Source: CALIFORNIA STATE UNIVERSITY submitted to
IMPACT OF UNCULTURED BACTERIA ON FREE-LIVING NEMATODE POPULATIONS IN SOIL
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0219368
Grant No.
2009-65106-05644
Project No.
CALR-2009-02606
Proposal No.
2009-02606
Multistate No.
(N/A)
Program Code
91113
Project Start Date
Sep 1, 2009
Project End Date
Jun 30, 2011
Grant Year
2009
Project Director
Orwin, P. M.
Recipient Organization
CALIFORNIA STATE UNIVERSITY
5500 UNIVERSITY PKY.
SAN BERNADINO,CA 92407
Performing Department
(N/A)
Non Technical Summary
This work will fundamentally address the mechanism by which nematode populations are suppressed in certain soils. It will allow for the detection of novel factors that inhibit nematode function, that are produced by bacteria that cannot yet be grown in the laboratory. This work will help illuminate the fundamental question of interactions between environmental bacteria and potential predatory nematodes. The discovery of new toxins that inhibit nematode growth may allow for the development of novel products for agricultural use. More broadly, understanding the mechanism by which nematodes are inhibited in soils may allow for the development of techniques to enhance naturally occuring nematotoxicity. Finally, this work will establish techniques for discovering functional genes from uncultured soil bacteria using a soil bacterium as a host for gene expression.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2153130110050%
2154010112050%
Knowledge Area
215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
3130 - Nematodes; 4010 - Bacteria;

Field Of Science
1120 - Nematology; 1100 - Bacteriology;
Goals / Objectives
Purpose: We will examine the role of uncultured bacteria in the structure of suppressive soils. This analysis will be conducted by constructing metagenomic libraries from nematode suppressive soils and screening them against free-living soil nematodes to identify potential factors derived from uncultured bacteria that affect nematode population structure. Objectives 1. To make a metagenomic library of soil and rhizosphere genes cloned into V. paradoxus EPS 2. Screen the library for novel factors that influence nematode viability in the free living soil species of agricultural significance. 3. Test for factors that disrupt oocysts or reduce their viability as well as factors which reduceviability of the adult worms 4. Identify these factors by subcloning and sequence analysis Outputs 1.Presentation of methods at American Society for Microbiology General Meeting (by a student associated with the project) 2.Professional growth by the PI in nematode culture techniques and metagenomic DNA purification and analysis 3.Publication of screening results and genes identified 4.Preparation of research grants with Orwin, DeLey, and Crowley as potential collaborators to further analyze this system.
Project Methods
1) Based on work done in our lab, we propose to use the soil gram negative bacterium V. paradoxus strain EPS for this work. In addition to the attractive qualities cited above, the complete draft genome sequence of this organism should be available soon (undertaken with several co-PIs and the Joint Genome Institute). 2) We will clone by PCR the hydrogen cyanide synthase gene from Pseudomonas aeruginosa PAO1. This gene has been identified in deletion studies as responsible for nematocidal activity (15). The expression of this gene will be controlled using the tetON/OFF system (Invitrogen) which allows for very tight control of gene expression, and will allow for determination of HCN toxicity in vitro. Some of this work will be done prior to the beginning of the sabbatical period, in the PDs lab at CSUSB. Initial testing of this construct against C. elegans will be performed at CSUSB as well. These experiments will establish the baseline for analysis of genes from the uncultured metagenome. 3) We will collect soil from a suppressive soil plot at UCR. The genomic DNA from this soil(including rhizosphere soil) will be cloned into the pEZ BAC system(Lucigen) for cloning large genomic DNA fragments into E. coli. The DNA from these BAC clones will be subcloned into pBBR1MCS2 with an expression construct promoter (based on tetON/OFF)(kanR) for transfer into V. paradoxus EPS by conjugation. This work will be done in the laboratory of Dr. David Crowley at UC Riverside. 4) The constructs in Variovorax paradoxus will be tested in co-culture with a selection of free living bacteriovorous nematodes from the collection of Dr. Paul De Ley at UC Riverside. Based on evaluation of these organisms from the Cephalobids, we will select one or more model free living bacteriovores. Dr. De Ley has suggested Acrobeles, Acrobeloides andZeldia species as potential test organisms, based on ease of cultivation, ecological relevance,and phylogenetic analysis. Tranconjugants derived from each BAC will be tested for nematocidal and nematotoxic activities in multi-well test plates. Simple testing for nematocidal activity will be complemented by video microscopy to assess the health of the nematodes challenged with strains from the library, ovicidal activity will be assessed as well. Testing of transconjugants will be performed in Dr. De Ley's lab. 5) Nematocidal and nematotoxic strains will be retested, and the gene(s) will be identified by subcloning and sequencing.

Progress 09/01/09 to 06/30/11

Outputs
OUTPUTS: Training - this funding for this project was used to train three students at University of California Riverside. The three students, Brian Thater, Jean-Paul Baquiran, and Sammy Sedky, were mentored and trained in DNA purification from environmental samples, cultivation of nematodes, enrichment and isolation culture of environmental bacteria, genomic DNA amplification, cloning, sequencing, and analysis. Events- the experimental results from this work were presented at three different scientific meetings. The results were presented at the 18th International Conference on Microbial Genomics in September 2010 at Lake Arrowhead, CA, by Jean-Paul Baquiran, and a separate set of results were presented at the NemaSym conference in November 2010 in Tuscon, Arizona. The poster presented by Jean-Paul Baquiran was also presented by the PI at the NIFA PIs meeting during the ESA annual meeting in San Diego, CA PARTICIPANTS: Jean Paul Baquiran was a graduate student at UC Riverside who worked on this project during his final year of his Ph.D work. He is currently a post-doc at USC working on marine microbiology. His work with me involved substantial training in DNA extraction as well as bacterial culture work from unusual environments. Similar training was provided to Sammy Sedky, who is currently a UCR Ph.D. candidate in Nematology, and Brian Thater, who graduated with a bachelors degree in environmental science. This work was performed in collaboration with two faculty at UC Riverside, David Crowley and Paul De Ley. I continue to collaborate with them on this project and other projects relating to my interests in microbial ecology and plant growth promotion. TARGET AUDIENCES: This work is primarily to be disseminated to the academic scientific community, including faculty colleagues, research scientists with interests in nematology as well as more generally in host-microbe interactions. It is also a project that can be used to disseminate information about the complexities of microbial ecology and soil ecosystems to a wider scientifically literate audience. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This work resulted in significant change in knowledge and action on the part of the investigators. We discovered that several species of bacteria specifically associate with this nematode in a direct and repeatable way. In particular, we identified members of three genera, Ochrobactrum, Pedobacter, and Chitinophaga, that were present in every sample associated with Acrobeloides maximus and were either minimal or absent in bulk soil or C. elegans associated bacterial populations based on 16s survey approaches. We followed up on these findings by isolating two of the three genera from nematode-bacteria mixed cultures, and also by demonstrating the specificity of the interaction with those two bacterial genera, Ochrobactrum and Pedobacter, by fluorescence in situ hybridization (FISH). This work also resulted in a significant change in actions because we were able to develop a novel technique for identifying nematode associated microbial populations based on a microcosm exposure strategy. We were also able to apply FISH to worms treated in this manner, which had not previously been reported in the literature. This project adds to a growing body of evidence that suggests that host-microbe interactions with substantial specificity are widespread in the phylum Nematoda, and that these interactions play important roles in diverse environment. It is possible that future work in this vein will result in new approaches to nematode biocontrol, or to thinking about the environmental impacts of nematode mitigation strategies.

Publications

  • Baquiran JP, Thater B, Sedky S, De Ley P, Crowley DE, and Orwin PM.Culture-Independent Investigation of the Microbiome associated with the Nematode Acrobeloides maximus. Presented at the 18th International Conference on Microbial Genomics in September 2010.
  • Sedky S, Baquiran JP, Thater B, Crowley DE, De Ley P, and Orwin PM. Investigations of symbiotic interactions between soil bacteria and the free-living nematode Acrobeloides maximus. Presented at the NEMASYM: The Second Nematode-Bacteria Symbioses Research Coordination Network Meeting in Nov. 2010
  • Baquiran JP, Thater B, Sedky S, De Ley P, Crowley DE, and Orwin PM. Culture-Independent Investigation of the Microbiome associated with the Nematode Acrobeloides maximus. Submitted to PlosONE in November 2012 for expected publication in 2013