endangered species of plants and animals.

Publications

• Bert, W., I.T. De Ley, R. Van Driessche, H. Segers, and P. De Ley. 2003 - In press. Baujardia mirabilis gen. n. sp. n. from pitcher plants and its phylogenetic position within Panagrolaimidae (Nematoda: Rhabditida). Nematology, : -.
• De Ley, P., and M. Blaxter. 2003 - In press. A new system for Nematoda: combining morphological characters with molecular trees, and translating clades into ranks and taxa. Nematology Monographs and Perspectives, 2: -.
• Moens, T., G.W. Yeates and P. De Ley. 2003 - In press. Carbon and energy sources for nematodes. Nematology Monographs and Perspectives, 2: -.
• Holovachov, O., S. Bostrom, I. Tandingan De Ley, P. De Ley and A. Coomans. 2003. Morphology and systematics of the genera Wilsonema Cobb, 1913, Ereptonema Anderson, 1966 and Neotylocephalus Ali, Farooqui & Tejpal, 1969 (Leptolaimina: Wilsonematinae). Journal of Nematode Morphology and Systematics, 5: 73-106 (2002).
• De Ley, P. and M. Mundo-Ocampo. 2003 - In press. The cultivation of nematodes. In: Z.X. Chen, S.Y. Chen and D.W. Dickson (eds.) Nematology: Advances and Perspectives. Tsinghua University Press, Tsinghua: - .

Progress 01/01/02 to 12/31/02

Outputs
The purpose of my research program is to apply new and traditional methods to the ecological characterisation of a taxonomically diverse range nematode species, in order to characterize their feeding behavior, dispersal and reproduction. This will allow more factual approaches to nematode community analysis. Because of the enormous diversity and numbers of nematodes occurring in rhizospheres, both aspects are essential to an improved understanding of rhizosphere ecology. In the past two years, I have focused on (i) developing the tools required for these goals, (ii) establishing cultures for initial studies, (iii) starting up research projects addressing specific questions of nematode diversity while providing sources of a wide range of nematode species from natural and agricultural ecosystems. (i) Methodological innovations and improvements: The combination of Video Capture and Editing (VCE) equipment with microscopy allows for the recording of nematode morphology prior to other procedures. The principles of VCE microscopy are being published (De Ley and Bert, in press), and the method has been applied to species descriptions in order to illustrate its usefulness (Stock et al., 2002; Bert et al., in press). We have further improved our tools to allow video capture of individual live nematodes prior to PCR, and have begun construction of a combined video archive and sequence database. The VCE+PCR data will allow us to integrate morphological and molecular markers into studies of population genetics of rhizosphere nematodes. (ii) In vitro studies of nematode reproduction and feeding: A number of species have been brought into culture to start comparative investigations. These include: Goodeyus ulmi, a species in which development of the female reproductive system is strongly influenced by environmental factors; two strains of the genus Brevibucca, used in crossing experiments and in development of suitable molecular markers for characterizing populations and closely related species; six species species from the families Bunonematidae, Cephalobidae, Plectidae and Rhabditidae, all bacterial-feeding nematodes with complex cephalic appendages and with different functionally linked feeding behavior. (iii) Specific projects and sources of specimens: We have obtained funding for the following studies of nematode diversity: a nematode survey of vernal pools from the Santa Rosa Plateau Ecological Reserve (Murietta - support by UC Water Resources Center); a project on application of organic mulches for thrips control in avocado orchards (Temecula - support by UC Specialty Crops and Exotic Pests and Diseases programs); a study of genetic diversity and pathogenicity of ring nematodes from five vineyards in central and southern California (support by DANR); and a national and international collaboration on nematode phylogeny (support by NSF Assembling the Tree of Life program). These projects provide complementary resources in the pursuit of my overall research program, they involve applications in crop protection and conservation biology, and they also allow me to access a taxonomically and ecologically diverse pool of nematode species from rhizospheres.

Impacts
This project sets out to explore nematode diversity and to analyze its relevance to rhizosphere ecology. In the past two years, I have developed video capture methods to record and distribute nematode morphology as seen through the light microscope, preliminary to molecular analysis of each individual nematode archived in this manner. The methods in question are affordable, highly versatile and directly applicable to a wide range of research settings. This has allowed me to promulgate this new approach among my colleagues, and to initiate a range of research projects with potential applications to control of pests of grapevine and avocado, and to management of vernal pools and their endangered species of plants and animals.

Publications

• De Ley, I. T., P. De Ley, A. Vierstraete, G. Karssen, M. Moens and J. Vanfleteren. 2002. Phylogenetic Analyses of Meloidogyne SSU rDNA. Journal of Nematology (In press).
• De Ley, P. and M. Mundo-Ocampo. 2002. The cultivation of nematodes. In: Z.X. Chen, S.Y. Chen and D.W. Dickson (eds.) Nematology: Advances and Perspectives. Tsinghua University Press, Tsinghua (In press).
• Bert, W., I.T. De Ley, R. Van Driessche, H. Segers, and P. De Ley. 2002. Baujardia mirabilis gen. n. sp. n. from pitcher plants and its phylogenetic position within Panagrolaimidae (Nematoda: Rhabditida). Nematology (In press).
• De Ley, P., and M. Blaxter. 2002. A new system for Nematoda: combining morphological characters with molecular trees, and translating clades into ranks and taxa. Nematology (In press).
• Subbotin, S.A., A. Vierstraete, P. De Ley, J. Rowe, L. Waeyenberge, M. Moens, and J.R. Vanfleteren. 2001. Phylogenetic relationships within the cyst-forming nematodes (Nematoda, Heteroderidae) based on analysis of sequences from the ITS regions of ribosomal DNA Molecular Phylogenetics and Evolution 21: 1-16.
• Wu, J., P. De Ley and Y. Liang. 2001. Description of a new species of the genus Tylopharynx (Nemata: Diplogasteroidea), T. clariamphida sp. n., with a redescription of T. foetida (Butschli, 1874) Goffart, 1930. Journal of Nematology, 33: 83-90.
• Holovachov, O. and P. De Ley. 2001. Description of Pseudacrobeles (Bunobus) bostromi sp. n. from rotting wood from Roztochya, Ukraine (Rhabditida: Cephalobidae). Journal of Nematode Morphology and Systematics, 4: 21-29.
• Elbadri, G.A.A., P. De Ley, L. Waeyenberge, A. Vierstraete, M. Moens and J. Vanfleteren. 2002. Intraspecific variation in Radopholus similis isolates assessed with restriction fragment length polymorphism and DNA sequencing of the internal transcribed spacer region of the ribosomal RNA cistron. International Journal of Parasitology, 32: 199-205.
• Baldwin, J.G., I.T. De Ley, M. Mundo-Ocampo, P. De Ley, S.A. Nadler and M. Gebre. 2002. Acromoldavicus mojavicus n. sp. (Nematoda: Cephaloboidea) from the Mojave Desert, California. Nematology, 3: 343-353.
• Stock, S. P., P. De Ley, I. T. De Ley, M. Mundo-Ocampo, J.G. Baldwin and S. A. Nadler. 2002. Plectonchus hunti n. sp. and Panagrobelus stammeri (Ruhm, 1956): Implications of new morphological observations for characterization of these closely related genera (Nematoda: Panagrolaimoidea). Nematology, 4: 403-419.
• De Ley, P. and M.L. Blaxter. 2002. Systematic position and phylogeny. In: D.L. Lee (ed.) The Biology of Nematodes. Taylor and Francis, London: 1-30.
• De Ley, P. and W. Bert. 2002. Video Capture and Editing as a tool for the storage, distribution and illustration of morphological characters of nematodes. Journal of Nematology (In press).