Progress 01/15/14 to 01/14/18
Outputs
Target Audience:Findings obtained in this project were presented in an invited talk at BARD Workshop: "Chemosensation of Plant - and Insect-Parasitic Nematodes and its Role in Pest Control", 25-29 June, 2017 in Ma'ale Hachamisha, Israel. Protocols and findings were also discussed with scientists visiting the University of California campus. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided professional development and training for two postdoctoral researchers to conduct research with plant parasitic nematodes and to study nematode behavior. Experience and training in giving professional presentations and in supervision of personnel was also provided. Three undergraduate students gained experience in laboratory and greenhouse research. Two of these students have presented their findings at UC Davis Undergraduate Research Conferences. Procedures and materials for behavioral studies on plant parasitic nematodes were provided to visitors from private industry and academic laboratories. How have the results been disseminated to communities of interest?The results of this research have been presented at several national and international meetings. The project PD gave a plenary presentation of the findings supported by this project at the annual meeting of the Society of Nematologists in East Lansing, Michigan in July 2015. Presentations on the findings obtained in this project were also presented by the PD as an invited presentation at a US-India bilateral workshop held in New Delhi, India, in March 2016, an oral presentation at the European Society of Nematologists meeting, Portugal, August 2016, and a presentation at the joint Southern Nematologists (S-1046) and Western Nematologists (W-3186) regional meeting in Davis, CA. A postdoctoral scientist supported by this project gave an oral presentation at the 2016 joint meeting of the Society of Nematologists and the Organization of Nematologists of Tropical America. In 2017, findings obtained in this project were presented in an invited talk at BARD Workshop: "Chemosensation of Plant - and Insect-Parasitic Nematodes and its Role in Pest Control'", in Israel. The PD also presented results of this project at NIFA project director meetings that were held during the course of this project. Results of this work and protocols have also been discussed with and presented to industry scientists working to develop alternative strategies for nematode management. Personnel from local companies have visited our laboratory to learn procedures for nematode culture and behavioral assays (eg, Marrone Bio Innovations, Davis, CA). Protocols and findings were also discussed with scientists visiting the University of California campus. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact of project: Root-knot nematodes (RKN) can parasitize over 2,000 plant species and are generally considered to be the most agriculturally damaging group of plant-parasitic nematodes worldwide. Toxic chemicals have been widely applied to protect crops from these pests. Safer management strategies are needed, but this requires more knowledge of nematode biology. These parasites feed only on living plant cells, and the infective larvae must locate and invade a host before their reserves are depleted. However, what attracts them to host roots is not known. Identification of the host chemicals that attract RKN to roots, repel them or otherwise modify their behavior has the potential to provide tools for novel and safe control strategies. This project has shown that substances produced by plant root tips are highly attractive to nematodes and has studied the properties of the attractants. Using mutant plant lines, we have also shown that the response of the plant to the hormone ethylene affects the attractiveness of host plants to nematodes. We showed that exudates from plant root tips contain a potent attractant to RKN and have chemically characterized this attractant. In addition, we showed that certain pheromone signals produced by nematodes affect their response to the root exudates. Accomplishments: Using assays developed under this project, we have shown that host plant mutants defective in ethylene signaling are more attractive to root-knot nematodes (RKN) than wild type plants. We demonstrated this difference using three different species of RKN and for three different plant species (tomato, Medicago truncatula, and Arabidopsis). In addition, in collaboration with us, a group headed by Dr. Wang, a former postdoc in my group, extended this finding to the interaction between soybean cyst nematodes and soybean. Based on our results showing that the root tip is most attractive region of the plant to nematodes, we developed a procedure to collect root exudate from root tips of tomato and the legume M. truncatula. For both plants, the exudate was highly attractive to the nematodes. This attraction activity was heat stable and did not bind to ion exchange resins. We developed a microassay to quantitatively assess attraction activity in a small amount of material. Using this assay coupled with column chromatography, we determined that the attractive components from both plant species were hydrophilic and not highly charged. Size-exclusion chromatography indicated a mass of ~400 for both plant species. This finding indicates that the attractive component is most likely a plant secondary metabolite rather than a simple sugar or sugar alcohol as we previously thought. In addition, we did not find attraction to the major sugars or sugar alcohols that were determined to be present in tomato root exudate by primary metabolite analysis. Together these findings demonstrate that a potent attractant with the same or similar properties is released from root tips of two evolutionarily distant host species. Mass spectrometry analysis has been carried out on active fractions. However, so far, the active component as not been identified. Many nematode species exude a class of compounds called ascarosides that have been shown to act as conserved pheromones to modulate their behavior in diverse ways. However, nothing has been reported about how plant parasitic nematodes respond to ascarosides. Together with the group of our collaborators (Schroeder lab, Boyce Thomas Inst.), we found that ascr#18 was the most abundant ascaroside in the exometabolome of RKN larvae. We have been carrying out exploratory assays to identify responses of RKN to the presence of this and other ascarosides. In preliminary experiments, males of the root-knot nematode species Meloidogyne hapla were found to be attracted to ascr#18, but juveniles were not. Using our pluronic gel assay, we have found that Meloidogyne javanica larvae were repelled by as little as 0.1 pmol of ascr#18, but did not respond to ascr#9, a pheromone produced by other nematode species, but not RKN. Application of ascr#18 near aggregated nematodes caused the aggregate to move away from the application site. Addition of ascr#18 to MRE resulted in loss of attraction of nematodes to the MRE. Taken together, these results indicate that ascr#18 may serve as a dispersal signal or repellent for RKN larvae.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2017
Citation:
Williamson, V.M., and Cepulyte, R. 2017. Assessing attraction of nematodes to host roots using pluronic gel medium. In: Ethylene Signaling Methods and Protocols, Brad M. Binder and G. Eric Schaller, eds., Humana Press, pp. 261-268.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Cepulyte, R., Danquah, W.B, Bruening, G., and Williamson, V.M. Potent attractant for root-knot nematodes in exudates from seedling root tips of two host species. Scientific Reports. Revision under review 4/11/18.
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Progress 01/15/16 to 01/14/17
Outputs
Target Audience:The findings obtained in this project have been presented by the PD at several US and international venues including: an invited presentation at a US-India bilateral workshop held in New Delhi, India, in March 2016; an oral presentation at the European Society of Nematologists meeting, Portugal, August 2016; a presentation at the joint Southern Nematologists (S- 1046) and Western Nematologists (W-3186) regional meeting in Davis, CA; a poster presentation at the NIFA project director meeting, Orlando, Florida, 2016. A postdoctoral scientist supported by this project gave an oral presentation at the 2016 joint meeting of the Society of Nematologists and the Organization of Nematologists of Tropical America. Audiences at these presentation included nematologists, students, soil ecologists, and industrial representatives. Personnel from local companies have also visited our laboratory to learn procedures for nematode culture and behavioral assays (eg, Marrone Bio Innovations, Davis, CA). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided professional development and training for a postdoctoral researcher to conduct research with plant parasitic nematodes and nematode behavior. Experience and training in professional presentations and supervision of personnel was also provided. Two undergraduate students gained experience in laboratory and greenhouse research. Procedures and materials for behavioral studies on plant parasitic nematodes were provided to visitors from private industry and academic laboratories. How have the results been disseminated to communities of interest?Results have been presented in several venues (see Target audience section). What do you plan to do during the next reporting period to accomplish the goals?Additional fractionation of TRE and MRE coupled with activity assays and mass spectroscopy will be carried out to identify the major attractants. The current limitation may be the small amount of active fraction available so we will scale up the production of root exudate with the aim of identifying the active chemical species. Additional types of assays to define the role of ascr#18 and other ascarosides in nematode behavior will be employed. These include assays to test the role of ascarosides in various nematode behaviors (attraction, dispersal, clumping, mating) as well as the influence that these compounds have on response of nematodes to root exudates. Two manuscripts are currently in preparation and should be completed during the next year. The first describes the work done to obtain attractive root exudates from tomato and Medicago and characterization of the properties of the attractive fractions. The second manuscript describes the role of ascarosides on root-knot nematode behavior
Impacts
What was accomplished under these goals?
We previously showed that host plant mutants defective in ethylene signaling were more attractive to root-knot nematodes than wild type plants. In collaboration with us, a group headed by Dr. Wang, a former postdoctoral scientist in my group, has extended this to the interaction between soybean cyst nematodes and soybean. In the previous period, we developed a strategy to collect tomato seedling root exudate (TRE) that is highly attractive rootknot nematodes. We showed that the activity is heat stable and does not bind to ionic exchange resins. During this period, we developed a new microassay to assess attraction activity more quantitatively and with a smaller amount of material. Using this assay, we have determined that the attractive components of TRE do not bind to a C18 column indicating that they arehydrophilic. Analysis of fractions from a size exclusion column that separates molecules in the 200 to 2000 Dalton range indicated that the major attractive component was approximately 1400 Daltons. This finding indicates that the attractive component is likely a plant secondary metabolite rather than a simple sugar or sugar alcohol as we previously thought. In addition, we did not find attraction to the major sugars or sugar alcohols that were determined to be present by primary metabolite analysis. We have also collected exudate from seedling root tips of Medicago, which is a host of root-knot nematodes but is evolutionarily distant from tomato, and determined that this exudate is highly attractive to root-knot nematodes. Fractionation coupled with activity assays indicated that the attractant in the Medicago root exudate (MRE) has properties indistinguishable from TRE and suggests that the same attractant(s) are shared by diverse plant species. Mass spectrometry analysis has been carried out on active fractions of both TRE and MRE. While a few mass peaks consistent with the 1400 Dalton size range are detected, so far no peak corresponding to the active compound has been unambiguously identified. Ascarosides are the main class of pheromones found in nematodes. Together with our collaborators, we found that ascr#18 was the most abundant ascaroside in the exometabolome of root-knot nematode juveniles. There appear to be life stage and species-specific differences in nematode response to this ascaroside. Males of the root-knot nematode species M. hapla are attracted to ascr#18, but juveniles are not. However, juveniles of a different species, M. javanica are attracted to ascr#18 at picomolar concentrations. Root-knot nematodes do not appear to respond to ascr#7, an ascaroside produced by the freeliving nematode Caenorhabditis elegans.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Cepulyte-Rakauskiene, R., and Williamson, V.M. 2016. Investigation of root-knot nematode male behavior in pluronic gel.
J. Nematol. 48:308-309 (abst)
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2017
Citation:
Hu, Y., You, J., Li, C. Williamson, V.M., and Wang, C. 2017. Ethylene response pathway modulates attractiveness of plant
roots to soybean cyst nematode Heterodera glycines. Scientific Reports 7:41282.
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Progress 01/15/15 to 01/14/16
Outputs
Target Audience:The project PD gave a plenary presentation of the findings supported by this project at the annual meeting of the Society of Nematologists in East Lansing, Michigan in July 2015. Nematologists, students, soil ecologists, and industrial representatives were addressed in this presentation. This work has also been presented in various on campus venues to students and extension personnel and has been discussed with and presented to industry scientists working to develop alternative strategies for nematode management. With the increasing restriction on chemical controls, alternative strategies for management of plant parasitic nematodes have become increasingly urgent targets for agriculture support industries. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?See target audience section. What do you plan to do during the next reporting period to accomplish the goals?Additional primary and secondary metabolite analysis of TRE and fractions will be carried out. Samples to be analyzed include exudates of roots from tomato seedlings that differ in ethylene signaling capability due to carrying ethylene signaling mutations or treatment with AVG. These exudates differ in attraction activity to nematodes and differential levels of specific metabolites and should provide information on molecules that are attractants or repellents to nematodes. Metabolite analysis will also be carried out on volatile fractions collected from tomato roots. Activity-guided fractionation of deionized TRE will be continued to resolve active components. Candidate compounds, including those that we have already identified, will be tested for activity in Pluronic gel-based assays. Assays to define the role of ascr#18 and other ascarosides in nematode behavior will be pursued. The optimal concentrations, specificity of chemical structure, and effect of ascr#18 on rate of movement of juveniles and adult males will be measured. To address the final goal of the project, we will test the hypotheses that we have developed by combining and adding, under different timing regimes, the components (seedling roots, tested chemicals, ascarosides, etc) identified in our work as contributing to host attraction. Two manuscripts are currently in preparation and are targeted for completion during the next funding period. The first describes the work done to obtain attractive tomato root exudates and characterize the attractive fractions. The second manuscript describes the role of ascarosides on root-knot nematode behavior.
Impacts
What was accomplished under these goals?
In the previous period, we showed that host plant mutants defective in ethylene signaling were more attractive to nematodes than wild type plants. We have now assessed the effect of exposing tomato seedlings to 2-aminoethoxyvinylglycine (AVG), a compound that reduces ethylene synthesis in plants by inhibiting the enzyme ACC synthase, on their attractiveness to root-knot nematodes (RKN). When Pluronic F-127 (PF127) gel was augmented with 1 µM AVG prior to the start of the assay, roots were significantly more attractive to M. hapla and M. javanica J2 than when the assay was carried out without AVG. Roots pretreated with 1 µM AVG for 1 hr prior to placement in the PF127 gel with suspended J2 were also more attractive to both nematode species. This finding provides us a new tool for collecting root exudate with differential levels of attraction to be used for metabolite analysis (see below). In the previous period, we developed a strategy to collect tomato seedling root exudate (TRE) that is highly attractive root-knot nematodes. We have shown using our Pluronic gel assay activity is retained following filtration through a 10,000 kD filter, indicating that active component is a relatively small chemical. Also, the TRE remains attractive following heating to 100 C, indicating that the active component is heat stable. When TRE was passed through a mixed-bed resin ion exchange, activity was found in the flow through fraction suggesting that the active components were uncharged but highly water-soluble. GC-MS analysis of exudate fractions was carried out to assess the primary metabolites present. This analysis revealed that TRE contains a complex complement of primary metabolites with 193 known and 318 unknown metabolites detected. Amino acids were the most abundant category of known primary metabolites identified followed by organic acids, sugars and sugar alcohols. Following passage of the TRE through a mixed bed resin column, amino acids and organic acids were largely depleted and sugar alcohols were the most abundant known components remaining together make up >70% to total metabolites detected. Eight of the 10 most abundant primary metabolites in the deionized TRE were identified by GC-MS, and all of these were sugars or sugar alcohols. Activity assays on the most abundant sugars and sugar alcohols present in the deionized TRE are currently in progress. To more accurately measure the activity of TRE fractions and individual chemicals, we are developing a more quantitative assay based in injection of a small amount (10 microliters) of the test fraction/chemical into the Pluronic gel. Ascarosides are the main class of pheromones found in nematodes. Together with our collaborators, we found that ascr#18 was the most abundant ascaroside found in the exometabolome of root-knot nematode juveniles. Prexposure of nematode juveniles to nanomolar levels of ascr#18 increases their rate of accumulation at seedling roots and also the rate at which they aggregate into tight clumps. However, plants also appear to perceive ascarosides to initiate a defense response. In a greenhouse assay, we have found that tomato seedlings pretreated with ascr#18 before inoculation with nematodes supported lower levels of root knot nematode reproduction than untreated control plants. We have also documented used a pluronic gel-based assay that male nematodes of the Northern root knot nematode are attracted to ascr#18. For both plant and nematode responses, the sensitivity to ascr#18 is high, requiring nanomolar levels, and dose dependent. The findings described above and in the previous progress report have led to the following testable hypotheses to address the fourth goal of this project. Multiple, simple compounds excreted from plant root tips may be perceived by the nematode's complex receptor system to direct the juveniles to a healthy root tip. Sugars and/or sugar alcohols appear to be key components of the active secretions. Root-knot nematodes are social animals and communication between individuals is key to mounting a successful infection. This communication is initiated by perception of an appropriate host entry site and transmitted by ascaroside blends and possibly other intraspecies signals.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Manosalva, P., Manohar, M., von Reuss, S.H., Chen, S., Koch, A., Kaplan, F., Choe, A., Micikas, R.J., Wang, X., Kogel, K.H., Sternberg, P.W., Williamson, V.M., Schroeder, F.C., and Klessig, D.F. 2015. Conserved nematode signaling molecules elicit plant defenses and pathogen resistance. Nature Commun. 6:7795 doi: 10.1038/ncomms8795
- Type:
Book Chapters
Status:
Under Review
Year Published:
2016
Citation:
Williamson, V.M. and ?epulyt?-Rakauskien?, R. 2016. Assessing attraction of nematodes to host roots using Pluronic gel medium. In Methods in Molecular Biology,B. M. Binder and G. E. Schaller, eds.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Williamson, V.M., W.B. Danquah, and F. Schroeder. J. 2015. Root-knot nematode behavior in response to plant and nematode semiochemicals. J. Nematol. 17:278 (abst)
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Progress 01/15/14 to 01/14/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One post doc and two undergraduates learned techniques in my lab and greenhouse. One undergraduate student has now graduated and obtained a position in Agricultural Biotechnology How have the results been disseminated to communities of interest? Poster presented atXVI International Congress on Molecular Plant-Microbe Interactions" in Rhodes, Greece. What do you plan to do during the next reporting period to accomplish the goals? Activity-guided fractionation of water-soluble root exudates and identification of specific attractants for root-knot nematodes is planned. Assays to date suggest that repellents may also be present in the exudates so it is possible that we will also identify specific repellants. Attempts to obtain VOC fractions with stable activity and to identify the active components will continue. The novel finding that infective juveniles preincubated in worm supernatant or in synthetic ascarosides accumulate at roots at a higher rate will be pursued. Rate of movement of the worms will be compared using in vitro assays. Also, we will test the possibility that incubation in the ascaroside causes developmental changes in the nematode using microscopy, specific dyes, and RNA analysis. Experiments reported above suggest that a component of the nematode supernatant promotes aggregation. Fractionation of the supernatant will be carried out in order to identify the active components. Combinations of exudate fractions applied with different timing regimes will be initiated in order to begin to gain an understanding of how nematodes integrate these signals for appropriate responses as proposed in Goal 4. PD Williamson is scheduled to present an invited talk at Society of Nematologists annual meeting (2015) on this work. Two manuscripts are targeted for completion during the next period, one on the activity-based fractionation of root extracts and the second on nematode behavior and development in response to ascarosides and other nematode-generated semiochemicals.
Impacts
What was accomplished under these goals?
The first goal of this project was to compare the attraction of different root-knot nematode (RKN) species to different host plant roots in order to find differences that would be useful for identifying semiochemicals that modulate attraction of RKN to their hosts. We compared attraction to Medicago truncatula and Solanum lycopersicum (tomato) seedlings of infective juveniles of three species of RKN, Meloidogyne javanica, M. incognita and M. hapla. Attraction to tomato cv Rutgers of M. hapla strain VW9 was significantly higher than M. javanica strainVW4 or M. incognita strain VW6. Also, attraction was significantly higher to tomato roots than to M. truncatula roots for all RKN species tested. We also compared attraction of RKN to plant mutants affected in ethylene perception as we had previously found that roots of mutants in ethylene perception were more attractive than wild type Arabidopsis. We multiplied seed for the following mutants and parental lines: M. truncatula wild type A17 and hypernodulation mutants sunn and skl; S. lycopersicum cv Rutgers and corresponding ethylene perception mutant Never ripe (Nr). In our in vitro attraction assay, seedling roots of mutants deficient in ethylene signaling in tomato and M. truncatula, Nr and skl, respectively, were more attractive than wild type for all three RKN species. This indicates that ethylene-signaling mutants in both species as well as in Arabidopsis either produced more attractant or less repellent than wild type and that the chemical differences were due to downstream signal produced following ethylene perception. Cell-free exudate from tomato roots of the tomato mutant Nr collected in pluronic gel showed higher attraction than the wild type. Comparison of exudate profiles of wild type and Nr should be useful for identification of host semiochemicals contributing to attraction or repellency as proposed in Goal 2. The second goal is to identify plant-generated semiochemicals critical for RKN attraction to host roots. Both dissolved components (DC) and volatile organic compounds (VOC) are thought to be involved in directing nematode juveniles to root tips of appropriate hosts. Previous experiments had found that exudates secreted from root tips of tomato seedlings are highly attractive to RKN. An apparatus was designed to collect aqueous exudate from the terminal 10 mm of 500 six-day old tomato root tips for 24 hrs. This tomato root exudate (TRE) was highly attractive to M. javanica VW4, but not M. hapla VW9. Size exclusion filtration of the TRE indicated that the attractant(s) was <10,000 kDaltons. Analysis by the UC Davis Metabolomics Center (GC-TOF) revealed a complex mixture of organic compounds was present in the TRE. The attraction activity could be extracted into ethyl acetate in a pH dependent manner. When TRE was acidified to pH3, attraction activity was partitioned into ethyl acetate. This activity could be re-extracted into the aqueous phase by increasing the pH, suggesting that attractants include organic acids. Additional activity-guided fractionation is in progress. Several strategies were tested to collect VOC from tomato root systems. Most successful was a design in which VOCs were collected from roots of 8-weeks-old tomato plants using a cold trap. Attraction assays demonstrated that the collected fraction was highly attractive to all three RKN species. Attraction to VOC appeared to be more rapid than for the DC. However, this activity is unstable so far. Many species of nematodes secrete small-molecules or pheromones that have been shown to mediate communication between individuals of that species. The best-characterized nematode pheromones are ascarosides, a family of compounds based on the sugar ascarylose with various side chains and decorations. Specific ascarosides or blends can act as attractants or otherwise modulate behavior and development of the nematode. Ascaroside#18 was previously identified as the most abundant ascaroside in aqueous supernatants following incubation with root-knot nematode juveniles. We found that incubating juveniles of three different RKN species in 10 nM ascaroside#18 for 48 hr prior to exposure to tomato seedlings resulted in a significantly increased rate of accumulation at root tips compared to control treatments. Treatment with ascaroside#7, which was not detected in RKN juvenile exudates, also increased to rate of accumulation, but to a lesser extent. These experiments suggest that incubation in the ascaroside increases movement rate of the juveniles or causes them to be more sensitive to root exudates. Experiments to differentiate these possibilities are being developed. While investigating the effect of the ascarosides on attraction to roots, we noted that the J2 pre-soaked in the worm supernatant aggregated into clumps overnight whereas worms presoaked in comparable concentrations of water, ascr#18 or ascr#7 did not aggregate. This suggests that compounds in the worm supernatant other than ascr#18 are responsible for the aggregation. Activity-guided fractionation of the supernatant will be carried out to identity the component responsible for the enhanced aggregation. Work on the fourth goal, to discover the order and contributions of chemical events that direct arrival and accumulation of root-knot nematodes at the root tip site of entry, will be carried out in the following years once more basic information is obtained.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
A poster with the title "The Chemical Ecology of Roo-knot Nematode Targeting to Plant Root Entry Points" was presented at the XVI International Congress on Molecular Plant-Microbe Interactions" in Rhodes, Greece. July 4-7, 2014. An abstract for this poster is in the program for the meeting.
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